Phytochemical characterization of bioactive compounds composition of Rosmarinus eriocalyx by RP–HPLC–ESI–QTOF–MS

Rosmarinus eriocalyx (rosemary or Elyazir) is an endemic species growing in arid steppe and rocky mountain in the South-West Algeria. This plant is well known in Algeria and Morocco due to its medicinal properties. However, little is known about its phytochemical composition. For this purpose, natural antioxidant compounds from R. eriocalyx were recovered by solid-liquid extraction and characterized by reversed-phase high-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry using negative and positive ionization modes. This analytical methodology enabled the characterization of 101 compounds, which were distributed in five major categories namely hydroxycinnamic acid derivatives, hydroxybenzoic acid derivatives, flavonoids, phenolic diterpenes and phenolic triterpenes. Moreover, the studied extract generally showed free radical-scavenging and reductive abilities in the range of butylated hydroxyanisole, butylated hydroxytoluene, α-tocopherol^, and ascorbic acid. Therefore, the result suggests that the aqueous-methanolic extract of R. eriocalyx could serve as a potential source of antioxidants.     

Characterization of phenolic acids in black carrots (Daucus carota ssp. sativus var. atrorubens Alef.) by high-performance liquid chromatography/electrospray ionization mass spectrometry

Phenolic acids were extracted from black carrot roots (Daucus carota ssp. sativus var. atrorubens Alef.) and a black carrot juice concentrate, and characterized by high-performance liquid chromatography/electrospray ionization mass spectrometry. Most of the compounds detected were identified as depsides composed of p-coumaric, caffeic and ferulic acids. Additionally, three hydroxybenzoic acid derivatives and one quercetin glycoside were detected. 5-O-Caffeoylquinic acid (chlorogenic acid) represented the predominant compound amounting to 657 mg/kg in the roots and 5815 mg/kg in the concentrate. The specific fragmentation patterns of mono- and dihydroxycinnamoylquinic acids allowed the distinction of several stereoisomers. The presence of 4-O-caffeoylquinic acid and several further hydroxycinnamic acid esters, together with compounds not belonging to the depside type, is reported for the first time. The present study reveals that the phenolic profile of black carrots is even more complex than hitherto assumed and may contribute to pigment stability of extracts derived from the roots.     

Analysis of phenolic compounds from different morphological parts of Helichrysum devium by liquid chromatography with on‐line UV and electrospray ionization mass spectrometric detection

A simple and rapid method has been used for the screening and identification of the main phenolic compounds from Helichrysum devium using high‐performance liquid chromatography with on‐line UV and electrospray ionization mass spectrometric detection (LC‐DAD/ESI‐MSⁿ). The total aerial parts and different morphological parts of the plant, namely leaves, flowers and stems, were analyzed separately. A total of 34 compounds present in the methanolic extract from Helichrysum devium were identified or tentatively characterized based on their UV and mass spectra and retention times. Three of these compounds were positively identified by comparison with reference standards. The phenolic compounds included derivatives of quinic acid, O‐glycosylated flavonoids, a caffeic acid derivative and a protocatechuic acid derivative. The characteristic loss of 206 Da from malonylcaffeoyl quinic acid was used to confirm the malonyl linkage to the caffeoyl group. This contribution presents one of the first reports on the analysis of phenolic compounds from Helichrysum devium using LC‐DAD/ESI‐MSⁿ and highlights the prominence of quinic acid derivatives as the main group of phenolic compounds present in these extracts. We also provide evidence that the methanolic extract from the flowers was significantly more complex when compared to that of other morphological parts. Copyright © 2009 John Wiley & Sons, Ltd.     

Phenolic compounds of mountain tea from the Balkans: LC/DAD/ESI/MSn profile and content

Twenty-one samples of Sideritis species (S. scardica, S. raeseri, S. taurica, S. syriaca and S. perfoliata) from various locations on the Balkan Peninsula were evaluated for their chemical constituents. Chemical analyses were focused on secondary metabolites, particularly phenolic compounds, which have several roles in the plant physiological processes and have demonstrated significant health beneficial effects. The occurrence of hydroxycinnamic acids, phenylethanoid glycosides and flavonoids has been investigated in taxonomically related taxa of the genus Sideritis. A systematic method for phenolic compounds identification was developed using tandem mass spectrometry coupled to high performance liquid chromatography with diode array detection. Scanning for precursor ions of commonly found phenolics in Sideritis species using LC/MS11 with an ion trap instrument permitted the specific determination of hydroxycinnamic acid derivatives, and phenylethanoid and flavonoid glycosides. Further characterization of each phenolic compound was performed using MS/MS product-ion analysis and common-neutral-loss analysis. This on-line technique allowed identification of three hydroxycinnamic acid derivatives, eight phenylethanoid glycosides, and twenty-four flavonoid glycosides. All the taxa analysed produced very similar phenolic patterns characterized by the presence of 5-caffeoylquinic acid, lavandulifolioside, verbascoside, hypolaetin 7-O-[6'-O-acetyl]-allosyl(1-->2)glucoside, apigenin 7-(4"-p-coumaroylglucoside), 4'-O-methylisoscutellarein 7-O-[6'-O-acetyl]-allosyl(1-->2)glucoside, and minor amounts of isoverbascoside, apigenin 7-O-allosyl(1-->2)glucoside, isoscutellarein 7-O-allosyl-(1-->2)-[6"-O-acetyl]-glucoside, hypolaetin 7-O-allosyl-(1-->2)-[6"-O-acetyl]-glucoside and 4'-O-methylhypolaetin 7-O-[6'-O-acetyl]-allosyl-(1-->2)-[6"-O-acetyl]-glucoside. These results show that the investigated species are systematically very closely related. Phenylethanoid glycosides and flavonoid acetylglycosides are dominant and constitute 90% of the total phenolic compounds compared with hydroxycinnamic acid and flavonoid 7-O-glycosides. Principal component analysis (PCA) was performed for the nature and content of the different compounds to be correlated to the particular Sideritis species and also to the locations.     

Determination of the Free Radical Scavenging Activity of Lycium Extracts

Lycium species growing in Turkey have not so far been studied sufficiently. For this reason, non-polar and polar extracts obtained from the fruits of Lycium barbarum L. and L. Ruthenicum Murray (Solanaceae) were assessed both in vitro for their potential as free radical scavenger crude extracts and their phenolic composition. Fruits of Lycium species were sequentially extracted with petroleum ether, ethyl acetate, methanol, n-butanol, and water in a Soxhlet extractor. All the extracts were assessed for the scavenging of the nitrogen-centered free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH^.) by in vitro method. Furthermore, the composition of each extract was investigated both in terms of its Folin-Ciocalteau reactive components and its qualitative content. The phenolic compounds within the extracts were determined as benzoic acid and hydroxycinnamic acid derivatives, flavonoids, and anthocyanins according to their retention time and UV spectral data by HPLC-DAD system.     

Characterization of phenolic compounds in Helichrysum melaleucum by high‐performance liquid chromatography with on‐line ultraviolet and mass spectrometry detection

Helicrysum melaleucum is a medicinal plant traditionally used in the islands of the Macaronesia region for the treatment of respiratory diseases. In this work, the phenolic compounds of Helicrysum melaleucum plants collected in different geographical locations of Madeira Island and their morphological parts (total aerial parts, leaves, flowers and stems) were extracted and analyzed separately by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC‐DAD/ESI‐MSⁿ). A total of 68 compounds were characterized based mainly on their UV and mass spectra. These included derivatives of O‐glycosylated flavonoids (flavonol and flavones type), quinic acid, caffeic acid, lignans and polyphenols. The flowers were found to be the morphological part with higher variety of phenolic compounds. The large differences in the phenolic composition of plants collected from different geographical locations allowed the identification of a few components, such as pinoresinol and methoxylated flavone derivatives, likely to be useful as geographical markers. Also, these results promote further comparison of the bioactivities of the different samples analyzed. This paper marks the first report on the chemical analysis of Helichrysum melaleucum species. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of Phlebodium decumanum Fronds by High-Performance Liquid Chromatography by Ultraviolet-Visible and Quadrupole Time-of-Flight Tandem Mass Spectrometry (HPLC–UV–VIS–QTOF–MS/MS)

Phlebodium decumanum (P. decumanum), together with other tropical ferns commonly known as Calaguala, have been empirically applied since ancient times to ameliorate inflammatory disorders, skins diseases and even cancer. There are evidences of antineoplastic potential and anti-inflammatory, immunomodulatory and antioxidant properties of the extract. Preliminary studies have also shown direct antitumor activity of the extracts. In the present article, the phytochemical composition of a hydro-ethanolic extract of P. decumanum is investigated using high-performance liquid chromatography (HPLC) with ultraviolet–visible (UV–vis) diode array detection (DAD) and electrospray ionization (ESI) quadrupole time-of-flight tandem mass spectrometry (QTOF–MS/MS) detection. First, the chromatographic profile is established as a representative fingerprint of the compounds extracted from the fern. Then, a total of 122 chemicals, including 23 flavonoids, 47 phenolic acids (34 hydroxycinnamic acids and 13 hydroxybenzoic acids), 9 amino and amino-sugar derivatives, 24 organic acids and their derivatives, and other metabolites, have been characterized. Moreover, 12 unknown compounds were also detected, providing the first comprehensive characterization available on the phytochemical composition of the leaves of P. decumanum.     

Characterization of acylated flavonoid-O-glycosides and methoxylated flavonoids from Tagetes maxima by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Liquid chromatography coupled to negative electrospray ionization (ESI) tandem mass spectrometry (MS/MS) employing a triple quadrupole mass spectrometer was used in the structural determination of acylated flavonoid-O-glycosides and methoxylated flavonoids occurring in Tagetes maxima. The compounds were identified by experiments in full scan mode (MS), and tandem mass experiments (MS/MS) of precursor ion scan, product ion scan, and neutral loss scan modes. In order to characterize the aglycones of the flavonoid glycosides, in-source fragmentation of the deprotonated molecule [M-H]- followed by product ion scan of the resulting aglycone [A-H]- were performed. This combined approach allowed the identification of 51 phenolic compounds, including flavonoid-O-glycosides acylated with galloyl, protocatechuoyl, coumaroyl or caffeoyl groups, methoxylated flavonoids, and hydroxycinnamic acid and phenolic acid derivatives, none of them previously reported in Tagetes maxima.     

Combined mass spectrometric and chromatographic methods for in‐depth analysis of phenolic secondary metabolites in barley leaves

Structural analysis via HPLC‐ESI‐MSn, UPLC‐HESI‐MS/MS and NMR reported 152 phenolic secondary metabolites in spring barley seedlings (Hordeum vulgare L.). Flavonoids with various patterns of glycosylation and acylation, as well as hydroxycinnamic acid glycosides, esters and amides, were identified in methanolic extracts from leaves of nine varieties of barley originating from different regions of the world. Hordatines derivatives, flavones acylated directly on the aglycone, and hydroxyferulic acid derivatives deserve special attention. Preparative chromatography enabled characterization of a number of compounds at trace levels with the 6‐C‐[6″‐O‐glycosyl]‐glycosides and the 6‐C‐[2″,6″‐di‐O‐glycosides]‐glucoside structure of flavones. Derivatives of flavonols, quercetin and isorhamnetin were observed only in Syrian varieties. The ultra performance liquid chromatography profiles of UV‐absorbing secondary metabolites were used for chemotaxonomic comparison between nine varieties of barley from different climatic conditions. The hierarchical clustering of bred lines from the Fertile Crescent and European and American varieties indicates a great diversity of chemical phenotypes within barley species. Copyright © 2015 John Wiley & Sons, Ltd.     

HPLC/QTOF‐MS/MS application to investigate phenolic constituents from Ficus pandurata H. aerial roots

Ficus pandurata H. aerial roots are used as a traditional Chinese medicine for the treatment of uarthritis, indigestion and hyperuricemia. However, the bioactive constituents responsible for the pharmacological effects of F. pandurata H. are unclear. A simple and efficient HPLC/QTOF‐MS/MS (high‐performance liquid chromatography/electrospray ionization with quadrupole time‐of‐flight tandem mass spectrometry) method was established to detect and identify active constituents in the n‐butanol extract of F. pandurata H. aerial roots. Chemical constituents were separated and investigated by HPLC/QTOF‐MS/MS in the negative‐ion mode. Thirty‐seven compounds, including hydroxycinnamic acid derivatives, hydroxybenzoic acid derivatives, hydroquinone glycosides, flavonoid glycosides, etc., were identified or tentatively characterized in the n‐butanol extract of F. pandurata H. aerial roots by comparing the UV spectra, accurate mass spectra and fragmentation pathways and retrieving the reference literatures. Moreover, the flavonoid trisaccharides and hydroxybenzoic acid derivatives were tentatively characterized in F. pandurata H. for the first time. The analytical tool used here is very valuable in the rapid separation and identification of the multiple and minor constituents in the n‐butanol extract of F. pandurata H. aerial roots. Copyright © 2014 John Wiley & Sons, Ltd.     

Investigation on the phenolic constituents in Hamamelis virginiana leaves by HPLC-DAD and LC-MS/MS

Aqueous and acetone/water extracts from Hamamelis virginiana leaves were investigated to obtain a thorough insight into their phenolic composition. To secure compound integrity, a gentle extraction method including the exclusion of light was used. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses yielded a fingerprint including 27 phenolic constituents. Quantification of the key compounds on an equivalent basis by high-performance liquid chromatography diode-array detection (HPLC-DAD) showed that gallotannins consisting of six to 11 galloyl units constitute the main fraction, whereas procyanidins and catechin represented only a minor part. Closer inspection revealed that both extracts possess virtually the same galloyl hexose distribution, and the octagalloyl hexose represents the major tannin constituent. Additionally, eight flavonol glycosides and their corresponding aglycones quercetin and kaempferol, as well as three chlorogenic acid isomers and other hydroxycinnamic acids, were identified. Moreover, stability studies on the aqueous extract (5 °C, dark; room temperature, dark; room temperature, light) revealed that the phenolic profile underwent changes when exposed to light. Especially the gallotannins proved to be considerably unstable which may result in phytochemically altered Hamamelis leaf extracts upon transport and storage.     

Fragmentation characteristics of hydroxycinnamic acids in ESI‐MSn by density functional theory

This work aims to analyze the electrospray ionization multistage mass spectrometry (ESI‐MSⁿ) fragmentation characteristics of hydroxycinnamic acids (HCAs) in negative ion mode. The geometric parameters, energies, natural bond orbitals and frontier orbitals of fragments were calculated by density functional theory (DFT) to investigate mass spectral fragmentation mechanisms. The results showed that proton transfer always occurred during fragmentation of HCAs; their quasi‐molecular ions ([M − H]⁻) existed in more than one form and were mainly with the lowest energy. The fragmentation characteristics included the followings: (1) according to the different substitution position of phenolic hydroxyl group, the ring contraction reaction by CO elimination from benzene was in an increasingly difficult order: m‐phenolic hydroxyl > p‐phenolic hydroxyl > o‐phenolic hydroxyl; and (2) ortho effect always occurred in o‐dihydroxycinnamic acids (o‐diHCAs), i.e. one phenolic hydroxyl group offered H⁺, which combined with the other one to lose H₂O. In addition, there was a nucleophilic reaction during ring contraction in diHCAs that oxygen atom attacked the carbon atom binding with the other phenolic hydroxyl to lose CO₂. The fragmentation characteristics and mechanism of HCAs could be used for analysis and identification of such compounds quickly and effectively, and as reference for structural analogues by ESI‐MS. Copyright © 2017 John Wiley & Sons, Ltd.     

Phenolic characterization of Northeast Portuguese propolis: usual and unusual compounds

In this study, an ethanolic extract from Portuguese propolis was prepared, fractionated by high-performance liquid chromatography, and the identification of the phenolic compounds was done by electrospray mass spectrometry in the negative mode. This technical approach allowed the identification of 37 phenolic compounds, which included not only the typical phenolic acids and flavonoids found in propolis from temperate zones but also several compounds in which its occurrence have never been referred to in the literature. Four of the novel phenolic compounds were methylated and/or esterified or hydroxylated derivatives of common poplar flavonoids, although six peculiar derivatives of pinocembrin/pinobanksin, containing a phenylpropanoic acid derivative moiety in their structure, were also identified. Furthermore, the Portuguese propolis sample was shown to contain a p-coumaric ester derivative dimer.     

Simultaneous identification of glucosinolates and phenolic compounds in a representative collection of vegetable Brassica rapa

Brassica raparapa group is widely distributed and consumed in northwestern Spain. The consumption of Brassica vegetables has been related to human health due to their phytochemicals, such as glucosinolates and phenolic compounds that induce a variety of physiological functions including antioxidant activity, enzymes regulation and apoptosis control and the cell cycle. For first time in Brassica crops, intact glucosinolates and phenolic compounds were simultaneously identified and characterized. Twelve intact glucosinolates, belonging to the three chemical classes, and more than 30 phenolic compounds were found in B. rapa leaves and young shoots (turnip greens and turnip tops) by LC–UV photodiode array detection (PAD)–electrospray ionization (ESI). The main naturally occurring phenolic compounds identified were flavonoids and derivatives of hydroxycinnamic acids. The majority of the flavonoids were kaempferol, quercetin and isorhamnetin glycosylated and acylated with different hydroxycinnamic acids. Quantification of the main compounds by HPLC-PAD showed significant differences for most of compounds between plant organs. Total glucosinolate content value was 26.84 μmol g⁻¹ dw for turnip greens and 29.11 μmol g⁻¹ dw for turnip tops; gluconapin being the predominant glucosinolate (23.2 μmol g⁻¹ dw). Phenolic compounds were higher in turnip greens 51.71 μmol g⁻¹ dw than in turnip tops 38.99 μmol g⁻¹ dw, in which flavonols were always the major compounds.     

Fragmentation of trimethylsilyl derivatives of 2-alkoxyphenols: A further violation of the ‘even-electron rule’

The mass spectra of trimethylsilyl (TMS) ethers of 2-methoxyphenols show abundant [M–30]⁺˙ ions originating from consecutive loss of two methyl radicals. This is illustrated by comparison of the accurate mass-measured and linked-scan spectra of the TMS derivatives of 2-methoxyphenol (guaiacol), 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid methyl ester (ferulic acid methyl ester) with those of the TMS derivatives of phenol, 4-hydroxybenzaldehyde, 3-(4-hydroxyphenyl)-2-propenoic acid methyl ester (p-coumaric acid methyl ester), 3-methoxyphenol and 4-methoxyphenol. This distinctive ortho effect is valuable in the identification of isomeric phenolic compounds. In the spectra of the TMS derivatives of 2-ethoxyphenol and 2-propoxyphenol the sequential loss of two radicals is less pronounced, because elimination of the side-chain and a methyl group with rearrangement and hydrogen migration is competitive.     

Rapid isocratic HPLC analysis of caffeic acid derivatives from <Emphasis Type="Italic">Echinacea purpurea</Emphasis> cultivated in Iran

Cichoric acid and caftaric acid are the main phenolic compounds in Echinacea purpurea tops. The level of these phenolic compounds in E. purpurea extracts is affected by different factors such as seasonal variations, drying methods, extraction methods, and growing location of the plant. HPLC analysis of caffeic acid derivatives in extracts of Echinacea purpurea (Cultivar) aerial parts, produced by boiling water extraction and ethanol-water extraction methods, showed various levels of the derivatives. Our findings revealed that the Iranian cultivated E. purpurea had a high level of cichoric acid (3.5–5.7 %). Caftaric acid was also the main phenolic compound in E. purpurea tops (3.1–4.5 %). After 2 h of boiling water extraction, the level of cichoric acid was 5.7 %, whereas the level of this acid in 60:40 ethanol-water extraction did not exceed 3.9 %. Published in Khimiya Prirodnykh Soedinenii, No. 2, pp. 150–152, March–April, 2008.     

Simultaneous determination of polyphenolic compounds in red and white grapes grown in Sardinia by high performance liquid chromatography–electron spray ionisation-mass spectrometry

A new method for the identification and the quantification of nonanthocyanin phenolic compounds from six Vitis Vinifera grape varieties native to Sardinia (three native: Vermentino, Malvasia and Cannonau and three non-native types: Chardonnay, Sauvignon and Cabernet Sauvignon; Argiolas vineyard) was developed. This rapid and selective method employs LC/ESI-MS in negative mode. Different solvents extraction and different sorbents for purification were compared to the direct analysis of the initial extracts without further sample preparation. A total of 54 phenolic compounds were identified either in the freeze-dried skins or seeds, including nonflavonoids (hydroxybenzoic and hydroxycinnamic acids and their derivatives, stilbenes) and flavonoids (flavanols, flavonols, dihydroxyflavonols).     

HPLC determination of organic acids, sugars, phenolic compositions and antioxidant capacity of orange juice and orange wine made from a Turkish cv. Kozan

Organic acids, sugars, phenolic compositions and antioxidant capacities of orange juice and orange wine obtained from the cv. Kozan of Turkey were determined. High-performance liquid chromatographic methods were used to identify and quantify of these compounds. Three organic acids (citric, malic and ascorbic acids) and three sugars (sucrose, glucose and fructose) were determined. The major organic acid was found as citric acid. With regard to sugars, sucrose was present in the largest amounts for orange juice and wine. A total of 13 phenolic compounds were identified and quantified in orange juice and wine, including hydroxybenzoic acids (2), hydroxycinnamic acids (5), and flavanones (6). Hesperidin, narirutin and ferulic acid were the most abundant phenolic compounds in orange juice and wine. Antioxidant activities of orange juice and wine were measured using the DPPH^• (2,2-diphenyl-1-picrylhydrazyl) assay, and the antioxidant capacity of orange juice was found to be higher than that of orange wine.     

Ultra‐fast liquid chromatography coupled with electrospray ionization time‐of‐flight mass spectrometry for the rapid phenolic profiling of red maple (Acer rubrum) leaves

The red maple (Acer rubrum) species is economically important to North America because of its sap, which is used to produce maple syrup. In addition, various other red maple plant parts, including leaves, were used as a traditional medicine by the Native Americans. Currently, red maple leaves are being used for nutraceutical and cosmetic applications but there are no published analytical methods for comprehensive phytochemical characterization of this material. Herein, a rapid and sensitive method using liquid chromatography with electrospray ionization time‐of‐flight tandem mass spectrometry was developed to characterize the phenolics in a methanol extract of red maple leaves and a proprietary phenolic‐enriched red maple leaves extract (Maplifa™). Time‐of‐flight mass spectrometry and tandem mass spectrometry experiments led to the identification of 106 phenolic compounds in red maples leaves with the vast majority of these compounds also detected in Maplifa™. The compounds included 68 gallotannins, 25 flavonoids, gallic acid, quinic acid, catechin, epicatechin, and nine other gallic acid derivatives among which 11 are potentially new and 75 are being reported from red maple for the first time. The developed method to characterize red maple leaves phenolics is rapid and highly sensitive and could aid in future standardization and quality control of this botanical ingredient.     

Die stufenweise Syntheseo,o′-methylenverbrückter Cyclohexamerer mitp-Kresol- oderP-Kresol- und 4-tert-Butylphenol-Bausteinen Vergleich mit ähnlich strukturierten Kettenoligomeren

Two phenolic alcohols with six phenolic units in their molecules have been obtained by stepwise syntheses starting from simple phenolic derivatives. The phenolic alcohols were cyclized in a one step cyclization by boiling in a very diluted acetic acid solution. A two step cyclization of a phenolic alcohol with three phenolic units to a cyclohexamer proceeds only with poor yields. The cyclic compounds were compared with chainlike oligomers containing six phenolic units in their molecules.

Herrn Prof. Dr.H. F. Mark mit allen guten Wünschen zum 85. Geburtstag gewidmet.