Comparative study of the flavonoids of some Verbena species cultivated in Egypt by using high-performance liquid chromatography coupled with ultraviolet spectroscopy and atmospheric pressure chemical ionization mass spectrometry

Verbena rigida L., Verbena tenera Spreng. and Verbena venosa L. were investigated for their flavonoid content. Analysis was carried out by high-performance liquid chromatography coupled to diode array UV detection (LC–UV), using different techniques, also using post-column addition of shift reagents, afforded precise structural information about the position of the free hydroxyl groups in the flavonoid nucleus. LC–MS using atmospheric pressure chemical ionization (APCI) in the positive mode provided the molecular weight, the number of hydroxyl groups, the number of sugars and an idea about the substitution pattern of the flavonoid. On-line UV and MS data demonstrated the presence of orientin, vitexin, isovitexin, luteolin, luteolin 7-O-glucoside, apigenin 7-O-glucoside in addition to luteolin, chryseriol and apigenin aglycones in the three Verbena species with different concentrations. Quantitative determination of flavonoid content revealed the presence of 69.84 mg/g dry sample, 88.26 mg/g dry sample and 85.82 mg/g dry sample total flavonoid compounds in V. rigida L., V. tenera Spreng. and V. venosa L., respectively. The method developed for identification is useful for further chromatographic fingerprinting of plant flavonoids.     

On-line identification of sugarcane (Saccharum officinarum L.) methoxyflavones by liquid chromatography-UV detection using post-column derivatization and liquid chromatography-mass spectrometry

Sugarcane (Saccharum officinarum L., Gramineae) bagasse and leaves were investigated for their flavonoid content and transgenic sugarcane ("Bowman-Birk" and "Kunitz") was compared with non-modified ("control") plants. Analyses were carried out by high-performance liquid chromatography coupled to diode array UV detection (LC/UV), also using post-column addition of shift reagents, and tandem MS (atmospheric pressure chemical ionization-MS/MS and collision-induced dissociation-MS). On-line UV and MS data demonstrated the presence of methoxyflavone glycosides and aglycones in a total of seven compounds. Three naturally occurring flavones glycosides and two unusual erythro- and threo-diastereoisomeric flavolignan 7-O-glucosides were identified together with their aglycones.     

Liquid chromatography-tandem mass spectrometry analysis allows the simultaneous characterization of C-glycosyl and O-glycosyl flavonoids in stingless bee honeys

The analysis of the phytochemicals present in stingless bee honey samples has been a difficult task due to the small amounts of samples available and to the complexity of the phytochemical composition that often combines flavonoid glycosides and aglycones. Honey samples produced in Venezuela from Melipona species were analyzed using a combination of solid-phase extraction and HPLC-DAD-MSn/ESI methodologies with specific study of the fragment ions produced from flavonoid glycosides. The analyses revealed that flavonoid glycosides were the main constituents. The honey samples analyzed contained a consistent flavonoid pattern composed of flavone-C-glycosides, flavonol-O-glycosides and flavonoid aglycones. The HPLC-DAD-MSn/ESI analysis and the study of the fragment ions obtained allowed the characterization and quantification for the first time of five apigenin-di-C-glycosides, and ten quercetin, kaempferol and isorhamnetin O-glycosides (di- and tri- glycosides), and the aglycones pinobanksin, quercetin, kaempferol and isorhamnetin in the different samples. This is the first report of flavonoid-C-glycosides in honey. The results show that the content of flavonoid-glycosides (mean values of 2712 μg/100 g) in stingless bee honeys is considerably higher than the content of flavonoid aglycones (mean values of 315 μg/100 g). This differs from previous studies on Apis mellifera honeys that consistently showed much higher aglycone content and smaller flavonoid glycoside content. The occurrence of relevant amounts of flavonoid glycosides, and particularly C-glycosides, in stingless bee honeys could be associated with their putative anticataract properties.     

Selective separation of flavonoid glycosides in Dalbergia odorifera by matrix solid-phase dispersion using titania

Dalbergia odorifera contains high concentrations of flavonoid aglycones and trace flavonoid glycosides. In this study, trace flavonoid glycosides were separated from D. odorifera by titania with matrix solid-phase dispersion (MSPD). Before the MSPD experiment, four standards, including two isoflavone glycosides (genistin and formononetin-8-C-apiosyl (1-6)-glucoside) and their aglycones (genistein and formononetin), were used to compare their retention on a titania column. The effect of acetonitrile concentration and pH on their retention was investigated and a conclusion was drawn that high acetonitrile concentration and pH lead to the greatest difference in the retention of flavonoid as glycosides and aglycones. Besides hydrophilic interaction and ligand-exchange interaction may exist between sugar moiety of flavonoid glycoside and titania, so that flavonoid glycosides have stronger retention than that of aglycones. Based on the chromatographic rule of flavonoid as glycosides and aglycones on the titania column, the MSPD method was optimized to elute high concentration flavonoid aglycones first with 90% acetonitrile and 10% water containing 100 mM ammonium acetate buffer, and then to elute trace flavonoid glycosides with 20% acetonitrile and 80% water containing 1% trifluoroacetate (TFA). Isolated flavonoid glycosides were further analyzed by UPLC-MS/MS, and their fragmentation in MS(2) showed they are C-glycosyl flavonoids.     

Internal glucose residue loss in protonated O-diglycosyl flavonoids upon low-energy collision-induced dissociation

The low-energy collision-induced dissociation of protonated flavonoid O-diglycosides, i.e., flavonoid O-rutinosides and O-neohesperidosides, containing different aglycone types has been studied. The results indicate that the unusual [M + H - 162]+ ion formed by internal glucose residue loss, which in a previous study was shown to be a rearrangement ion, is strongly dependent upon the aglycone type. For 7-O-diglycosides, the internal glucose loss is very pronounced for aglycones of the flavanone type, but is completely absent for aglycones of the flavone and flavonol types. Internal glucose residue loss was found to correspond to a minor fragmentation pathway for flavonol 3-O-diglycosides. A plausible mechanism is proposed based on proton mobilization from the aglycone to the disaccharidic part of the flavonoid O-diglycosides which is supported by theoretical calculations and model building.     

Saponins with neuroprotective effects from the roots of Pulsatilla cernua

Four new oleanene-type triterpenoid saponins together with six known saponins were isolated from the roots of Pulsatilla cernua and their structures were elucidated on the basis of spectroscopic data, including 2D NMR spectra and chemical evidence. Among these one of the aglycones (gypsogenin) is reported for the first time from this genus. Some of these compounds showed significant neuroprotective effects against the cytotoxicity induced by β-amyloid(25-35) (Aβ(25-35)) on human neuroblastoma SH-SY5Y cells.     

Characterization and quantification of flavonoid aglycones and phenolic acids in the hydrolyzed methanolic extract of <Emphasis Type="Italic">Caucalis platycarpos</Emphasis> using HPLC-DAD-MS/MS

The water extract of burr parsley (Caucalis platycarpos L.) showed remarkable antitumor activity in rats and mice. Phenolic compounds, including phenolic acids and flavonoids, are considered to be the major bioactive compounds. The aim of this work was to develop a reverse phase HPLC-DAD method for the simultaneous quantification of flavonoid aglycones and phenolic acids, and an HPLC-DAD-MS/MS method for structural characterization of phenolic compounds, obtained after hydrolysis of C. platycarpos methanolic extract. Caffeic acid was the predominant phenolic acid, and luteolin was the predominant flavonoid aglycone. The optimized and validated method for the determination of the five phenolic acids and four flavonoid aglycones ensured reliable results and could be used for the quality control of raw plant material.     

Quantitative HPTLC separation of flavonoid glycosides in the taxonomy of elm (Ulmus spp.)

Summary The flavonoid glycosides of elm (Ulmus spp.) leaves have been separated by means of HPTLC on silica gel and quantitative analysis carried out by densitometry. The results obtained were used for discriminant analysis in order to investigate whether the flavonoid pattern could be used for the classification of elms. The results readily showed the different provenance of four species and the attribution of an unknown sample to a species can be carried out. This result is important as it offers a chemical tool in the taxonomy of the elm. The classification method is compared with one based on qualitative data from two-dimensional chromatography of the flavonoid aglycones. It was found to exhibit much better results than those obtained from the cluster analysis of these qualitative data.     

Determination of isoflavones in red clover and related species by high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection

High-performance liquid chromatography-UV-electrospray ionization-mass spectrometric detector (HPLC-UV-ESI-MSD) method for determination of isoflavones in red clover (Trifolium pratense L.) and related species has been developed. The separated isoflavones including aglycones, glycosides and glycoside malonates, were individually analyzed and identified by their molecular ions and characteristic fragment ion peaks using LC-MSD under MS and MS-MS mode, and in comparison with the standard isoflavones. A total of 31 isoflavones were detected in red clover. Several isoflavones were also identified for the first time in related species, T. repense L. (white clover), T. hybridum L. (alsike clover) and T. campestre Schreber (hop trefoil). Based on reversed phase HPLC, all 10 isoflavone aglycones, daidzein, formononetin, genistein, pseudobaptigenin, glycitein, calycosin, prunetin, biochanin A, irilone and pratensein in acidic hydrolyzed extracts were successfully separated within 40 min and quantified individually by UV and MS detectors. For the 10 target compounds, the investigated concentrations ranged from approximately 24 to approximately 12500 ng/ml for UV detection and approximately 6 to approximately 3125 ng/ml for MS detection, and good linearities (r2 > 0.999 for UV and r2 > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and repeatability (n = 10) were within 15% for these 10 compounds. This is the first method reported that enables the simultaneous quantitation of all 10 isoflavone aglycones in red clover and related species.     

Identification of Epilobium Species and Willow-herbs (Onagraceae) by HPLC Analysis of Flavonoids as Chemotaxonomic Markers

The aim of this work was to compare macro- and micromorphological characteristics and flavonoid composition of some Epilobium species and commercial willow-herbs for their authentication and quality control. In order to determine the flavonoid composition of extracts, the flavonol-glycosides were also hydrolised quantitatively to their aglycones. Before and after hydrolysis flavonoid fingerprints were studied by high-performance liquid chromatography using two different gradient mobile phase systems. Flavonoid composition was found to be suitable for the authentication of Epilobium species and commercial willow-herbs as an alternative to the current phytochemical study and time-consuming microscopic procedures [1]. The combined HPLC analysis of free flavonoid-aglycones and glycosides, developed in our laboratory can be used for the quality control of willow-herb samples and various commercial preparations.     

Evaluation of flavonoids binding to DNA duplexes by electrospray ionization mass spectrometry

In this study, electrospray ionization mass spectrometry (ESI-MS) was used to investigate the binding interactions of ten flavonoid aglycones and ten flavonoid glycosides with DNA duplexes. Relative binding affinities of the flavonoids toward DNA duplexes were estimated based on the fraction of bound DNA. The results revealed that the 4'-OH group of flavonoid aglycones was essential for their DNA-binding properties. Flavonoid glycosides with sugar chain linked on ring A or ring B showed enhanced binding toward the duplexes over their aglycone counterparts, whereas glycosylation of the flavonol quercetin on ring C exhibited a less pronounced effect. The aglycone skeletons and other hydroxyl substitutions on the aglycone also have an effect on the fractions of bound DNA. Upon collision-induced dissociation, the complexes containing flavonoid aglycones underwent the predominant ejection of a neutral ligand molecule, suggesting an intercalative DNA-binding mode. However, for complexes containing flavonoid glycosides, the loss of nucleobase increased to different extents, indicating a stronger binding or different binding mode. The results may provide not only a deeper insight into the DNA-binding properties of flavonoids but also a useful guideline for the design of efficient DNA-binding agents for chemotherapy.     

Collision-induced fragmentation of deprotonated methoxylated flavonoids, obtained by electrospray ionization mass spectrometry

Electrospray operated in the negative mode was used to analyse methoxylated flavonoids. They were found to produce radical anions by collision-induced fragmentation of the aglycones. Loss of a methyl group from the deprotonated molecule corresponding to [M - H - 15]-* ions, as well as [M - H - 15-28]-* and [M - H - 15-29]- fragment ions, were found to constitute the characteristic fragmentation for the monomethoxylated species, whereas [M - H - 15]-*, [M - H - 30]- and [M - H - 30-28]- were predominant for the polymethoxylated species. Obtained under similar conditions, the product-ion spectra of isomeric compounds were characteristically different. It is therefore possible to distinguish between methoxylated flavonoids with identical molecular mass, e.g. when screening plant extracts for flavonoid composition. However, comparison with standard compounds is necessary for the identification of unknown flavonoid aglycones.     

LC-DAD UV and LC-MS for the Analysis of Isoflavones and Flavones from In Vitro and In Vivo Biomass of Genista tinctoria L.

Conditions were determined for the separation of a complex set of isoflavones and flavones (free aglycones, monoglucosides, diglucosides and esters) by HPLC-DAD UV and MS detection. A good separation of all analytes was obtained and satisfactory peak shapes were achieved by gradient elution on an RP C18 column. The method was then applied to carry out qualitative and quantitative analysis of bioflavonoids present in the herb and in vitro cultures of Genista tinctoria L. Electrospray ionisation mass spectrometry in the negative mode was used to identify individual flavonoids in G. tinctoria plant material. Photodiode array detection was also utilised in flavonoid characterisation. All flavonoid compounds were then quantified using an internal standard. The method developed is useful for monitoring the process of flavonoid biosynthesis in biomass obtained through in vitro cultures.     

Leptopyrine,new alkaloid from Leptopyrum fumarioides L. (Ranunculaceae)

A new type of isoquinoline alkaloid leptopyrine was isolated from the aerial parts of Leptopyrum fumarioides L. (Ranunculaceae) of Mongolian origin. The known alkaloids protopine and thalifoline were isolated for the first time from this the species. All structures were established by physical and spectral analyses.     

An efficient,scalable approach to hydrolyze flavonoid glucuronides via activation of glycoside bond

Hydrolyzing flavonoid glucuronides into corresponding aglycones posed some significant challenges. To improve acid-catalyzed hydrolysis process of flavonoid glucuronide, structures of glucuronide, hydrolysis parameters and post-processing were optimized. The optimized condition was performed by hydrolysis flavonoid glycoside methyl ester in a mixed solvent consisting of 2 mol/L H₂SO₄/EtOH/H₂O (1/8/1, v/v/v) at 95 °C for 7 h and resulted in up to 90% aglycone yields, minimal byproduct formations and milder hydrolysis conditions. Furthermore, the optimized method avoids tedious purification steps and is easily conducted on a relatively large-scale using economical and commercially available reagents.     

A Study of Interaction between Flavonoids and the Parallel Quadruplex Structure [d(TGGGGT)]4 by Electrospray Ionization Mass Spectrometry

In this study, electrospray ionization mass spectrometry (ESI‐MS) was used to investigate interaction of 21 flavonoids (10 aglycones and 11 glycosides) with the parallel quadruplex structure [d(TGGGGT)]₄. Relative binding affinities of flavonoids toward [d(TGGGGT)]₄ were estimated based on the fraction of bound DNA. It was found that [d(TGGGGT)]₄ showed a binding preference to the flavonoid glycosides over flavonoid aglycones. It was deduced that glycosylation played a key role for the [d(TGGGGT)]₄‐binding properties of flavonoid glycosides. Upon collision‐induced dissociation, complexes of flavonoid/[d(TGGGGT)]₄ underwent the loss of flavonoids, suggesting an end‐stacking binding mode. The current work demonstrates that ESI‐MS is a powerful tool in the study of interaction between drugs and nucleic acids.     

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.     

Mass spectra of trimethylsilyl derivatives of naturally occurring flavonoid aglycones and chalcones

Electron impact mass spectra of the trimethylsilyl derivatives of a series of flavonoid aglycones and chalcones are reported. The spectra show prominent ions arising from fragmentation of the trimethylsilyl (TMS) groups. Inter-actions between adjacent TMS groups, and between TMS groups in the 3- or 5-position (6′-position for the chalcones) and the C-ring carbonyl, yield structurally significant ions. Few fragments associated with the retro-Diels-Alder cleavage of the C-ring characteristic of the underivatized compounds, are observed. The TMS derivatives thus provide complementary information for the identification of flavonoid aglycones and chalcones in biological systems.     

Neutral loss scan in complement with high-resolution MS/MS: Combination of detection methods for flavonoid and limonoid glycosides analysis

In this study, neutral loss scan and high-resolution MS/MS were used in combination to detect and tentatively identify various flavonoid and limonoid glycosides in navel orange albedo, juice, peel and pulp. These compound classes are of research interest due to their flavour and bioactive properties, and although flavonoid glycosides have been previously studied in other food matrices, to the best of our knowledge, neutral loss scans have not been used for the elucidation of limonoid glycosides. Neutral loss masses of 120, 162 and 308 Da were selected for the detection of hexose, rutinose and neohesperidose-substituted flavonoids, whereas 197 Da was explored for limonoid glycosides due to their tendency to form ammonium adducts. Fragmentation patterns obtained from targeted MS/MS were then used to differentiate rutinose and neohesperidose substituents as well as flavonoid subclasses of flavones, flavanones and flavonols. Additionally, high-resolution MS/MS was also used for the identification of aglycones by accurate mass (to four decimal places), allowing for the differentiation of aglycones with similar unit masses but different chemical formulas. In total, 19 flavonoid glycosides and six limonoid glycosides were detected. This workflow allows for a rapid screening of flavonoid and limonoid glycosides in citrus, which can be further extended to other food products such as tea.     

Mass spectrometric profiling of flavonoid glycoconjugates possessing isomeric aglycones

In fields such as food and nutrition science or plant physiology, interest in untargeted profiling of flavonoids continues to expand. The group of flavonoids encompasses several thousands of chemically distinguishable compounds, among which are a number of isobaric compounds with the same elemental composition. Thus, the mass spectrometric identification of these compounds is challenging, especially when reference standards are not available to support their identification. Many different types of isomers of flavonoid glycoconjugates are known, i.e. compounds that differ in their glycosylation position, glycan sequence or type of interglycosidic linkage. This work focuses on the mass spectrometric identification of flavonoid glycoconjugate isomers possessing the same glycan mass and differing only in their aglycone core. A non‐targeted HPLC‐ESI‐MS/MS profiling method using a triple quadrupole MS is presented herein, which utilizes in‐source fragmentation and a pseudo‐MS³ approach for the selective analysis of flavonoid glycoconjugates with isomeric/isobaric aglycones. A selective MRM‐based identification of the in‐source formed isobaric aglycone fragments was established. Additionally, utilizing the precursor scanning capability of the employed triple quadrupole instrument, the developed method enabled the determination of the molecular weight of the studied intact flavonoid glycoconjugate. The versatility of the method was proven with various types of flavonoid aglycones, i.e. anthocyanins, flavonols, flavones, flavanones and isoflavones, along with their representative glycoconjugates. The developed method was also successfully applied to a commercially available sour cherry sample, in which 16 different glycoconjugates of pelargonidin, genistein, cyanidin, kaempferol and quercetin could be tentatively identified, including a number of compounds containing isomeric/isobaric aglycones. Copyright © 2015 John Wiley & Sons, Ltd.