Characterisation of an Aromatic Plant-based Formula using UV-Vis Spectroscopy, LC-ESI(+)QTOF-MS and HPLC-DAD Analysis

Abstract. It is known for a long time that seasoning/condimentary herbs have antioxidant activity and antibacterial properties, being good natural alternatives for disease prevention. The different efficiency of these plants is assigned to their bioactive molecules, stability and bioavailability. In the present study seven aromatic herbs (basil, thyme, oregano, rosemary, clove, cinnamon and sage) were investigated individually. A new product was developed using basil, thyme, oregano, rosemary, clove, cinnamon and sage, according to a default recipe. The characterization of each plant aimed to identify the specific “fingerprint†by its main bioactive molecules and the “traceability†of these molecules in the new product, made by mixing the selected plants according to a default recipe. In order to determine the main bioactive compounds of the individual plants composition, in comparison with the new plant-based (EPC) formula, high throughput techniques like UV-Vis spectroscopy and LC-QTOF-MS  spectrometry were used. The most important bioactive compounds determined in the studied herbs, which may exert antioxidant activity and antibacterial properties, were phenolic compounds (phenolic acids, flavonoids), quinones, clorophylls as well some polar terpenoids. The fingerprints are providing comprehensive and accurate information about the compounds that may exert antimicrobial properties. In order to assure the biological effects and the bioavailability of the polyphenols and the secondary metabolites we have to consider the antagonistic and synergistic effect that the metabolites can exert on each other

SERUM LIPIDOMIC BIOMARKERS FROM PATIENTS WITH PROSTATE PATHOLOGY, IDENTIFIED BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY COUPLED WITH MASS SPECTROMETRY

Introduction: Lipidomics can offer an instant picture of the lipophilic metabolites from tissues and biofluids and can indicate the evolution of different pathologies, such as hyperplasia or different types of cancers. Related to these pathologies, Prostate Serum Antigen (PSA), proved to have a low grade prediction for an accurate diagnosis. Meanwhile, untargeted or targeted metabolomics became a useful advanced technology to discover new biomarkers for a better diagnosis.Aims: To  realize an adequate procedure based on liquid chromatography coupled with mass spectrometry (HPLC-MS) to determine the profile of lipids from blood serum, followed by adequate biostatistics.Materials and Methods: Blood samples, obtained from healthy men and patients with prostate benign hyperplasia,  post-biopsy cancer and post-surgery cancer were processed for lipid extraction and subjected to HPLC-ESI(+)QTOF-MS measurements, followed by the multivariate analysis (PCA and Cluster Analysis) with Unscrambler 10.1 software. TofControl 3.2 and Data Analysis 4.2 software (BrukerDaltonics) were used for the control of the instrument and data processing.Results: The molecules responsible for such discriminations were identified to be mainly represented by lyso-phospatidylcholines. By Cluster Analysis, the dendograms showed good statistical clustering of samples, especially for cancer patients agains controls and less clustered for hyperplasia.Conclusion: One can consider that molecules belonging to phospholipid family and diacyl /triacylglycerides or ceramides or carnitines can be considered potential biomarkers for hyperplasia and prostate cancer

Comparative Phenolic Fingerprint and LC-ESI+QTOF-MS Composition of Oregano and Rosemary Hydrophilic Extracts in Relation to their Antibacterial Effect

Rosemary (Rosmarinus officinalis) and oregano (Origanum vulgare) are known aromatic plants used as spice, with good flavoring, preservative, antioxidant and antibacterial activity. Beside their known terpenoid content responsible for the antibacterial activity, the water-soluble compounds (phenolic derivatives) are of high interest not only for their antioxidant activity but as a good alternative or as a hydrophilic new antibacterial solution. Two hydrophilic extracts from each plant were obtained (15% plant in hot water) and the phytochemicals were fingerprinted by UV-Vis and FTIR spectrometry and quantified. The total phenolic content was higher in case of oregano (54.2 mg GAE/g DW) comparing to rosemary (54.25 vs 15.35 mg GAE/g dry matter). By LC-ESI+QTOF-MS analysis there were identified mainly, in both extracts, flavonoid and diterpene derivatives, mainly carnosol, carnosic acid, rosmarinic acid, kaempferol 3-O-glucuronide. Other flavonoid glucuronides were more specific to one or the other plant, e.g. Luteolin 3'-(4''-acetylglucuronide) for rosemary and Apigenin 7-O-glucuronide for oregano. Water favorized increased extraction of flavonoid derivatives and soluble diterpenes, but not non-soluble  terpenes. The antibacterial activity of both extracts was tested against B.cereus, L. monocytogenes, Salmonella, S. aureus and E.coli. Both oregano and rosemary extracts showed a slight antibacterial activity, which can be related to the low concentration of terpenoids, known to have the most important antibacterial activity in these plants. Nevertheless, the antibacterial activity seems to be strain dependent, Bacillus cereus being the most sensitive bacterial strain comparing with the other four bacteria, the oregano extract having a slightly superior activity comparing to the rosemary extract

Preparation and Comparative Characterization of Alginate-Made Microcapsules and Microspheres Containing Tomato, Seabuckthorn Juices and Pumpkin Oil

Recent studies have shown the benefits of tomatoes, seabuckthorn juices and pumpkin oil, rich in bioactives with antioxidant capacity, in the prevention of prostate diseases. To stabilize their antioxidant activity, microencapsulation represent a good technological alternative, improving the stability and bioavailability of bioactive molecules ( phenolic derivatives, carotenoids, phytosterols, vitamins).   The aim of the study was to prepare and characterize microspheres and microcapsules based on emulsions made of natural polymers like Natrium alginate mixed with tomato and/or seabuckthorn juices, with or without pumpkin oil.  The viscosity of emulsions, the morphology of microcapsules and microspheres were characterized comparatively and the bioactives were monitored by UV-Vis spectrometry.  In the lipophilic extract there were identified, before and after encapsulation, different classes of compounds, from lipids, to phenolic acid derivatives, flavonoids and carotenoids. Carotenoids were the major components having concentrations from 9.16 up to 19.71 mg/100 g sample. The viscosity of  each emulsion including juices, oil and natrium alginate 2%, before encapsulation, showed differences, dependent on the oil addition and speed of homogenization. The macroscopic and microscopic structure of microspheres and microcapsules were comparatively evaluated. Both microspheres and microcapsules had external diameters  ranging from 750 to 900 μm and the microcapsules’ oily core of 150-180 μm. The results obtained from emulsion’s viscosity will be correlated with the rigidity and optimal release rate of bioactive molecules from microcapsules and microspheres.  Further studies are directed towards these aspects

Untargeted LC-QTOF (ESI +) MS Analysis of Small Serum Metabolites Related to Prostate Cancer and Prostate Specific Antigen

Prostate cancer has an increasing incidence and there is an urgent need for development of new serum biomarkers for early diagnostic as the ones known are ineffective. The aim of the study was to use untargeted metabolomics in order to identify and characterize small metabolite fingerprints in patients with normal vs pathologic values of PSA ( previously determined by electrochemiluminiscence). A cohort of one hundred patients with different Prostate Specific amtigen values were investigated by untargeted metabolomics. The serum small metabolite profile determined by high performance liquid chromatography coupled with mass spectrometry, LC-QTOF(ESI+)MS in order to identify specific biomarkers, for normal patient group (PSA = 0-4 ng.ml) and four pathologic groups, having PSA values from 4 to >1000 ng/ml. The major molecules identified in the samples were polar phospholipids, maily lysophosphatidyl choline derivatives, having m/z values from 496 to 524, like LPC(O-16:0/O-1:0), LPC(18:1/2:0) or PS(18:1(9Z)/0:0), LPC(18:2(9Z,12Z)/0:0 and their isomers and  LPC(O-18:1(11Z)/2:0), respectively. Also, small molecules (free fatty acids and prostaglandin derivatives) were identified and are significantly different in pathologic vs normal serum samples. Generally the pathologic samples had increased concentrations of all above mentioned molecules. The Principal Component analysis showed , by plot and loadings scores, significant clustering of normal vs pathological groups