From functional food to medicinal product: Systematic approach in analysis of polyphenolics from propolis and wine

In the last decade we have been working on standardization of propolis extract and determination of active constituents of wine those are rich in polyphenolics and have nutritional as well as therapeutic value. Here we are summarizing our results and providing overview on systematic approach how to analyse natural products rich in flavonoids and phenolic acids

Chromatographic Methods for the Analysis of Polyphenols in Wines

Vino je bogat izvor različitih skupina polifenola koje uključuju fenolne kiseline, flavonoide i trihidroksistilben-resveratrol. U posljednje vrijeme zanimanje za te supstancije potaknuto je brojnim dokazima o njihovim pozitivnim učincima na zdravlje čovjeka. Do sada su primjenjivane različite metode analize polifenola u vinu uključujući kromatografske, spektrofotometrijske i elektrokemijske metode. U ovom članku opisane su ukratko metode priprave uzoraka i najnovija dostignuća u analizi polifenola u vinu, temeljnim kromatografskim metodama: tankoslojnom kromatografijom (TLC), tekućinskom kromatografijom visoke djelotvornosti (HPLC) uz različite tehnike detekcije te plinskom kromatografijom (GC). Kao posebna tehnika spomenuta je i kapilarna elektroforeza, koja se odnedavno primjenjuje za analizu polifenola u vinu.Wine is an excellent source of various classes of polyphenols, including phenolic acids, flavonoids, and trihydroxystilbene resveratrol (Fig.1). Polyphenols play a major role in wine quality since they contribute to the sensory characteristics of wine, particularly color and astringency. A recent interest in these substances has been stimulated by abundant evidence of their beneficial effects on human health, such as anticarcinogenic, antiinflamatory and antimicrobial activities. Therefore, numerous studies have been performed in the attempt to analyze polyphenols in wine. This paper reviews the current advances in the determination of polyphenols in wine by the major chromatographic techniques such as thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The great complexity of the polyphenolic content of wine and the difficulty in obtaining some of the standards usually require sample preparation before analysis. Two methods for sample preparation, liquid-liquid extraction and solid-phase extraction, are most commonly applied. Hydrolysis is applied frequently, but not exclusively, to remove the sugar moieties from glycosides. TLC on silica gel plates is useful for the rapid and low-cost separation and identification of the polyphenols present in wine (Fig. 2). Densitometric quantitative analysis of polyphenols in wine extracts is usually performed by scanning the TLC plates with UV light at wavelengths of 350–365 nm or 250–260 nm (Fig. 3). For the evaluation of the most efficient mobile phase and an optimal choice of the combination of two or more mobile phases, two methods may be applied: information theory and numerical taxonomy. HPLC currently represents the most popular technique for the analysis of polyphenols in wine. For this purpose, a reversed-phase HPLC method that uses gradient elution with binary elution system is usually employed. Routine detection is based on measurement of UV-Vis absorption with a diode array detector (DAD). Enhancing selectivity and sensitivity for the determination of certain polyphenols requires the application of different detection techniques, such as fluorimetry (Fig. 4), electrochemistry, chemiluminescence, and/or mass spectrometry coupled with ionization techniques: electrospray (ESI), matrix-assisted laser desorption/ionization (MALDI), and atmospheric pressure chemical ionization (APCI). Gas chromatography (GC) methods developed for the analysis of polyphenols require derivatization to the volatile compounds and mass-spectrometric detection in the selective ion-monitoring mode (GC/MS-SIM). A special technique, capillary electrophoresis (CE), is a powerful tool for the analysis of polyphenol contents of white and red wines, with an opportune sample preconcentration step. Micellar electrokinetic capillary chromatography (MECC) has extended the utility of capillary electrophoresis to the separation of neutral analytes under the influence of an electric field. Fractionation of monomeric and polymeric pigments of higher molecular mass by gel permeation chromatography (GPC) improved the analysis of these compounds by CE

Evaluation of antiaggregatory activity of flavonoid aglycone series

<p>Abstract</p> <p>Background</p> <p>Among natural compounds, present in every day diet, flavonoids have shown beneficial effect in prevention of cardiovascular diseases that can be attributed, at least partially to the described antiaggregatory activity i.e. antiplatelet effects of flavonoids. Due to the ever increasing pharmacological interest in antiplatelet agents a systematic experimental evaluation of large flavonoid series is needed.</p> <p>Methods</p> <p>A set of thirty flavonoid aglycones has been selected for the evaluation. All measurements of aggregation were done under standardized and firmly controlled <it>in vitro </it>conditions. The whole blood samples, multiple platelet functional analyzer and adenosine diphosphate (ADP) as a weak agonist of aggregation were selected for this purpose.</p> <p>Results</p> <p>The results were expressed as minimal concentration of flavonoid that can significantly lower the platelet aggregation compared to the corresponding untreated sample (minimal antiaggregatory concentration - <it>MINaAC</it>). All analyzed flavonoids exhibited antiaggregatory activity <it>MINaAC </it>ranging from 0.119 μM to 122 μM, while the most potent representatives were 3,6-dihydroxyflavone (0.119 μM) and syringetin (0.119 μM).</p> <p>Conclusions</p> <p>Measurable antiplatelet activity established at submicromolar flavonoid concentrations suggests that even a dietary consumption of some flavonoids can make an impact on <it>in vivo </it>aggregation of platelets. These findings also point out a therapeutical potential of some flavonoids.</p

Propolis ethanolic extracts reduce adenosine diphosphate induced platelet aggregation determined on whole blood

Abstract Background Propolis is a well-known bee product containing more than 2000 identified compounds. It has many beneficial effects on human health that include antibacterial, antiviral, anticancer and hepatoprotective justifying its use as a dietary supplement. Platelet aggregation plays crucial role in thrombus formation that can cause stroke or heart attacks. As cardiovascular diseases, including those caused by thrombus formation, are related to 50% of deaths of Western population, the objective of this study was to determine antiaggregatory activity of propolis on platelet aggregation on the whole blood samples. Methods Twenty one propolis samples from Southeast Europe were characterized by spectrophotometric methods to determine content of the total flavonoids and phenolic acids. High performance liquid chromatography coupled with diode array detection was used to identify and quantify individual polyphenols. Platelet aggregation was tested by impedance aggregometry on the whole blood samples of ten healthy volunteers. Results The mean content of total polyphenols was 136.14 mg/g and ranged from 59.23 to 277.39 mg/g. Content of total flavonoids ranged between 6.83 and 55.44 mg/g with the mean value of 19.28 mg/g. Percentage of total phenolic acids was in the range 8.79 to 45.67% (mean 26.63%). Minimal antiaggregatory concentration, representing the lowest concentration of propolis extract sample that can cause statistically significant reduction of aggregation, ranged from 5 μM to 10.4 mM. Samples of propolis with lower content of luteolin and higher content of pinocembrin-7-methyleter showed better antiplatelet activity i.e. lower values of minimal antiaggregatory concentration. Conclusions This is the first study that shows antiaggregatory potential of propolis ethanolic extracts on the whole blood samples in the low micromolar concentrations suggesting that propolis supplementation may influence platelet aggregation and consequently thrombus formation. Further in vivo studies are needed to confirm the beneficial effects in prevention of cardiovascular diseases