Ágnes Alberti

Characterisation of Diarylheptanoid- and Flavonoid-type Phenolics in Corylus avellana L. Leaves and Bark by HPLC/DAD–ESI/MS

INTRODUCTION The leaves of Corylus avellana L. (common hazel, Betulaceae), a plant with a wide distribution in Europe, have been used in folk medicine for various diseases, but phytochemical exploration of C. avellana is still incomplete. To the best of our knowledge there is no previous report concerning diarylheptanoids in C. avellana, although these compounds show a frequent occurrence among Betulaceae plants. OBJECTIVE To improve existing online chromatographic methods for the investigation of the phenolic compounds in C. avellana leaves and bark, focusing on diarylheptanoid-type molecules. METHODS Dried and powdered leaves and bark of C. avellana were extracted with increasing polarity solvents (n-hexane, chloroform, ethyl acetate and methanol) in Soxhlet extractor apparatus. For the characterisation of the phenolic compounds in the ethyl acetate and methanolic extracts, UV spectral data, obtained by LC with a diode-array detector (DAD), accurate molecular mass and formula, acquired by LC and electrospray ionisation (ESI) with time-of-flight (TOF) MS and fragmentation pattern, given by LC-ESI/MS/MS analyses were used. Quantitation of the compounds was performed by LC-MS/MS. RESULTS In the methanolic and ethyl acetate extracts of C. avellana bark four flavonoid glycosides and a caffeoyl hexoside derivative were detected and characterised, while in C. avellana leaves, seven diarylheptanoid-type molecules were tentatively identified in addition to six flavonoid components. As far as we know this is the first study where the presence of diarylheptanoids in C. avellana is reported. CONCLUSION The improved HPLC/DAD-ESI/MS method was successfully utilised for the characterisation and quantitation of the phenolic compounds in C. avellana bark and leaves extracts.

First characterisation of flavonoid- and diarylheptanoid-type antioxidant phenolics in Corylus maxima by HPLC-DAD-ESI-MS

Corylus maxima Mill. (Betulaceae) leaves have been used in traditional medicine both internally and externally, nevertheless phytochemical exploration of the plant remains incomplete. In this study, the in vitro antioxidant activity and polyphenolic composition of the ethyl acetate and methanolic extracts of C. maxima leaves and bark are reported for the first time. The radical scavenging activities of the extracts were investigated by the ABTS and DPPH assays. All the extracts of C. maxima possessed notable antioxidant activity. By mean of a HPLC-DAD-ESI-TOF and a HPLC-DAD-ESI-MS/MS method, altogether twenty-two phenolics were tentatively characterised: one flavan derivative (1), seven flavonol derivatives (4, 6, 12, 13, 16, 20 and 21) and fourteen diarylheptanoids (2, 3, 5, 7-11, 14, 15, 17-19 and 22). The amount of the two main flavonoids - myricetin-3-O-rhamnoside (6) and quercetin-3-O-rhamnoside (13) - and two diarylheptanoids - oregonin (3) and hirsutenone (15) - in the extracts were determined by a validated HPLC-ESI-MS/MS method in multiple reaction monitoring (MRM) mode. Our results showed that C. maxima could be considered as a valuable source of pharmacologically important natural products that might contribute to the revaluation of the phytotherapeutical potential of the plant.

Effect-Directed Discovery of Bioactive Compounds Followed by Highly Targeted Characterization, Isolation and Identification, Exemplarily Shown for Solidago virgaurea

A nontargeted, effect-directed screening (bioprofiling) and a subsequent highly targeted characterization of antibacterial compounds from plant matrices is demonstrated on the example of Solidago virgaurea root extracts. The procedure comprises high-performance thin-layer chromatography (HPTLC) coupled with six bacterial bioassays including two plant pathogens, a radical scavenging assay, an acetylcholinesterase assay as well as in situ and ex situ mass spectrometric analyses. In situ mass spectra were directly recorded from the adsorbent using the Direct Analysis in Real Time interface (HPTLC-DART-MS), whereas ex situ mass spectra were recorded using an elution head-based interface (HPTLC-ESI-MS). For further bioassay-guided isolation of the main antimicrobial compounds, flash chromatographic fractionation and semipreparative high-performance liquid chromatographic purification were used and nuclear magnetic resonance data allowed the identification of the unknown antimicrobial compounds as 2Z,8Z- and 2E,8Z-matricaria esters. The discovered antibacterial activity was confirmed and specified by a luminometric assay and as minimal inhibitory concentration in the liquid phase.

In-situ clean-up and OPLC fractionation of chamomile flower extract to search active components by bioautography

Bioassay-guided isolation of antibacterial components of chamomile flower methanol extract was performed by overpressured layer chromatography (OPLC) with on-line detection, fractionation combined with sample clean-up in-situ in the adsorbent bed after off-line sample application. The antibacterial effect of the eluted fractions and of those compounds remaining on the adsorbent layer after separation was tested with direct bioautography (DB) against the bioluminescent Pseudomonas savastanoi pv. maculicola and Vibrio fischeri. The fractions with high biological activity were analyzed by solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and liquid chromatography and tandem mass spectrometry (LC-MS/MS). Two active uneluted compounds were characterized by off-line OPLC-MS using a thin-layer chromatography (TLC)-MS interface. Mainly, essential oil components, coumarins, flavonoids, phenolic acids, and fatty acids were identified in the active fractions.

Characterization of phenolic compounds and antinociceptive activity of Sempervivum tectorum L. leaf juice.

Sempervivum tectorum L. (houseleek) leaf juice has been known as a traditional herbal remedy. The aim of the present study was the chemical characterization of its phenolic compounds and to develop quantitation methods for its main flavonol glycoside, as well as to evaluate its antinociceptive activity. Lyophilized houseleek leaf juice was studied by HPLC-DAD coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to identify flavonol glycosides, hydroxy-benzoic and hydroxy-cinnamic acids. Ten flavonol glycosides and sixteen phenolic acid compounds were identified or tentatively characterized. Structure of the main flavonol compound was identified by nuclear magnetic resonance spectroscopy. Three characteristic kaempferol glycosides were isolated and determined by LC-ESI-MS/MS with external calibration method, using the isolated compounds as standard. The main flavonol glycoside was also determined by HPLC-DAD. Validated HPLC-DAD and LC-ESI-MS/MS methods were developed to quantify kaempferol-3-O-rhamnosyl-glucoside-7-O-rhamnoside and two other kaempferol glycosides. Antinociceptive activity of houseleek leaf juice was investigated by writhing test of mice. Sempervivum extract significantly reduced pain in the mouse writhing test.

Contribution of individual flavonoids in Lysimachia species to the antioxidant capacity based on HPLC-DPPH assay

Abstract Quantitative phytochemical characterisation of the chief flavonoid aglycones in the hydrolysed Lysimachia extracts revealed the dominance of kaempferol, quercetin and myricetin in L. vulgaris, L. nummularia, L. punctata, L. christinae, L. ciliata and L. clethroides, respectively. Due to the significant radical scavenging capacity of the samples, the contribution of the individual aglycones to the total antioxidant activity became of interest. Therefore, a HPLC method coupled to pre-column DPPH scavenging assay was developed. Differences in the six Lysimachia species’ phenolic composition regarding their participation to the antioxidant activity were revealed. The participation of the three investigated flavonoids to the radical quenching activity was the highest (91.2%) in the L. vulgaris sample, the lowest in L. christinae sample with 29.6%. In L. vulgaris sample, the 76.3% contribution of quercetin to the scavenger capacity was the highest peak area decrement ratio among the investigated samples.

Characterization of diarylheptanoids: An emerging class of bioactive natural products

HighlightsOverview of linear and cyclic diarylheptanoids phytochemistry was presented.Natural sources of diarylheptanoids were summarized.Main extraction and isolation strategies were reviewed.Analytical techniques used for diarylheptanoid characterization were discussed. ABSTRACT Diarylheptanoids are a class of secondary plant metabolites with a wide variety of bioactivity. Research on their phytochemistry and phytoanalysis is rapidly growing and the number of identified structures bearing the aryl‐C7‐aryl skeleton is at present approaching 500. Historically, the yellow pigment curcumin has been characterized as the first diarylheptanoid and the extensive research on naturally occurring analogues is still ongoing. In this review, studies dealing with the characterization of linear and cyclic derivatives are discussed from the phytoanalytical point of view. Isolation, fractionation and purification strategies from natural sources along with their chromatographic behavior and structural characteristics are discussed. The role of various techniques used for the extraction (such as Soxhlet extraction, sonication, maceration/percolation, microwave‐assisted extraction, supercritical carbon dioxide extraction); isolation (liquid–liquid extraction, column chromatographic techniques, preparative thin‐layer and high‐performance liquid chromatography, centrifugal partition chromatography, counter‐current chromatography); separation (thin‐layer chromatography, high‐performance liquid chromatography, gas chromatography, capillary electrophoresis) and structural characterization (UV/Vis spectroscopy, infrared spectroscopy, X‐ray crystallography, mass spectrometry, nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy) are critically reviewed.

A new ultra-high pressure liquid chromatography method for the determination of antioxidant flavonol aglycones in six Lysimachia species

Abstract UPLC-DAD method was developed and validated for the quantitative determination of free flavonol aglycones (kaempferol, quercetin and myricetin) after acidic hydrolysis in six Lysimachia species. Quantitative analyses showed that the amounts of various flavonol aglycones were significantly different in Lysimachia vulgaris, Lysimachia nummularia, Lysimachia punctata, Lysimachia christinae, Lysimachia ciliata and Lysimachia clethroides. The L. clethroides sample was found to be the richest in kaempferol (25.77 ± 1.29 μg/mg extract) and quercetin (97.67 ± 4.61 μg/mg extract), while the L. nummularia sample contained the highest amount of myricetin (20.79 ± 1.00 μg/mg extract). The antioxidant capacity of hydrolysed extracts was evaluated using in vitro DPPH• (2,2-diphenyl-1-picrylhydrazyl) and ABTS•+ [2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)] decolourisation tests. The observed radical scavenging capacities of the extracts showed a relationship with the measured flavonol aglycone content and composition. The acidic treatment resulted in an increased free radical scavenging activity compared to the untreated methanol extract.

Three newly identified lipophilic flavonoids in Tanacetum parthenium supercritical fluid extract penetrating the Blood-Brain Barrier

HighlightsOptimized supercritical fluid extraction of lipohilic flavonoids and parthenolid from Tanacetum parthenium L.Three newly identified lipophilic flavonoids (aceronin, sudachitin, nevadensin) in Tanacetum parthenium L.PAMPA‐BBB penetration of flavonoids and sesquiterpene lactones from feverfew. ABSTRACT Feverfew (Tanacetum parthenium L.) as a perennial herb has been known for centuries due to its medicinal properties. The main sesquiterpene lactone, parthenolide is considered to be responsible for the migraine prophylactic effect, however the pharmacological benefits of the lipophilic flavonoid components can not be neglected. Supercritical fluid extraction (7% ethanol, 22 MPa, 64 °C) was carried out on the leaves of Tanacetum parthenium L. from which the presence of methylated flavonoids beside parthenolide and other sesquiterpene lactones were indicated by preliminary LC–MS analyses. Specific Parallel Artificial Membrane Permeability Assay (PAMPA) was applied to identify the components capable to cross the Blood‐Brain Barrier (BBB). Three lipophilic flavonoids were detected on the acceptor side, that were isolated (Prep‐HPLC) and identified as sudachitin, aceronin and nevadensin (LC–MS/MS, NMR). These flavonoids were also characterized individually by PAMPA‐BBB model. The presence of sudachitin and nevadensin was proven in the Asteraceae family, but neither of the three flavonoids were reported in Tanacetum parthenium L.

Layer chromatography-bioassays directed screening and identification of antibacterial compounds from Scotch thistle

The antibacterial profiling of Onopordum acanthium L. leaf extract and subsequent targeted identification of active compounds is demonstrated. Thin-layer chromatography (TLC) and off-line overpressured layer chromatography (OPLC) coupled with direct bioautography were utilized for investigation of the extract against eight bacterial strains including two plant and three human pathogens and a soil, a marine and a probiotic human gut bacteria. Antibacterial fractions obtaining infusion-transfusion OPLC were transferred to HPLC-MS/MS analysis that resulted in the characterization of three active compounds and two of them were identified as, linoleic and linolenic acid. OPLC method was adopted to preparative-scale flash chromatography for the isolation of the third active compound, which was identified after a further semi-preparative HPLC purification as the germacranolide sesquiterpene lactone onopordopicrin. Pure onopordopicrin exhibited antibacterial activity that was specified as minimal inhibitory concentration in the liquid phase as well.