Blood-brain barrier permeability study of ginger constituents.

Ginger, the rhizome of Zingiber officinale Roscoe is of great importance in the traditional medicine for the treatment of various diseases. More than 400 constituents have been reported in the plant, the most important ones being the gingerol and shogaol derivatives. Positive effects of ginger extracts and isolated [6]-gingerol have been proved in animal models of anxiety, Alzheimer's disease, Parkinson's disease and epilepsy. Taken in consideration these promising positive effects of ginger and its constituents in the central nervous system, the isolation of gingerol and shogaol derivatives ([6]-gingerol (1), [8]-gingerol (2), [10]-gingerol (3), [6]-shogaol (4), [10]-shogaol (5), 1-dehydro-[6]-gingerdione (6), 1-dehydro-[10]-gingerdione (7)) and investigation of their transcellular passive diffusion across the blood-brain barrier (BBB) were carried out. For this purpose, a Parallel Artificial Membrane Permeability Assay for the Blood-Brain Barrier (PAMPA-BBB) was chosen that had previously been validated for natural compounds. Based on our results, [6]-gingerol, [8]-gingerol and [6]-shogaol were found to be able to penetrate the BBB via passive diffusion, suggesting them to contribute to the positive effects of ginger extracts in the central nervous system.

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

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.

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

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, 22MPa, 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.

Evaluation of Radical Scavenging Activity of Sempervivum tectorum and Corylus avellana Extracts with Different Phenolic Composition.

Semnpervivum tectorum L. and Corylus avellana L. are traditional herbal remedies exhibiting antioxidant activity and representing diverse phenolic composition. The aim of this study was to reveal the contribution of certain compounds to total radical scavenging activity by studying S. tectorum and C. avellana extracts prepared with solvents of different selectivity for diverse classes of phenolics. Antioxidant activity of S. tectorum and C. avellana samples was determined in the ABTS and DPPH radical scavenging assays, and phenolic composition was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Correlations between antioxidant activity and phenolic content of houseleek extracts have been revealed. Significant differences regarding antioxidant activity have been shown between S. tectorum 80% (v/v) methanol extract and its fractions. Additionally, synergism among the constituents present together in the whole extract was assumed. Significantly higher radical scavenging activity of hazel extracts has been attributed to the differences in phenolic composition compared with houseleek extracts.

HPLC-DPPH Screening Method for Evaluation of Antioxidant Compounds in Corylus Species.

Corylus avellana L., C. colurna L. and C. maxima Mill. have been used in traditional medicine for a long time for the treatment of various diseases, such as phlebitis, varicose veins, haemorrhoidal symptoms and eczema. Our previous studies indicated the presence of flavonol-3-O-glycosides, diarylheptanoids and caffeic acid derivatives in the bark and leaf extracts of the three species mentioned above. Comparison of in vitro DPPH scavenging activity of the Corylus extracts prepared with ethyl acetate and methanol to those of well-known antioxidant phenolics was performed. The contribution of certain compounds to the total antioxidant activity of the extracts was investigated by developing a HPLC method coupled to the DPPH radical scavenging assay.

Convergence chromatographic determination of camphor in the essential oil of Tanacetum parthenium L.

Feverfew (Tanacetum parthenium L., Asteraceae) is a perennial medicinal plant which has been used to alleviate the symptoms of migraine, headache and rheumatoid arthritis and possesses numerous pharmacological activities. An ultra-high-performance supercritical fluid chromatographic method (UHPSFC) was developed and validated in accordance with the International Conference on Harmonization guidelines in order to determine the camphor content of the volatile oil, which was accurate, precise, robust and selective. The method was validated for specificity, accuracy (100.2%), repeatability and intermediate precision, linearity (r2 > 0.999), limit of detection (2.055 µg/mL), limit of quantification (6.228 µg/mL) and robustness. The common range of accuracy and linearity was between 0.125 and 1.000 mg/mL. Steam distillation was carried out in order to study the essential oil yield of three different T. parthenium L. samples originating from Hungarian medicinal herb collections. The camphor content of the essential oils from the aerial parts of feverfew samples from different origin was compared. Although the composition of the essential oil is well reported, a validated quantitative UHPSFC method for the determination of the constituents is presented herein for the first time.

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

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.

Characterization of antioxidant phenolics in Syringa vulgaris L. flowers and fruits by HPLC-DAD-ESI-MS.

In this study the polyphenolic composition of lilac flowers and fruits was determined for the first time. For the identification of compounds, accurate molecular masses and formulas, acquired by LC and ESI-TOF-MS and fragmentation pattern given by LC-ESI/MS/MS analyses, were used. Our chromatographic system in conjunction with tandem MS was found to be valuable in the rapid separation and determination of the multiple constituents in methanolic extracts of lilac flowers and fruits. Altogether 34 phenolics, comprising 18 secoiridoids, seven phenylpropanoids, four flavonoids and five low-molecular-weight phenols, were identified. As marker compounds two secoiridoids (oleuropein and nuzhenide), two phenylpropanoids (acteoside and echinacoside) and rutin were quantified by validated methods. As a result of quantitative analysis, it was confirmed that flowers contain significant amounts of phenylpropanoids (acteoside, 2.48%; echinacoside, 0.75%) and oleuropein (0.95%), while in fruits secoiridoid oleuropein (1.09%) and nuzhenide (0.42%) are the major secondary metabolites. The radical scavenging activities of the extracts and the constituents were investigated by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] assays. Both extracts show remarkable antioxidant activities. Our results clearly show that lilac flowers and fruits are inexpensive, readily available natural sources of phenolic compounds with pharmacological and cosmetic applications. Copyright © 2015 John Wiley & Sons, Ltd.

[Occurence of diarylheptanoids in Corylus species native to Hungary]. / Diarilheptanoidok elofordulása a mogyoró nemzetség Kárpát-medencében honos fajaiban.

Since the last decade naturally occurring diarylheptanoids have been in the focus of scientific interest due to their various. beneficial biological effects. Besides the outstanding importance of the curcuminoids isolated from members of the Curcuma genus (Zingiberaceae), several different diarylheptanoids identified in Alnus species (Betulaceae) have been proved to possess notable pharmacological effects. Chemoprotective, neuroprotective, hepatoprotective, antiviral, antibacterial, antiinflammatory and antioxidant activities suggest their potential role in clinical practice. The aim of our study was the phytochemical investigation of the Corylus (Betulaceae) species native to Hungary: the Common hazel (Corylus avellana L.), the Turkish hazel (Corylus colurna L.) and the Filbert (Corylus maxima Mill.) in order to characterise their phenolic-profile. Although these plants have been used in traditional medicine for long time, literature data regarding their phytochemical composition is limited to the flavonoid and hydroxycinnamic-acid derivatives of C. avellana leaves. No previous studies have been published reporting the presence of diarylheptanoid compounds in any of the Corylus species. Soxhlet extraction with solvents of increasing polarity was performed on the bark and leaves of the mentioned three Corylus species. The phenolic-profile of the methanolic and ethyl acetate extracts was investigated by HPLC-DAD-ESI-TOF-MS and HPLC-DAD-ESI-MS/MS methods. Altogether 37 different phenolic compounds were detected in the extracts: twenty diarylheptanoids (1-20), nine flavonols (21-29) and eight other phenolics: caffeic and quinic acid derivatives and flavanones (30-37). The main compounds of the extracts were identified as myricetin- quercetin- and kaempferol-3-O-rhanmosides.

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.

Polyphenol composition and antioxidant capacity of three Lysimachia species.

The polyphenol composition and antioxidant properties of three Lysimachia species (L. nummularia L., L. vulgaris L. and L. punctata L.) and their column chromatographic fractions were investigated. The antioxidant activity of herb extracts and 54 different column chromatographic fractions was evaluated using in vitro DPPH and ABTS+ decolorization tests. The total polyphenol content was determined by spectrophotometric methods. The phenolic compounds of extracts of different Lysimachia species and their bioactive fractions were characterized by online chromatographic methods. For identification of the compounds, UV spectral data, accurate molecular mass and formula, as well as MS and fragmentation patterns given by LC-DAD-ESI/MS/MS and LC-ESI- TOF analyses were used. Quantification of the compounds was performed by LC-DAD using an external standard method. In the extracts, caffeic acid derivatives, chlorogenic acid, free flavonoid aglycones, and 11 various flavonoid glycosides were identified. Flavonoid composition of Lysimachia extracts showed significant differences. L. punctata extracts had the strongest DPPH and ABTS+ radical scavenger activity (IC50=43.3 µg/mL and 21.3 µg/mL), due to their high myricitrin and quercetin-hexoside content. Correlation between polyphenol content and radical scavenging activity of each column chromatographic fractions is also included.

Antioxidant activity and phenolic composition of Corylus colurna.

Corylus colurna L. (Turkish hazel), a common hazel species in Europe, could be considered as a source of pharmacologically important natural compounds, since other members of the Corylus genus have been reported to contain several constituents with beneficial biological activity. Nevertheless, its phytochemical exploration is still incomplete. In this study the potent antioxidant phenolic compounds in different parts of the plant were investigated. The phenolics in the leaves, bark, catkins and involucre of C. colurna possess notable scavenger activity on both DPPH and ABTS free radicals. The total polyphenol content shows significant correlation with the antioxidant activity of the samples. Twenty-three phenolic compounds - hydroxycinnamic acid derivatives, flavonoid derivatives and diarylheptanoids - were characterized in the C. colurna extracts by LC-DAD, LC-ESI-TOF and LC-ESI-QQQ-MS.

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.

Applicability of a blood-brain barrier specific artificial membrane permeability assay at the early stage of natural product-based CNS drug discovery.

While numerous natural products (NPs) possess activity on central nervous system (CNS) targets, there has been no analytical approach to effectively identify compounds with high brain penetration potential in complex mixtures at the early stage of drug discovery. To overcome this issue, the performance of an in vitro parallel artificial membrane permeability assay for the blood-brain barrier (PAMPA-BBB) for natural products and for plant extracts has been validated and characterized. It was found that the PAMPA-BBB assay preserves its predictive power in the case of natural products and provides high phytochemical selectivity, which enables its use as a unique filtering tool in terms of selecting brain-penetrable compounds from plant extracts. Moreover, the present study has demonstrated that simple modifications in the assay design allow the direct use of PAMPA-BBB filtered samples in a dereplication process, as performed by NMR and LC-MS. The applicability of this procedure was demonstrated using extracts prepared from Tanacetum parthenium, Vinca major, Salvia officinalis, and Corydalis cava, representing different types of chemical diversity and complexity. Thus, the proposed protocol represents a potentially valuable strategy in the NP-based CNS drug discovery environment with a high-throughput screening platform.

Applicability of preparative overpressured layer chromatography and direct bioautography in search of antibacterial chamomile compounds.

In situ sample preparation and preparative overpressured layer chromatography (OPLC) fractionation on a 0.5 mm thick adsorbent layer of chamomile flower methanol extract prepurified by conventional gravitation accelerated column chromatography were applied in searching for bioactive components. Sample cleanup in situ on the adsorbent layer subsequent to sample application was performed using mobile phase flow in the opposite direction (the input and output of the eluent was exchanged). The antibacterial effect of the fractions obtained from the stepwise gradient OPLC separation with the flow in the normal direction was evaluated by direct bioautography against two Gram-negative bacteria: the luminescence gene tagged plant pathogenic Pseudomonas syringae pv. maculicola, and the naturally luminescent marine bacterium Vibrio fischeri. The fractions having strong activity were analyzed by SPME-GC/MS and HPLC/MS/MS. Mainly essential oil components, coumarins, flavonoids, phenolic acids, and fatty acids were tentatively identified in the 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.

[Epilobium parviflorum--in vitro study of biological action]. / Az Epilobium parviflorum kivonat hatásmechanizmusának in vitro vizsgálata.

Epilobium parviflorum Schreb. (willow-herb) is used for the treatment of benign prostatic hyperplasia (BPH), but its biological action is not entirely identified. This paper aims to report data on willow-herbs probable biological effect. In vitro studies have been made to investigate different aspects of its antioxidant capacity, anti-inflammatory (COX-inhibitory) action, steroid-receptor -agonistic and -antagonostic effect as well as aromatase-inhibitory effect. Based on our results, willow-herb possess remarkable antioxidant and COX-inhibitory action.

[Antioxidant activity of different phenolic fractions separated from Euphrasia rostkoviana Hayne]. / Az Euphrasia rostkoviana Hayne fenoloid tartalmú frakcióinak antioxidáns értékelése.

Euphrasia rostkoviana Hayne (Eyebright) is a valuable part of the traditional folk medicine as an anti-inflammatory herb since centuries. Since oxidative stress may underlie as a key feature of inflammatory process, the methanolic extract ofEuphrasia rostkoviana Hayne (eyebright) and its methanolic fractions were evaluated for antioxidant activity by the use of ABTS and DPPH decolorization assays. Fractionation of the Soxhlet extract was accomplished by polyamide column chromatography. For chemical characterisation HPLC-DAD-MS/MS was used. Fraction 1., dominated by a glycosilated caffeic acid derivative, exhibited the strongest antioxidant activity in DPPH and ABTS assays, IC50: 11.88 microg/ml and 4.24 microg/ml, respectively, which are notable results if compared to the investigated standards. Fractions of flavonoid glycosides proved to have similarly strong, but lower effect. The scavenging reaction of the two radical cations were comparable, IC50 results were differing, while precision not.

Polyphenol composition and antioxidant capacity of Epilobium species.

Epilobium species (Onagraceae) are commonly used herbal remedies in traditional, adjuvant therapy of benignus prostate hyperplasia (BPH), however the pharmacological and clinical standardization of commercially available Epilobii herba (willow-herb) remains difficult. Willow-herb products usually consist of mixtures from various species, with different phenoloid content, often only partially identified. The present study reports comprehensive LC-MS/MS investigation on the polyphenol composition of the most common Epilobium species, emphasizing the pharmaceutical importance of a uniform standardization protocol in case of their products. The antioxidant capacity of species was evaluated by a simple spectrophotometric method, using ABTS(+) (2,2'azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)). High ratio of macrocyclic tannins, mainly oenothein B was identified in all Epilobium species examined. Flavonoid composition of Epilobium extracts showed several differences, especially comparing E. angustifolium to other species. Myricetin, quercetin, kaempferol and their various glycosides were dominant in samples, but their combination and ratio were distinctive in all cases. Epilobium extracts showed high radical-scavenger activity, comparable to that of well-known antioxidants, Trolox and ascorbic acid. Among species examined, extract of Epilobium parviflorum possessed the highest antioxidant capacity (EC(50)=1.71+/-0.05 microg/ml).

Analysis of heartsease (Viola tricolor L.) flavonoid glycosides by micro-liquid chromatography coupled to multistage mass spectrometry.

Micro-liquid chromatography (microLC) in conjunction with multistage mass spectrometry (MSn) was introduced to study several major heartsease flavonoid glycosides. High-resolution microLC separation was achieved by using a monolithic poly(p-methylstyrene-co-1,2-bis(p-vinylphenyl)ethane) column under reversed-phase conditions. The MS/MS and MS3 analysis of the flavonoid components of interest provided data about their glycosylation type and position, nature of their aglycones, and the structure/linkage information of their glycan moieties. With our microLC-MSn approach, four flavonol O-glycosides, nine flavone-C-glycosides, and three flavone C,O-glycosides were characterized in heartsease methanol extract. All of these glycoconjugates were found to be the derivatives of six aglycones: apigenin, chrysoeriol, isorhamnetin, kaempferol, luteolin, and quercetin.