ANTIAGE-DB: A Database and Server for the Prediction of Anti-Aging Compounds Targeting Elastase, Hyaluronidase, and Tyrosinase.

Natural products bear a multivariate biochemical profile with antioxidant, anti-inflammatory, antibacterial, and antitumoral properties. Along with their natural sources, they have been widely used both as anti-aging and anti-melanogenic agents due to their effective contribution in the elimination of reactive oxygen species (ROS) caused by oxidative stress. Their anti-aging activity is mainly related to their capacity of inhibiting enzymes like Human Neutrophil Elastase (HNE), Hyaluronidase (Hyal) and Tyrosinase (Tyr). Herein, we accumulated literature information (covering the period 1965-2020) on the inhibitory activity of natural products and their natural sources towards these enzymes. To navigate this information, we developed a database and server termed ANTIAGE-DB that allows the prediction of the anti-aging potential of target compounds. The server operates in two axes. First a comparison of compounds by shape similarity can be performed against our curated database of natural products whose inhibitory potential has been established in the literature. In addition, inverse virtual screening can be performed for a chosen molecule against the three targeted enzymes. The server is open access, and a detailed report with the prediction results is emailed to the user. ANTIAGE-DB could enable researchers to explore the chemical space of natural based products, but is not limited to, as anti-aging compounds and can predict their anti-aging potential. ANTIAGE-DB is accessed online.

Simultaneous determination of artemisinin and its analogs and flavonoids in Artemisia annua crude extracts with the use of NMR spectroscopy.

Artemisia annua is a promising and potent antimalarial herbal drug. This activity has been ascribed to its component artemisinin, a sesquiterpene lactone. The ability to determine artemisinin and its known analogs in plant extracts is an especially difficult task because the compounds are present in low concentrations, are thermolabile, and lack ultraviolet or fluorescent chromophores. We report herein a facile and rapid 1-D 1 H, 1-D total correlation spectroscopy, 2-D 1 H-13 C heteronuclear single quantum coherence, and 1 H-13 C heteronuclear multiple bond correlation nuclear magnetic resonance techniques for the simultaneous identification and quantification of artemisinin and five of its analogs along with five flavonoids, an aromatic ketone, and camphor (in total, 13 compounds) in crude diethyl ether A. annua extract without the need of laborious isolation of the individual analytes. The above method was validated in terms of precision, linearity, and limit of detection. The analytical results were found to be in excellent agreement with those obtained with the use of the time consuming high-performance liquid chromatography with diode-array detection and liquid chromatography with tandem mass spectrometry for the compounds that standards were available.

Deconvoluting the Dual Antiplatelet Activity of a Plant Extract.

A thorough evaluation of the antiplatelet activity profile of hexane olive leaf extract in human platelets indicated a potent activity accomplished through a two axis inhibition of platelet activation triggered both by ADP and thrombin. To delineate the extract components responsible for this dual activity, an NMR based method was established to determine and quantify the triterpenoid content leading to the characterization of uvaol, erythrodiol, and oleanolic acid. The antiplatelet profile of the total extract and of the 3 determined triterpenoids was evaluated against in vitro platelet aggregation induced by several platelet agonists as also on PAC-1 binding and P-selectin membrane expression both in healthy volunteers and in platelets from patients with an acute coronary syndrome receiving dual antiplatelet therapy with aspirin and ticagrelor. The extract was identified to inhibit ADP-induced platelet activation due to its erythrodiol content and TRAP-induced platelet activation due to the activity of uvaol and oleanolic acid.

Ethyl Acetate Extract of Origanum vulgare L. ssp. hirtum Prevents Streptozotocin-Induced Diabetes in C57BL/6 Mice.

Type 1 diabetes (T1D) is an autoimmune disease that develops as a consequence of pancreatic ß-cell death induced by proinflammatory mediators. Because Origanum vulgare L. ssp. hirtum (Greek oregano) contains antiinflammatory molecules, we hypothesized that it might be beneficial for the treatment of T1D. An ethyl acetate extract of oregano (EAO) was prepared from the leaves by a polar extraction method. Phytochemical composition was determined by liquid chromatography-UV diode array coupled to ion-trap mass spectrometry with electrospray ionization interface (LC/DAD/ESI-MS(n) ). In vitro immunomodulatory effect of EAO was estimated by measuring proliferation (MTT) or cytokine secretion (ELISA) from immune cells. Diabetes was induced by multiple low doses of streptozotocin (MLDS) in male C57BL/6 mice and EAO was administered intraperitoneally for 10 d. Determination of cellular composition (flow cytometry) and cytokine production (ELISA) was performed on 12th d after diabetes induction. EAO suppressed the function of both macrophages and lymphocytes in vitro. In vivo, EAO treatment significantly preserved pancreatic islets and reduced diabetes incidence in MLDS-challenged mice. Besides down-modulatory effect on macrophages, EAO reduced the number of total CD4(+) and activated CD4(+) CD25(+) T cells. Furthermore, EAO affected the number of T helper 1 (Th1) and T helper 17 (Th17) cells through downregulation of their key transcription factors T-bet and RORγT. Because EAO treatment protects mice from development of hyperglycemia by reducing proinflammatory macrophage/Th1/Th17 response, this plant extract could represent a basis for future diabetes therapy.

PRESS: PRotEin S-Sulfenylation server.

MOTIVATION: Transient S-sulfenylation of cysteine thiols mediated by reactive oxygen species plays a critical role in pathology, physiology and cell signaling. Therefore, discovery of new S-sulfenylated sites in proteins is of great importance towards understanding how protein function is regulated upon redox conditions. RESULTS: We developed PRESS (PRotEin S-Sulfenylation) web server, a server which can effectively predict the cysteine thiols of a protein that could undergo S-sulfenylation under redox conditions. We envisage that this server will boost and facilitate the discovery of new and currently unknown functions of proteins triggered upon redox conditions, signal regulation and transduction, thus uncovering the role of S-sulfenylation in human health and disease. AVAILABILITY AND IMPLEMENTATION: The PRESS web server is freely available at http://press-sulfenylation.cse.uoi.gr/ CONTACTS: agtzakos@gmail.com or gtzortzi@cs.uoi.gr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

1H-NMR as a structural and analytical tool of intra- and intermolecular hydrogen bonds of phenol-containing natural products and model compounds.

Experimental parameters that influence the resolution of 1H-NMR phenol OH signals are critically evaluated with emphasis on the effects of pH, temperature and nature of the solvents. Extremely sharp peaks (Δν1/2≤2 Hz) can be obtained under optimized experimental conditions which allow the application of 1H-13C HMBC-NMR experiments to reveal long range coupling constants of hydroxyl protons and, thus, to provide unequivocal assignment of the OH signals even in cases of complex polyphenol natural products. Intramolecular and intermolecular hydrogen bonds have a very significant effect on 1H OH chemical shifts which cover a region from 4.5 up to 19 ppm. Solvent effects on -OH proton chemical shifts, temperature coefficients (Δδ/ΔT), OH diffusion coefficients, and nJ(13C, O1H) coupling constants are evaluated as indicators of hydrogen bonding and solvation state of phenol -OH groups. Accurate 1H chemical shifts of the OH groups can be calculated using a combination of DFT and discrete solute-solvent hydrogen bond interaction at relatively inexpensive levels of theory, namely, DFT/B3LYP/6-311++G (2d,p). Excellent correlations between experimental 1H chemical shifts and those calculated at the ab initio level can provide a method of primary interest in order to obtain structural and conformational description of solute-solvent interactions at a molecular level. The use of the high resolution phenol hydroxyl group 1H-NMR spectral region provides a general method for the analysis of complex plant extracts without the need for the isolation of the individual components.

Olive leaf extracts are a natural source of advanced glycation end product inhibitors.

Advanced glycation end products (AGEs), which are readily formed and accumulated with sustained hyperglycemia, contribute to the development of diabetic complications. As a consequence, inhibition of AGE formation constitutes an attractive therapeutic/preventive target. In the current study, we explored the phytochemical composition and the in vitro effect of two different olive leaf extracts (an aqueous and a methanolic) on AGE formation. The methanolic olive leaf extract inhibited fluorescent AGE formation in a bovine serum albumin (BSA)-ribose system, whereas the aqueous extract had no effect in both BSA-fructose and BSA-ribose systems. The phytochemical profile was investigated with liquid chromatography-ultraviolet-visible (UV-Vis) diode array coupled to electrospray ionization multistage mass spectrometry (LC/DAD/ESI-MS(n)). Quantification of the major phenolic compounds was performed with high performance liquid chromatography with UV-Vis diode array detection and nuclear magnetic resonance spectroscopy. Among the major phenolic components (luteolin, hydroxytyrosol, luteolin-4'-O-ß-D-glucopyranoside, luteolin-7-O-ß-D-glucopyranoside, and oleuropein), luteolin and luteolin-4'-O-ß-D-glucopyranoside were assigned as potent inhibitors of AGE formation. The extraction procedure greatly affects the composition and therefore the anti-glycation potential of olive leaves.

Phytochemical composition of "mountain tea" from Sideritis clandestina subsp. clandestina and evaluation of its behavioral and oxidant/antioxidant effects on adult mice.

PURPOSE: The goals of this study were to monitor the effect of drinking of herbal tea from Sideritis clandestina subsp. clandestina for 6 weeks on behavioral and oxidant/antioxidant parameters of adult male mice and also to evaluate its phytochemical composition. METHODS: The phytochemical profile of the Sideritis tea was determined by liquid chromatography-UV diode array coupled to ion-trap mass spectrometry with electrospray ionization interface. The effects of two doses of the herbal infusion (2 and 4% w/v, daily) intake on anxiety-like state in mice were studied by the assessment of their thigmotactic behavior. The oxidant/antioxidant status of brain (-Ce), liver and heart of adult male Balb-c mice following the consumption of Sideritis tea was also evaluated via the measurement of malondialdehyde (MDA) and reduced glutathione (GSH) levels using fluorometric assays. Our study was further extended to determine the antioxidant effects of the herbal tea on specific brain regions (cerebral cortex, cerebellum and midbrain). RESULTS: The identified compounds were classified into several natural product classes: quinic acid derivatives, iridoids, phenylethanol glycosides and flavonoids. Our results showed that only the 4% Sideritis tea exhibited anxiolytic-like properties as evidenced by statistically significant (p < 0.05) decrease in the thigmotaxis time and increase in the number of entries to the central zone in comparison with the control group. Consumption of both tea doses (2 and 4% w/v) elevated GSH (12 and 28%, respectively, p < 0.05) and decreased MDA (16 and 29%, p < 0.05) levels in brain (-Ce), while liver and heart remained unaffected. In regard to the effect of herbal tea drinking (2 and 4% w/v) on specific brain regions, it caused a significant increase in GSH of cerebellum (13 and 36%, respectively, p < 0.05) and midbrain (17 and 36%, p < 0.05). Similarly, MDA levels were decreased in cerebellum (45 and 79%, respectively, p < 0.05) and midbrain (50 and 63%, respectively, p < 0.05), whereas cerebral cortex remained unaffected. CONCLUSIONS: Mountain tea drinking prevents anxiety-related behaviors and confers antioxidant protection to rodent's tissues in a region-specific, dose-dependent manner, and its phytochemical constituents are shown for the first time.

Phytochemical profile of Rosmarinus officinalis and Salvia officinalis extracts and correlation to their antioxidant and anti-proliferative activity.

The goal of this study was to monitor the anti-proliferative activity of Rosmarinus officinalis and Salvia officinalis extracts against cancer cells and to correlate this activity with their phytochemical profiles using liquid chromatography/diode array detection/electrospray ion trap tandem mass spectrometry (LC/DAD/ESI-MS(n)). For the quantitative estimation of triterpenic acids in the crude extracts an NMR based methodology was used and compared with the HPLC measurements, both applied for the first time, for the case of betulinic acid. Both extracts exerted cytotoxic activity through dose-dependent impairment of viability and mitochondrial activity of rat insulinoma m5F (RINm5F) cells. Decrease of RINm5F viability was mediated by nitric oxide (NO)-induced apoptosis. Importantly, these extracts potentiated NO and TNF-α release from macrophages therefore enhancing their cytocidal action. The rosemary extract developed more pronounced antioxidant, cytotoxic and immunomodifying activities, probably due to the presence of betulinic acid and a higher concentration of carnosic acid in its phytochemical profile.

Rapid and direct low micromolar NMR method for the simultaneous detection of hydrogen peroxide and phenolics in plant extracts.

A rapid and direct low micromolar ¹H NMR method for the simultaneous identification and quantification of hydrogen peroxide and phenolic compounds in plant extracts was developed. The method is based on the highly deshielded ¹H NMR signal of H2O2 at ∼10.30 ppm in DMSO-d6 and the combined use of picric acid and low temperature, near the freezing point of the solution, in order to achieve the minimum proton exchange rate. Line widths of H2O2 below 3.8 Hz were obtained for several Greek oregano extracts which resulted in a detection limit of 0.7 µmol L⁻¹. Application of an array of NMR experiments, including 2D ¹H-¹³C HMBC, spiking of the samples with H2O2, and variable temperature experiments, resulted in the unequivocal assignment of H2O2 precluding any confusion with interferences from intrinsic phenolics in the extract.

Phenolic compounds and antioxidant activity of olive leaf extracts.

The total phenolic content and antioxidant activities of olive leaf extracts were determined. Plant material was extracted with methanol and fractionated with solvents of increasing polarity, giving certain extracts. The qualitative changes in the composition of the extracts were determined after the storage of leaves for 22 h at 37°C, before the extraction. Total polyphenol contents in extracts were determined by the Folin-Ciocalteu procedure. They were also analysed by liquid chromatography-mass spectrometry. Their antioxidant activities were evaluated using the diphenyl picrylhydrazyl method and the ß-carotene linoleate model assay. Moreover, the effects of different crude olive leaf extracts on the oxidative stability of sunflower oil at 40°C and sunflower oil-in-water emulsions (10% o/w) at 37°C, at a final concentration of crude extract 200 mg kg(-1) oil, were tested and compared with butylated hydroxyl toluene.

Unprecedented ultra-high-resolution hydroxy group (1)H NMR spectroscopic analysis of plant extracts.

A general method is demonstrated for obtaining ultra-high resolution in the phenolic hydroxy group 1H NMR spectroscopic region, in DMSO-d6 solution, with the addition of picric acid. Line-width reduction by a factor of over 100 was observed, which resulted in line-widths ranging from 1.6 to 0.6 Hz. This unprecedented resolution, in combination with the shielding sensitivity of the hydroxy group absorptions to substituent effects at least up to 11 bonds distant and the application of 2D 1H-13C HMBC techniques, allows the unequivocal structure analysis of natural products with phenolic hydroxy groups in complex plant extracts.

Contribution of flavonoids to the overall radical scavenging activity of olive (Olea europaea L.) leaf polar extracts.

The contribution of flavonoids to the overall radical scavenging activity of olive leaf polar extracts, known to be good sources of oleuropein related compounds, was examined. Off line and on line HPLC-DPPH(*) assays were employed, whereas flavonoid content was estimated colorimetrically. Individual flavonoid composition was first assessed by RP-HPLC coupled with diode array and fluorescence detectors and verified by LC-MS detection system. Olive leaf was found a robust source of flavonoids regardless sampling parameters (olive cultivar, leaf age or sampling date). Total flavonoids accounted for the 13-27% of the total radical scavenging activity assessed using the on line protocol. Luteolin 7-O-glucoside was one of the dominant scavengers (8-25%). Taking into consideration frequency of appearance the contribution of luteolin (3-13%) was considered important, too. Our findings support that olive leaf, except for oleuropein and related compounds, is also a stable source of bioactive flavonoids.

Rapid and novel discrimination and quantification of oleanolic and ursolic acids in complex plant extracts using two-dimensional nuclear magnetic resonance spectroscopy-Comparison with HPLC methods.

A novel strategy for NMR analysis of mixtures of oleanolic and ursolic acids that occur in natural products is described. These important phytochemicals have similar structure and their discrimination and quantification is rather difficult. We report herein the combined use of proton-carbon heteronuclear single-quantum coherence ((1)H-(13)C HSQC) and proton-carbon heteronuclear multiple-bond correlation ((1)H-(13)C HMBC) NMR spectroscopy, in the identification and quantitation of oleanolic acid (OA) and ursolic acid (UA)in plant extracts of the Lamiaceae and Oleaceae family. The combination of (1)H-(13)C HSQC and (1)H-(13)C HMBC techniques allows the connection of the proton and carbon-13 spins across the molecular backbone resulting in the identification and, thus, discrimination of oleanolic and ursolic acid without resorting to physicochemical separation of the components. The quantitative results provided by 2D (1)H-(13)C HSQC NMR data were obtained within a short period of time ( approximately 14min) and are in excellent agreement with those obtained by HPLC, which support the efficiency of the suggested methodology.

Phytochemicals in olive-leaf extracts and their antiproliferative activity against cancer and endothelial cells.

Olive oil compounds is a dynamic research area because Mediterranean diet has been shown to protect against cardiovascular disease and cancer. Olive leaves, an easily available natural material of low cost, share possibly a similar wealth of health benefiting bioactive phytochemicals. In this work, we investigated the antioxidant potency and antiproliferative activity against cancer and endothelial cells of water and methanol olive leaves extracts and analyzed their content in phytochemicals using LC-MS and LC-UV-SPE-NMR hyphenated techniques. Olive-leaf crude extracts were found to inhibit cell proliferation of human breast adenocarcinoma (MCF-7), human urinary bladder carcinoma (T-24) and bovine brain capillary endothelial (BBCE). The dominant compound of the extracts was oleuropein; phenols and flavonoids were also identified. These phytochemicals demonstrated strong antioxidant potency and inhibited cancer and endothelial cell proliferation at low micromolar concentrations, which is significant considering their high abundance in fruits and vegetables. The antiproliferative activity of crude extracts and phytochemicals against the cell lines used in this study is demonstrated for the first time.

1H NMR determination of hypericin and pseudohypericin in complex natural mixtures by the use of strongly deshielded OH groups.

The (1)H NMR spectra of the commercially available compounds hypericin and its derivative pseudohypericin in CD(3)OH solutions indicate significantly deshielded signals in the region of 14-15 ppm. These resonances are attributed to the peri hydroxyl protons OH(6), OH(8) and OH(1), OH(13) of hypericins which participate in a strong six-membered ring intramolecular hydrogen bond with CO(7) and CO(14), respectively, and therefore, they are strongly deshielded. In the present work, we demonstrate that one-dimensional (1)H NMR spectra of hypericin and pseudohypericin, in Hypericum perforatum extracts show important differences in the chemical shifts of the hydroxyl groups with excellent resolution in the region of 14-15 ppm. The facile identification and quantification of hypericin and its derivative compound pseudohypericin was achieved, without prior HPLC separation, for two H. perforatum extracts from Greek cultivars and two commercial extracts: a dietary supplement, and an antidepressant medicine. The results were compared with those obtained from UV-vis and LC/MS measurements.

Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS.

The newly established hyphenated instrumentation of LC/DAD/SPE/NMR and LC/UV/(ESI)MS techniques have been applied for separation and structure verification of the major known constituents present in Greek Hypericum perforatum extracts. The chromatographic separation was performed on a C18 column. Acetonitrile-water was used as a mobile phase. For the on-line NMR detection, the analytes eluted from column were trapped one by one onto separate SPE cartridges, and hereafter transported into the NMR flow-cell. LC/DAD/SPE/NMR and LC/UV/MS allowed the characterization of constituents of Greek H. perforatum, mainly naphtodianthrones (hypericin, pseudohypericin, protohypericin, protopseudohypericin), phloroglucinols (hyperforin, adhyperforin), flavonoids (quercetin, quercitrin, isoquercitrin, hyperoside, astilbin, miquelianin, I3,II8-biapigenin) and phenolic acids (chlorogenic acid, 3-O-coumaroylquinic acid). Two phloroglucinols (hyperfirin and adhyperfirin) were detected for the first time, which have been previously reported to be precursors in the biosynthesis of hyperforin and adhyperforin.

LC-NMR coupling technology: recent advancements and applications in natural products analysis.

An overview of recent advances in nuclear magnetic resonance (NMR) coupled with separation technologies and their application in natural product analysis is given and discussed. The different modes of LC-NMR operation are described, as well as how technical improvements assist in establishing LC-NMR as an important tool in the analysis of plant-derived compounds. On-flow, stopped-flow and loop-storage procedures are mentioned, together with the new LC-SPE-NMR configuration. The implementation of mass spectrometry in LC-NMR is also useful on account of the molecular weight and fragmentation information that it provides, especially when new plant species are studied. Cryogenic technology and capillary LC-NMR are the other important recent developments. Since the plant kingdom is endless in producing potential drug candidates, development and optimization of LC-NMR techniques convert the study of natural products to a less-time-consuming task, speeding up identification.