Characterization of Sideritis clandestina subsp. peloponnesiaca Polar Glycosides and Phytochemical Comparison to Other Mountain Tea Populations.

Sideritis clandestina (Bory & Chaub.) Hayek subsp. peloponnesiaca (Boiss. & Heldr.) Baden (SCP) is endemic to the mountains of the Northern Peloponnese (Greece). This and other Sideritis taxa, collectively known as mountain tea, are widely ingested as beverages for refreshment or medicinal purposes. We describe a methodology for the characterization of SCP. Four iridoid glycosides (monomelittoside, melittoside, ajugoside, and 7-O-acetyl-8-epiloganic acid), two phenolic acid glycosides (vanillic and salicylic acid glycosides), and three caffeoyl ester glycosides (chlorogenic acid, verbascoside, and isoverbascoside) were isolated from SCP for the first time. We used ultrasound-assisted extraction of 3 g of plant material to produce petroleum ether and aqueous extracts, which we then analyzed using GC/MS and LC/MS. This was applied to eight samples from four different taxa. In total, 70 volatile and 27 polar metabolites were determined. The S. clandestina samples had a lower phenolic content and weaker antioxidant properties than S. raeseri and S. scardica. However, S. clandestina ssp. clandestina seemed to be the most aromatic taxon, with almost double the number of volatiles as the others. Τhis study could contribute to authentication and chemotaxonomic studies of Sideritis taxa.

Nutritional Value of Eggplant Cultivars and Association with Sequence Variation in Genes Coding for Major Phenolics.

Eggplant is a widely consumed vegetable, with significant nutritional value and high antioxidant content, mainly due to its phenolic constituents. Our goal was to determine the levels of carbohydrates, proteins, total phenolics, anthocyanins, flavonoids, chlorogenic acid, and the antioxidant capacity in thirteen eggplant cultivars cultivated in Greece and to identify sequence polymorphisms in key regulating genes of the phenylpropanoid pathway (C4H, HCT, HQT, C3H, F3H, ANS, MYB1), which might relate to the phytochemical content of those cultivars. The carbohydrates' content differs among and within cultivars, while the rest of the phytochemicals differ only among cultivars. The cultivars 'EMI' and 'Lagkada' scored higher than the rest in phenolics, anthocyanins, ascorbic acid, caffeoylquinic acid, and antioxidant capacity. Moreover, significant correlations were observed between various ingredients and the antioxidant capacity (FRAP and DPPH). Sequence analysis revealed several SNPs in C4H, HQT, F3H, ANS, and MYB1 among the cultivars studied. According to chi-square and logistic regression analyses, the missense mutation C4H4-108 correlates significantly with flavonoids, anthocyanins, and proteins; the synonymous mutation HQT-105 correlates with anthocyanins and ascorbic acid; the missense mutation HQT-438 correlates with flavonoids and chlorogenic acid, while the missense mutation ANS1-65 correlates with anthocyanins and sugars. These polymorphisms can be potentially utilized as molecular markers in eggplant breeding, while our data also contribute to the study of eggplant's secondary metabolism and antioxidant properties.

GC-MS and LC-DAD-MS Phytochemical Profiling for Characterization of Three Native Salvia Taxa from Eastern Mediterranean with Antiglycation Properties.

Salvia fruticosa and S. pomifera subsp. calycina are native to Eastern Mediterranean and S. pomifera subsp. pomifera is endemic to Greece. The primary aim of this study was to develop an analytical methodology for metabolomic profiling and to study their efficacy in combating glycation, the major biochemical complication of diabetes. After sequential ultrasound-assisted extraction of 2 g of leaves with petroleum ether and 70% methanol, the volatile metabolites in the petroleum ether extracts were studied with GC-MS (Gas Chromatography-Mass Spectrometry), whereas the polar metabolites in the hydroalcoholic extracts were determined and quantified by UHPLC-DAD-ESI-MS (Ultra-High Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry). This methodology was applied to five populations belonging to the three native taxa. 1,8-Cineole was the predominant volatile (34.8-39.0%) in S. fruticosa, while S. pomifera had a greater content of α-thujone (19.7-41.0%) and ß-thujone (6.0-39.1%). Principal Component Analysis (PCA) analysis of the volatiles could discriminate the different taxa. UHPLC-DAD-ESI-MS demonstrated the presence of 50 compounds, twenty of which were quantified. PCA revealed that not only the taxa but also the populations of S. pomifera subsp. pomifera could be differentiated. All Salvia samples inhibited advanced glycation end-product formation in a bovine serum albumin/2-deoxyribose assay; rosmarinic and carnosic acid shared this activity. This study demonstrates the antiglycation activity of S. fruticosa and S. pomifera extracts for the first time and presents a miniaturized methodology for their metabolomic profiling, which could aid chemotaxonomic studies and serve as a tool for their authentication and quality control.

Protective antioxidant effects of saffron extract on retinas of streptozotocin-induced diabetic rats.

Objective: Oxidative stress plays an important role in the pathogenesis of diabetic retinopathy. The aim of the present study was to investigate the effect of Crocus sativus L. styles (saffron) extract on oxidative stress indices of retina in streptozotocin (STZ)-induced diabetic rats. Methods: Adult male Wistar rats (n=20) were randomized into the following 4 groups (n=6-7/ group): Control group (C): normal, Control + Saffron group (CS): non-diabetic rats treated with 60 mg/ kg of saffron extract, Diabetic group (D) and Diabetic + Saffron group (DS): diabetic rats treated with 60 mg/ kg saffron extract. We determined the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) as markers of antioxidant response, as well as malondialdehyde (MDA) as a marker of lipid peroxidation. Results: Induction of diabetes caused a significant decline in the activities of CAT (76.43%), SOD (53.43%) and GPx (77.58%). MDA levels were significantly lower in the DS group (0.878 ± 0.375 nmol MDA/ mg protein) as compared to D group (1.950 ± 0.299 nmol MDA/ mg protein, p<0.01) and in the CS group (0.503 ± 0.221) in comparison to C group (1.699 ± 0.454, p<0.01). Moreover, SOD and GPx activities were significantly higher (more than 1.5 and 3.5-fold respectively) after treatment with saffron (p<0.01). Regarding the retinas of non-diabetic animals, the administration of the extract caused an > 1.8-fold increase in the activity of CAT (p<0.05) and a 3-fold decrease in MDA levels (p<0.01). Conclusions: This study showed that saffron extract has a protective antioxidant action in retinas of diabetic rats. Abbreviations: C = Control group, CS = non-diabetic rats diabetic rats treated with 60 mg/ kg saffron extract, D = diabetic group, DS = diabetic rats treated with 60 mg/ kg saffron extract, SOD = superoxide dismutase, GPx = glutathione peroxidase, CAT = catalase, MDA = malondialdehyde, DM = diabetes mellitus, DR = diabetic retinopathy, ROS = reactive oxygen species, STZ = streptozotocin, GSH = reduced glutathione.

Variability in Bulb Organosulfur Compounds, Sugars, Phenolics, and Pyruvate among Greek Garlic Genotypes: Association with Antioxidant Properties.

In order to assess the diversity of Greek garlic (Allium sativum L.) landraces, 34 genotypes including commercial ones were grown in the same field and their content in organosulfur compounds, pyruvate, total sugars, and total phenolics, alongside antioxidant capacity, was determined. The organosulfur compounds were studied by Gas Chromatography-Mass Spectrometry (GC-MS) after ultrasound-assisted extraction in ethyl acetate, identifying 2-vinyl-4H-1,3-dithiin and 3-vinyl-4H-1,2-dithiin as the predominant compounds, albeit in different ratios among genotypes. The bioactivity and the polar metabolites were determined in hydromethanolic extracts. A great variability was revealed, and nearly one-third of landraces had higher concentration of compounds determining bioactivity and organoleptic traits than the imported ones. We recorded strong correlations between pyruvate and total organosulfur compounds, and between antioxidant capacity and phenolics. In conclusion, chemical characterization revealed great genotype-dependent variation in the antioxidant properties and the chemical characters, identifying specific landraces with superior traits and nutritional and pharmaceutical value.

High Resolution Melting (HRM) Genotyping in the Genus Origanum: Molecular Identification and Discrimination for Authentication Purposes.

Origanum L. (Lamiaceae) is an important genus of medicinal and aromatic plants used since ancient times as culinary herbs and remedies in traditional medicine. Although it is a relatively small genus, intra-generic species delineation, as well as its inter-generic relationships within tribe Mentheae, are still poorly understood. High resolution melting (HRM) analysis, coupled with microsatellite markers (SSRs), could facilitate the molecular identification and characterization of certain genotypes more efficiently and relatively faster when compared to other analytical methods. In this study, 38 Origanum samples corresponding to six Origanum taxa (O. dictamnus, O. majorana, O. onites, O. scabrum, O. sipyleum, and O. vulgare subsp. hirtum) were analyzed, using six microsatellite loci. Our goal was to molecularly identify and discriminate among the selected samples and to evaluate the ability of the HRM technique as an analytical tool for the discrimination of Origanum species from Greece. The temperature-shifted melting curves produced by the HRM analysis, resulted in 98 unique HRM profiles, which enabled the discrimination of the Origanum genotypes studied. According to the similarity dendrogram based on the HRM profiles, six unique clusters were formed, each one corresponding to a single taxon. In conclusion, HRM genotyping provided a fast, cost-effective method, well suited for the molecular characterization and identification of Origanum taxa and for the authentication of the original genetic material.

Metabolomic fingerprinting and genetic discrimination of four Hypericum taxa from Greece.

Hypericum perforatum has plenty of uses in traditional medicine and is the source of top-selling herbal drugs and food supplements. The secondary metabolite chemistry for most of the nearly 500 Hypericum taxa is still unknown, even though they are used interchangeably. In the present study, we characterized four Hypericum populations from Achaia, Greece, belonging to H. perforatum ssp. veronense, H. perfoliatum, H. triquetrifolium, and an uninvestigated taxon, H. empetrifolium ssp. empetrifolium, in terms of their essential oils and polar bioactives in methanolic extracts via GC-MS, LC-HRMS, LC-DAD-MS, and HPLC-DAD. We also performed sequence analysis of nrITS to explore the genetic profile of these taxa and to examine whether their genotype is correlated to the metabolome. Sixty-three non-volatile compounds, phloroglucinols in their majority, and over one hundred (113) volatiles, mostly sesqui- and mono- terpenes, were detected. The concentration of the major polar constituents varied greatly among samples. In particular, phloroglucinols' diversity and abundance in H. empetrifolium ssp. empetrifolium was remarkable. The PCA and Biplot analysis revealed the contribution of each compound to the total chemodiversity and also revealed certain compounds that contribute to the discrimination of the samples. Sequence analysis of nrITS revealed different genetic profiles and markers which can be used for the identification of the four Hypericum taxa. The Mantel test showed a relatively strong correlation between the genetic profile and the volatile compounds and low with the main polar metabolites.

Physical and chemical quality characteristics and antioxidant properties of strawberry cultivars (Fragaria × ananassa Duch.) in Greece: assessment of their sensory impact.

BACKGROUND: There are many factors determining the strawberry organoleptic profile and they are difficult to define. In this study, the sensory, physical, and chemical quality characteristics, the antioxidant properties as examined using ferric reducing antioxidant power (FRAP) and 1-diphenyl-2-picrylhydrazyl (DPPH) assays, the lactone concentration, and the FaFAD1 expression of ripe strawberries (cv. Camarosa, Florida Fortuna, and Sabrina) from Greece were evaluated and their interrelationships were investigated. RESULTS: 'Camarosa' had the highest antioxidant capacity and polyphenol content, although significant intra-cultivar variations of sugars, solid soluble content/titratable acidity (SSC/TA), red color intensity, sweetness, and hardness were recorded. In 'Sabrina' there was a constant lactone presence and FaFAD1 expression; it also had the lowest ascorbic acid content, the highest pH, SSC/TA index, firmness, and sweetness. 'Fortuna' showed the lowest sweetness and aroma indices, whereas 'Camarosa' had intermediate ones. Overall, firmness was correlated with hardness, while pH and SSC/TA index correlated with juiciness and sweetness. Both γ-decalactone and γ-dodecalactone concentrations were correlated with FaFAD1 expression and pH, but they did not solely determine the aroma sensory perception. In total, FRAP values were positively correlated with ascorbic acid and polyphenol content, and negatively with pH. CONCLUSIONS: Significant inter- and intra-cultivar variation was recorded, revealing the impact of the genotype and underlining the effect of microenvironmental and cultivation conditions on quality and sensory perception. © 2018 Society of Chemical Industry.

Optimized and Validated HPLC Analysis of St. John's Wort Extract and Final Products by Simultaneous Determination of Major Ingredients.

Aim of this work was to develop a validated high performance liquid chromatography method for the analysis of extracts and final products of St. John's wort, according to international guidelines for bioanalytical method validation. Chromatographic separation was performed on a C18 column with a combination of gradient and isocratic steps; the mobile phase composed of ammonium acetate solution (pH 4.5; 10 mM), acetonitrile and methanol. Quantification and method validation was performed using extract spiked with external reference standards of chlorogenic acid, rutin, hyperoside, isoquercitrin, quercetin and hypericin. Validation study revealed that trans-chlorogenic acid is partially transformed into its cis-isomer during analysis. The method showed good linearity, precision and accuracy. Hyperforin was completely unstable. All other ingredients were stable at -18°C and after three freeze-thaw cycles, while stability of most ingredients was limited at room temperature and 4 - 8°C; quercetin was the most unstable one. The major ingredients of methanolic extracts, infusions and final products of Hypericum perforatum were completely resolved and quantified. Beyond its potential usefulness in the analysis of St. John's wort products, this study addresses the issue of validation from the perspective of the field of bioanalysis and reveals the wealth of critical information which can be derived.

Transient winter leaf reddening in Cistus creticus characterizes weak (stress-sensitive) individuals, yet anthocyanins cannot alleviate the adverse effects on photosynthesis.

Under apparently similar field conditions individual plants of Cistus creticus turn transiently red during winter, while neighbouring plants remain green. These two phenotypes provide a suitable system for comparing basic photosynthetic parameters and assessing critically two hypotheses, i.e. anthocyanins afford photoprotection and anthocyanins induce shade characteristics on otherwise exposed leaves. With that aim, pigment levels and in vivo chlorophyll fluorescence parameters were monitored in dark-acclimated (JIP-test) and light-acclimated (saturation pulse method) leaves during both the green and the red period of the year. No evidence for actual photoprotection by anthocyanins was obtained. On the contrary, all fluorescence parameters related to yields and probabilities of photochemical energy conversion and electron flow, from initial light trapping to final reduction of ultimate electron acceptors in PSI, declined in the red phenotype after leaf reddening. Moreover, the pool sizes of final electron acceptors of PSII diminished, indicating that both photosystems were negatively affected. Vulnerability to winter stress was also indicated by sustained chlorophyll loss, inability to increase the levels of photoprotective xanthophylls and increased quantum yield of non-regulated energy loss during reddening. However, during the same period, the relative PSII antenna size increased, indicating an apparent shade acclimation after anthocyanin accumulation, while changes in the photosynthetic pigment ratios were also compatible to the shade acclimation hypothesis. All parameters recovered to pre-reddening values upon re-greening. It is concluded that the photosynthetic machinery of the red leaf phenotype has an inherently low capacity for winter stress tolerance, which is not alleviated by anthocyanin accumulation.