Apostolis Angelis

Application of pH-zone refining hydrostatic countercurrent chromatography (hCCC) for the recovery of antioxidant phenolics and the isolation of alkaloids from Siberian barberry herb

The development of a fast hCCC method tailored to recover phenolics of Siberian barberry (Berberis sibirica, Berberidaceae) responsible for the observed strong antioxidant activity was performed. Initially, the optimization of extraction procedure was evaluated based on the antiradical potential assessment (DPPH and Folin-Ciocalteu assays). 100 °C methanol ASE extract exhibited the highest antiradical activity (IC50=60 ± 4 μg/mL), and a significant TPC (159 ± 2 mgGAE/g). Thorough determination of phenolic content by UHPLC-DAD-ESI(-)HRMS revealed the presence of 10 phenolics as major constituents, and several groups of alkaloids. pH-zone refining hCCC was chosen as the most promising method for the extracts fractionation due to the ionizable character of its constituents. For this purpose a MtBE-H2O (1:1) system with 10mM TEA and HCl was applied leading to a phenolic fraction, free of alkaloids, with higher antioxidant capacity (IC50=25 μg/mL, TPC=178 mg GAE/g). Additionally, fractionation of alkaloids was achieved resulting isolation of pharmacologically important alkaloids: magnoflorine and berberine.

One-step isolation of γ-oryzanol from rice bran oil by non-aqueous hydrostatic countercurrent chromatography.

The value-added γ-oryzanol was purified in one step from crude rice bran oil (RBO) using a preparative hydrostatic countercurrent chromatography (hydrostatic CCC) method, operating in the dual mode. The fractionation was performed using a non-aqueous biphasic solvent system consisting of heptane-acetonitrile-butanol (1.8:1.4:0.7, v/v/v), leading rapidly to the target compounds. Transfer of the analytical CCC method to large-scale isolation was also carried out yielding a high quantity-high purity fraction of γ-oryzanol. In addition, a fraction of hydroxylated triterpene alcohol ferulates (polar γ-oryzanol) was clearly separated and obtained. Furthermore, a fast HPLC-APCI(±)-HRMS method was developed and applied for the identification of γ-oryzanol as well as the polar γ-oryzanol in RBO and the resulting fractions. The purity of γ-oryzanol fraction was estimated as 97% based on HPLC-APCI-HRMS analysis.

Bioactivity-guided identification of antimicrobial metabolites in Alnus glutinosa bark and optimization of oregonin purification by Centrifugal Partition Chromatography

Barks from conifers and broadleaved trees constitute abundant wastes generated from wood harvesting and logging activities. Extracts of such residues obtained from Alnus trees have been reported as interesting resources with potent antibacterial activities. The present study aims to determine the antimicrobial activity of a crude methanol extract prepared from the bark of Alnus glutinosa against a panel of 22 bacteria and yeasts and to optimize a purification method enabling the high production of the most active substances. Fractionation of the crude extract was performed by Centrifugal Partition Chromatography (CPC) using a three-phase solvent system composed of n-heptane, methyl-ter-butyl ether, acetonitrile and water. The major known compounds contained in the fractions produced by CPC were chemically profiled by (13)C NMR dereplication, resulting in the unambiguous identification of oregonin, hirsutanonol, betulinic acid, and alusenone 1a. The antibacterial evaluation of the fractions by bioautography on Staphylococcus aureus revealed that oregonin, in addition to being the major metabolite of the crude extract (∼32% w/w), was the most active with an antibacterial inhibitory effect comparable to antibiotics. The purification of oregonin was optimized at the laboratory-scale by CPC. A single injection of 3.7g of crude extract resulted in a recovery of 72% (850mg) of the available oregonin at purity higher than 94%.

A single-step isolation of squalene from olive oil deodorizer distillates by using centrifugal partition chromatography

ABSTRACT High added-value squalene (SQ) was purified in one step from olive oil deodorizer distillates (OODD) using preparative centrifugal partition chromatography (CPC) method, operating in the dual mode. The fractionation was performed using a non-aqueous biphasic solvent system consisting of heptane–acetonitrile–butanol (1.8:1.4:0.7, v/v/v), leading to the isolation of the target compound in 4 hours, using a preparative 1L column. Furthermore, a fast UHPLC-DAD method was developed and applied for the identification and quantification of SQ in both OODD and purified form. The content of SQ in the initial material was 23.4%, while the purity of the isolated SQ was 95.5%. The recovery of SQ was calculated at 76.3%. The productivity of the process was calculated at 234 mg/h/L.

γ-Oryzanol: An Attractive Bioactive Component from Rice Bran

Abstract Rice ( Oryza sativa seeds – Poaceae) is considered one of the most important grains, as it is consumed by the half of the world population. Various by-products of the rice industry, such as rice bran and the rice bran oil (RBO), have recently attracted much attention from both academia and industry due to their high content of high added-value phytochemicals such as tocopherols/tocotrienols, carotenoids, and γ-oryzanol, of nutritional, pharmaceutical, and cosmetic interest. In particular, γ-oryzanol, a mixture of triterpene alcohol and phytosterol ferulates, is characterized by a wide spectrum of health-beneficial effects, including anticarginogenic, anti-inflammatory, antihyperlipidemic, and neuroprotective, which are mainly attributed to its significant antioxidant capacity. This chapter presents the chemical characteristics of γ-oryzanol components, their classification and biosynthesis, as well as an overview regarding the traditional and modern methods for γ-oryzanol extraction, isolation, analysis, and identification. A summary regarding its biological profile and applications is also provided.

Antioxidant effects of an olive oil total polyphenolic fraction from a Greek Olea europaea variety in different cell cultures

BACKGROUND Numerous studies have been carried out concerning the advantageous health effects, especially the antioxidant effects, of olive oils (OO) individual biophenolic compounds, but none until now for its total phenolic fraction (TPF). Plenty of evidence, in research about nutrition and healthiness, points out that it is the complex mixture of nutritional polyphenols, more than each compound separate, which can synergistically act towards a health result. PURPOSE The aim of the present study was to examine the antioxidant properties of an extra virgin olive oil (EVOO) total polyphenolic fraction, from a Greek endemic variety of Olea europaea in cell lines. METHODS EVOO from a Greek endemic variety was used for the extraction of a total polyphenolic fraction, using a green CPE‑based method. The redox status [in terms of ROS, GSH, TBARS, protein carbonyls] was assessed at a cellular level, particularly in EA.hy926 endothelial, HeLa, HepG2 hepatic cells and C2C12 myoblasts. Moreover, the levels of glutamate-cysteine ligase catalytic subunit (γ-GCLc) of GSH, one of the most important antioxidant enzymes, were assessed by western blot. RESULTS According to the results, TPF improves the redox profile of all cell lines, mainly by increasing GSH and its catalytic subunit, while at low, not cytotoxic TPF concentrations there was a decrease in TBARS and carbonyls. Regarding ROS levels a reduction was observed only in the HepG2 cell line, contrary to the other cell lines, that there is no statistically significant difference. CONCLUSION The TPF appeared to protect cells from oxidative stress due to the strong antioxidant activity of its polyphenols. This could have interesting implications in development of new products based on this olive oil to provide protection and treatment against harmful effects of free radicals.

Rapid isolation and characterization of crocins, picrocrocin, and crocetin from saffron using centrifugal partition chromatography and LC-MS

This study demonstrates a simple method for one-step isolation of the main secondary metabolites of a hydroalcoholic extract of Crocus sativus stigmas (saffron) using step-gradient centrifugal partition chromatography. The analysis was performed in dual and elution-extrusion mode, using five biphasic systems of the solvents heptane/ethyl acetate/butanol/ethanol/water in ratios of 4:10:0:4:10, 1:13:0:4:10, 1:12:1:4:10, 1:10:3:4:10, and 1:7:6:4:10. Five major crocins, picrocrocin, and crocetin were directly isolated in one step. Scaling up to preparative level, allowed the recovery of significantly high quantities of pure compounds, especially trans-crocin-4, saffrons principal crocin. Comparing dual-mode and elution-extrusion, in dual-mode, the trans-crocin-4 containing fractions were co-eluted with a high amount of free β-d-glucose. In contrast, absence of free β-d-glucose was observed in the corresponding trans-crocin-4 fractions obtained by the second method denoting its superiority against dual-mode. Initiating analysis with the 4th solvent-system afforded selective isolation of trans-crocin-4, with reduction in experimental time and solvent consumption. Structure elucidation was performed by nuclear magnetic resonance spectroscopy, liquid chromatography with mass spectrometry, and high-resolution tandem mass spectrometry. The proposed methodology comprises an integrated approach for the purification and characterization of biologically active saffron components in a fast, selective, and environmentally friendly manner.

Identification of Novel Melanin Synthesis Inhibitors From Crataegus pycnoloba Using an in Vivo Zebrafish Phenotypic Assay

Zebrafish has emerged as a powerful model organism for high throughput drug screening. Several morphological criteria, transgenic lines and in situ expression screens have been developed to identify novel bioactive compounds and their mechanism of action. Here, we used the inhibition of melanogenesis during early zebrafish embryo development to identify natural compounds that block melanogenesis. We identified an extract from the Greek hawthorn Crataegus pycnoloba as a potent inhibitor of melanin synthesis and used activity based subfractionation to identify active subfractions and eventually three single compounds of the same family (dibenzofurans). These compounds show reversible inhibition of melanin synthesis and do not act via inhibition of tyrosinase. We also showed that they do not interfere with neural crest differentiation or migration. We identified via in silico modeling that the compounds can bind to the aryl hydrocarbon receptor (AHR) and verified activation of the Ahr signaling pathway showing the induction of the expression of target genes.