On-line hyphenation of centrifugal partition chromatography and high pressure liquid chromatography for the fractionation of flavonoids from Hippophaë rhamnoides L. berries

Centrifugal Partition Chromatography (CPC), a liquid–liquid preparative chromatography using two immiscible solvent systems, benefits from numerous advantages for the separation or purification of synthetic or natural products. This study presents the on-line hyphenation of CPC-Evaporative Light Scattering Detector (CPC-ELSD) with High Performance Liquid Chromatography-UV (HPLC-UV) for the fractionation of flavonols from a solvent-free microwave extract of sea buckthorn (Hippophaë rhamnoides L., Elaeagnaceae) berries. An Arizona G system was used for the fractionation of flavonoids by CPC and a fused core Halo C18 column allowed the on-line analyses of collected fractions by HPLC. The on-line CPC/HPLC procedure allowed the simultaneous fractionation step at preparative scale combined with the HPLC analyses which provide direct fingerprint of collected fractions. Thus the crude extract was simplified and immediate information on the composition of fractions could be obtained. Furthermore, this methodology reduced the time of post-fractionation steps and facilitated identification of main molecules by Mass Spectrometry (MS). Rutin, isorhamnetin-3-O-rutinoside, isorhamnetin-3-O-glucoside, quercetin-3-O-glucoside, isorhamnetin-rhamnoside, quercetin and isorhamnetin were identified. CPC-ELSD/HPLC-UV could be considered as a high-throughput technique for the guided fractionation of bioactive natural products from complex crude extracts.     

Reactive oxygen species scavenging activity of flavone glycosides from Melilotus neapolitana

One new and six known flavone glycosides were isolated from the MeOH extract of Melilotus neapolitana Ten. The new compound, identified as 7-O-beta-D-glucopyranosyloxy-4',5-dihydroxy-3-[O-alpha-L-rhamnopyranosyl-(1-->6)-3-O-beta-D-glucopyranosyloxy]flavone (1) by 1D and 2D NMR techniques and mass spectra, was isolated along with kaempferol-3-O-rutinoside (2), kaempferol-3-O-glucoside (3), rutin (4), quercetin-3-O-glucoside (5), isorhamnetin-3-O-rutinoside (6), and isorhamnetin-3-O-glucoside (7). The antioxidant and radical scavenging activities of these compounds and the whole crude methanol extract were evaluated. The organic extract can inhibit MDA marker's synthesis by 57%. All the metabolites displayed good reducing power, with the kaempferol (2,3) and isorhamnetin derivatives (6,7) being less active than the corresponding quercetin derivatives 4,5.     

Comparison of the contents of various antioxidants of sea buckthorn berries using CE

The increased interest in sea buckthorn (Hippophae rhamnoides L.) made it possible to investigate the antioxidant content in it. To address this issue, the presence of following antioxidant compounds were analyzed: trans-resveratrol, catechin, myricetin, quercetin, p-coumaric acid, caffeic acid, L-ascorbic acid (AA), and gallic acid (linear range of 50-150 micromol/L) in six different varieties of sea buckthorn berries extracts (sea buckthorn varieties: "Trofimovskaja (TR)," "Podarok Sadu (PS)," and "Avgustinka (AV),") received from two local Estonian companies. Trans-Resveratrol, catechin, AA, myricetin, and quercetin were found in extracts of sea buckthorn. Moreover, AA, myricetin, and quercetin contents were quantified. The biggest average AA content was found in TR (740 mg/100 g of dried berries, respectively). Furthermore, the same varieties gave the biggest quercetin content 116 mg/100 g of dried berries, respectively. For analysis, CZE was used and the results were partly validated by HPLC. Statistically no big differences in levels of antioxidants were consistently found in different varieties of sea buckthorn extracts investigated in this work.     

New acylated anthocyanins and other flavonoids from the red flowers of Clematis cultivars

Six new acylated cyanidin glycosides, cyanidin 3-O-beta-(2'-E-caffeoylglucopyranosyl)-(1 --> 2)-O-beta-galactopyranoside (1), cyanidin 3-O-beta-(2'-E-caffeoylglucopyranosyl)-(1 --> 2)-O-beta-(6'-malonylgalactopyranoside) (2), cyanidin 3-O-beta-(2'-E-caffeoylglucopyranosyl)-(1 --> 2)-O-beta-(6'-succinylgalactopyranoside) (3), cyanidin 3-O-beta-(2'-E-caffeoylglucopyranosyl)-(1 --> 2)-O-beta-galactopyranoside-3'- O-beta-glucuronopyranoside (4), cyanidin 3-O-beta-(2'-E-caffeoylglucopyranosyl)-(1 --> 2)-O-beta-(6'-malonylgalactopyranoside)-3'-O-beta-glucuronopyranoside (5), and cyanidin 3-O-beta-(2'-E-feruloylglucopyranosyl)-(1 --> 2)-O-beta-(6'-malonylgalactoside)-3' -O-beta-glucuronopyranoside (6), were isolated from the red flowers of two Clematis cultivars, 'Niobe'and 'Madame Julia Correvon'. The chemical structures of the isolated anthocyanins were determined by UV, LC-MS, HPLC, TLC, characterization of hydrolysates, and 1H and 13C NMR spectroscopy, including H-H COSY, C-H COSY, HMBC, HMQC and NOESY. The last three anthocyanins were widely distributed in 37 red flower Clematis cultivars. On the other hand, the first three compounds were found only in two cultivars. Five known flavonol glycosides, kaempferol 3-O-glucoside, kaempferol 3-O-rutinoside, quercetin 3-O-galactoside, quercetin 3-O-glucoside and quercetin 3-O-rutinoside, were isolated from the flowers of'Madame Julia Correvon'.     

Impact of Drying Methods on Phenolic Components and Antioxidant Activity of Sea Buckthorn (Hippophae rhamnoides L.) Berries from Different Varieties in China

Sea buckthorn berries are rich in bioactive compounds and can be used for medicine and food. The variety and drying method used have an important influence on quality. In this study, different sea buckthorn varieties from China were selected and dried with four common drying methods. The total phenolic content (TPC), total flavonoids content (TFC), contents of 12 phenolic compounds and antioxidant capacity in vitro were analyzed. The results showed that the TPC, TFC and antioxidant activity of two wild sea buckthorn berries were higher than those of three cultivated berries, and for the same varieties, measured chemical contents and antioxidant activity of the freeze-dried fruit were significantly higher than those obtained with three conventional drying methods. In addition, forty-one compounds in sea buckthorn berry were identified by UPLC-PDA-Q/TOF-MS, most of which were isorhamnetin derivatives. Multivariate statistical analysis revealed narcissin and isorhamnetin-3-O-glucoside varied significantly in sea buckthorn berries of different varieties and with different drying methods; they were potential quality markers. Strong correlations were found between TPC, gallic acid and antioxidant capacity (p < 0.05). The results revealed how components and antioxidant activity varied in different sea buckthorn, which provides a valuable reference for quality control and further development and utilization of sea buckthorn.     

Simultaneous determination of quercetin, kaempferol, and isorhamnetin in phytopharmaceuticals of Hippophae rhamnoides L. by high-performance liquid chromatography with chemiluminescence detection

A novel method based on reversed-phase high-performance liquid chromatography with chemiluminescence detection has been developed for the simultaneous determination of three flavonols including quercetin, kaempferol, and isorhamnetin. The procedure was based on the chemiluminescent enhancement by flavonols of the cerium(IV)-rhodamine 6G system in sulfuric acid medium. The effects of several parameters on the HPLC resolution and CL emission were studied systematically. Good separation was achieved with isocratic elution using a mixture of methanol and aqueous 1.0% acetic acid (37:63, v/v) within 25 min. Under optimized conditions, the linear working range covers 3 orders of magnitude with relative standard deviations below 4.5% for 11 replicate injected flavonol samples, and detection limits (S/N= 3) were 1.6 x 10(-8), 3.5 x 10(-9), and 6.5 x 10(-9) g mL(-1) for quercetin, kaempferol, and isorhamnetin, respectively. The chemiluminescence reaction was compatible with the mobile phase of high-performance liquid chromatography. The proposed method has been successfully applied to the determination of three active flavonols in phytopharmaceuticals of Hippophae rhamnoides L. After a simple extraction procedure, the repeatability and recovery were satisfactory.     

Simple quality assessment approach for herbal extracts using high performance liquid chromatography-UV based metabolomics platform

A lack of adequate or accepted research methodology has been a major obstacle to study herbal medicines. In this study, instead of the prevalent hyphenated chromatographies, common high performance liquid chromatography equipped with ultraviolet detector (HPLC-UV) and multivariate statistical analysis were utilized to assess the qualities of total flavones of sea buckthorn (TFS), an 85% ethanol extract of the sea buckthorn berries. Two complementary HPLC-UV methods were developed, validated and combined to comprehensively determine the ingredients in TFS. Principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) of the combined analytical data showed that the six batches of TFS could be well differentiated. Hierarchical cluster analysis (HCA) using Ward's minimum variance method of the PLS-DA loading matrix demonstrated the known ingredients (quercetin, kaempferol, isorhamnetin, oleanolic acid and ursolic acid) and three unknown ingredients in TFS significantly contributed to the quality differences. A PLS regression model indicated that the results of the present method correlated well with the content of total flavones, which is now the quality control approach of TFS. Results from this study indicated that the proposed method is reliable for the quality reassessment of some widely used herbal extracts.     

Application of 1-alkyl-3-methylimidazolium-based ionic liquids in separation of bioactive flavonoids by capillary zone electrophoresis

Room-temperature ionic liquids (ILs) are liquids that are constituted entirely of ions and can provide a solvent environment quite unlike any other available at room temperature. They continue to attract considerable interest in the chemistry research community as they are good solvents for a wide range of both inorganic and organic materials. In this study, a CZE method has been established for resolving natural flavonoids, quercetin, kaempferol and isorhamnetin in the Chinese herbal extract from Hippophae rhamnoides and its medicinal preparation (Sindacon Tablet). In this method, 1-alkyl-3-methyl-imidazolium-based ILs are used as the additive, and the effects of the alkyl group, imidazolium counterion (anionic part), along with the concentration of IL are investigated and discussed. Baseline separation, high efficiencies and symmetrical peaks of the three flavonoids were obtained. The separation mechanism seems to be the hydrogen-bonding interaction between the imidazolium cations of IL and the flavonoids.     

Study of the collision-induced radical cleavage of flavonoid glycosides using negative electrospray ionization tandem quadrupole mass spectrometry

Negative electrospray ionization tandem quadrupole mass spectrometry was used to study the collision-induced dissociation (CID) of the O-glycosidic bond from different commercially available flavonoid glycosides. Depending on the structure, flavonoid glycosides can undergo both a collision-induced homolytic and heterolytic cleavage of the O-glycosidic bond producing deprotonated radical aglycone ((Y(0) - H)(-*)) and aglycone (Y(0) (-)) product ions. The relative abundance of the radical aglycone to the aglycone fragment from flavonol-3-O-glycosides increased with increasing number of hydroxyl substituents in the B ring and in the order kaempferol - 相似文献   

Folate content in sea buckthorn berries and related products (Hippophaë rhamnoides L. ssp. rhamnoides): LC-MS/MS determination of folate vitamer stability influenced by processing and storage assessed by stable isotope dilution assay

A stable isotope dilution assay was adopted for quantitation of folate vitamers in sea buckthorn berries, juice, and concentrate using fourfold labeled folate isotopologues of the folate derivatives as the internal standards and reversed-phase liquid chromatography-tandem mass spectrometry with electrospray ionization (LC-ESI-MS/MS). Processing effects and storage stability were investigated during juice and concentrate production from sea buckthorn berries (Hippopha? rhamnoides). The technological processing of the berries caused a total degradation of tetrahydrofolate and 5-formyltetrahydrofolate in the generated juice. The content of the main folate vitamer 5-methyltetrahydrofolate remained approximately unchanged during the whole processing from the berries to the concentrate. Sea buckthorn juice was stored under two household storage conditions (6 degrees C, 25 degrees C), and also under accelerated aging conditions (40 degrees C) for up to 7 days to determine the effects of storage temperature on the stability of 5-methyltetrahydrofolate. The content of 5-methyltetrahydrofolate was nearly unchanged during the storage at 6 degrees C after 7 days. The juice showed almost identical degradation of 5-methyltetrahydrofolate of about 17-20% at 25 degrees C and 40 degrees C after 7 days of storage. [figure: see text]     

Separation of patuletin-3-O-glucoside, astragalin, quercetin, kaempferol and isorhamnetin from Flaveria bidentis (L.) Kuntze by elution-pump-out high-performance counter-current chromatography

Flaveria bidentis (L.) Kuntze is an annual alien weed of Flaveria Juss. (Asteraceae) in China. Bioactive compounds, mainly flavonol glycosides and flavones from F. bidentis (L.) Kuntze, have been studied in order to utilize this invasive weed, Analytical high-performance counter-current chromatography (HPCCC) was successfully used to separate patuletin-3-O-glucoside, a mixture of hyperoside (quercetin-3-O-galactoside) and 6-methoxykaempferol-3-O-galactoside, astragalin, quercetin, kaempferol and isorhamnetin using two runs with different solvent system. Ethyl acetate-methanol-water (10:1:10, v/v) was selected by analytical HPCCC as the optimum phase system for the separation of patuletin-3-O-glucoside, a mixture of hyperoside and 6-methoxykaempferol-3-O-galactoside, and astragalin. A Dichloromethane-methanol-water (5:3:2, v/v) was used for the separation of quercetin, kaempferol and isorhamnetin. The separation was then scaled up: the crude extract (ca 1.5 g) was separated by preparative HPCCC, yielding 12 mg of patuletin-3-O-glucoside at a purity of 98.3%, yielding 9 mg of a mixture of hyperoside and 6-methoxykaempferol-3-O-galactoside constituting over 98% of the fraction, and 16 mg of astragalin (kaempferol-3-O-glucoside) at a purity of over 99%. The pump-out peaks are isorhanetin (98% purity), kaemferol (93% purity) and quercitin (99% purity). The chemical structure of patuletin-3-O-glucoside and astragalin were confirmed by MS and 1H, 13C NMR.     

Polyphenolic profile characterization of Agrimonia eupatoria L. by HPLC with different detection devices

Liquid chromatography coupled to diode array and electrospray ionization mass spectrometry detection was used to establish the polyphenolic profile of an ethyl acetate fraction from Agrimonia eupatoria L. aqueous-alcoholic extract. Additionally, an HPLC technique with post-column derivatization by p-dimethylaminocinnamaldehyde was employed for the selective detection and quantification of flavan-3-ols. Important information was obtained by combining the data of these two HPLC techniques. Flavan-3-ols (catechin and procyanidins B1, B2, B3, B6, B7, C1, C2 and epicatechin-epicatechin-catechin), quercetin 3-O-glucoside, quercetin 3-O-galactoside, kaempferol 3-O-glucoside, kaempferol 3-O-(6'-O-p-coumaroyl)-glucoside, apigenin 6-C-glucoside and various phenolic acids were identified. Antioxidant activity of the Agrimonia eupatoria L. fraction containing these compounds was assessed through the 1,1-diphenyl-2-picrylhydrazyl, trolox equivalent antioxidant capacity and thiobarbituric acid reactive substances methods. Significant activity was observed for this fraction, where compounds with recognized antiinflammatory properties such as procyanidins, kaempferol 3-O-(6'-O-p-coumaroyl)-glucoside and quercetin glycosides were identified for the first time. These results are predictive of the beneficial effects of this fraction, or some of its compounds, in human health, as possible anti-inflammatory drug.     

A sensitive dispersive micro solid‐phase extraction coupled with high performance liquid chromatography for determination of three flavonoids in complex matrics by using crab shell as a sorbent

A sensitive dispersive micro solid‐phase extraction coupled with HPLC has been developed for preconcentration and determination of three flavonoids (quercetin, kaempferol, and isorhamnetin) in complex matrix samples. Parameters that affect extraction efficiency have been optimized. The optimal extraction conditions are using 2 μg/mL of crab shell as the sorbent, extraction for 2 min at pH 7, and then eluting with 100 μL of methanol. As a result, the method shows good linearity (R > 0.9994), low LODs (even 0.08 ng/ml) and satisfactory recovery in real honey and rat urine samples. As an eco‐friendly biomaterial, crab shell powder is used as sorbent in pretreatment of flavonoids, and its adsorption mechanism has been investigated for the first time. Compared with the other reported methods, the proposed strategy is time‐saving, eco‐friendly, and highly sensitive using HPLC (even achieving MS grade sensitivity).     

Rapid screening and evaluation of XOD inhibitors and O2•− scavenger from total flavonoids of Ginkgo biloba leaves by LC–MS and multimode microplate reader

Superoxide anion radical scavenger and xanthine oxidase inhibitor play an important role in the treatment of several relevant human diseases. In the present study, ultrafiltration liquid chromatography–mass spectrometry coupled to microplate reader was applied to screen and identify superoxide anion radical scavengers and xanthine oxidase inhibitors from total flavonoids of Ginkgo biloba leaves. As a result, four compounds (quercetin, apigenin, kaempferol and isorhamnetin) were screened as xanthine oxidase inhibitors by ultrafiltration LC–MS, and the 50% scavenging concentration values of the screened flavonoids were lower than those for allopurinol. Lineweaver–Burk plot results indicated that kaempferol was a competitive xanthine oxidase inhibitor; the other flavonoids were all anticompetitive inhibitors. Four flavonoids—rutin, quercetin, kaempferol and isorhamnetin—were screened as superoxide anion radical scavengers by LC–MS. The results demonstrate that the method for screening and evaluation of superoxide anion radical scavenger and xanthine oxidase inhibitor from a complex mixture system is feasible and efficient.     

Preparative Isolation and Purification of Flavonoids and Protocatechuic Acid from Sea Buckthorn Juice Concentrate (Hippophaë rhamnoides L. ssp. rhamnoides) by High-Speed Counter-Current Chromatography

High-speed counter-current chromatography (HSCCC)—a support free all liquid–liquid chromatography technique—has been successfully used for the preparative isolation of isorhamnetin 3-O-β-d-glucoside, isorhamnetin 3-O-β-rutinoside, quercetin 3-O-β-d-glucoside, syringetin 3-O-β-d-glucoside and protocatechuic acid from sea buckthorn juice concentrate (Hippophaë rhamnoides L. ssp. rhamnoides, Elaeagnaceae). The preparative HSCCC instrument was a multilayer coil planet centrifuge equipped with three preparative coils. Separation was performed with a two phase solvent system (n-hexane–n-butanol–water, 1:1:2 v/v/v) in ‘head-to-tail’ mode. Each injection of 4.1 g crude ethyl acetate extract yielded isorhamnetin 3-O-β-d-glucoside (95 mg), isorhamnetin 3-O-β-rutinoside (10 mg), quercetin 3-O-β-d-glucoside (5 mg), and protocatechuic acid (34 mg) with purities >98%. The flavonoid syringetin 3-O-β-d-glucoside (2 mg) was a novel compound for H. rhamnoides. Chemical structures of all compounds were determined by HPLC–ESI–MS–MS, 1D-NMR (¹H, ¹³C, DEPT 135) spectroscopy and for elucidation of glycosidic linkages 2D-NMR (HMBC) spectroscopy was used.     

Determination of flavonoids as complexes with Al3+ in microemulsion media by HPLC method with fluorescence detection

The scheme of postcolumn complexation reaction of flavonoids with aluminum (III) ions in various microemulsion media followed by fluorimetric detection is proposed. It is shown that fluorescence intensity of such complexes is five, three, and three times higher for quercetin, isorhamnetin, and kaempferol, respectively, when compared with the complexes obtained by dissolving flavonoids in MeCN/0.1 M HCl (30/70) and AlCl₃ in H₂O. The prospect of using microemulsions for quantitative quercetin extraction from plant raw material is demonstrated. Recovery of quercetin from onion peel is 98% with detection limits being 100, 45, and 15 ng/L for quercetin, isorhamnetin, and kaempferol, respectively. Quercetin content in three types of onion peel is determined. This content is found to be 34 ± 3 and 5 ± 1 mg/g for yellow and purple onion peel, respectively, while the content in white onion peel is below the detection limit.     

HPLC法测定不同施肥模式栽培的金线莲中黄酮类成分

HPLC法测定不同施肥模式栽培的金线莲中3种黄酮类成分:槲皮素、山奈酚和异鼠李素.超声-微波协同提取法提取4种不同施肥模式下金线莲中的黄酮类成分.HPLC-电喷雾离子化法/质谱(HPLC-ESI-MS)对黄酮类化合物进行定性分析.HPLC分析得槲皮素在0.25~20.0μg/m L(r=0.999 2)、山奈酚在0.25~20.0μg/m L(r=0.999 5)、异鼠李素在1.0~80.0μg/m L(r=0.999 0)范围内峰面积与浓度呈良好的线性关系,不同栽培方法的金线莲药材中槲皮素、山奈酚、异鼠李素的质量分数分别在151.8~240.0、47.2~88.2、16.8~29.8μg/g之间.方法简单、精确,快速,可为科学评价不同培养模式金线莲质量提供依据.栽培过程施加氮、磷、钾(NPK)复合肥及添加腐植素和微量元素,能提高金线莲中3种黄酮类成分的含量.栽培过程施加硫、磷(SP)复合肥,槲皮素、山奈酚和异鼠李素的质量分数分别下降了2.3%、17.2%和18.8%.结果表明采用不同施肥模式直接影响到药材中的药效物质的含量.     

Comparison of Electrochemical Oxidation of Flavonols and Calculated Proton Affinity and Electron Transfer Enthalpy in Water

Flavonols, a class of flavonoids, are present in flowers, fruits and vegetables. They are jointly responsible for antioxidant activity as free radical acceptors. The redox behaviour of myricetin, quercetin, isorhamnetin, fisetin, morin and kaempferol is investigated using cyclic and differential pulse voltammetry. Quantum chemical calculations of proton affinities and electron transfer enthalpies were performed to identify possible reactive sites and radical species to compare them with measured oxidation potentials of the flavonols. Regarding to their chemical structure, these flavonols showed an oxidation order: myricetin > quercetin > isorhamnetin > fisetin > morin > kaempferol.     

First report of non‐coloured flavonoids in Echium plantagineum bee pollen: differentiation of isomers by liquid chromatography/ion trap mass spectrometry

Apicultural products have been widely used in diet complements as well as in phytotherapy. Bee pollen from Echium plantagineum was analysed by high‐performance liquid chromatography/photodiode‐array detection coupled to ion trap mass spectrometry (HPLC‐PAD‐MSⁿ) with an electrospray ionisation interface. The structures have been determined by the study of the ion mass fragmentation, which characterises the interglycosidic linkage in glycosylated flavonoids and differentiates positional isomers. Twelve non‐coloured flavonoids were characterised, being kaempferol‐3‐O‐neohesperidoside the major compound, besides others in trace amounts. These include quercetin, kaempferol and isorhamnetin glycosides, with several of them being isomers. Acetylated derivatives are also described. This is the first time that non‐coloured flavonoids are reported from this pollen, with MS fragmentation proving to be most useful in the elucidation of isomeric structures. Copyright © 2010 John Wiley & Sons, Ltd.     

Flavonoids from Algerian endemic Centaurea microcarpa and their chemotaxonomical significance

Six flavonoids, namely 6-methoxykaempferol (1), 6-methoxykaempferol 7-O-glucoside (2), kaempferol 7-O-glucoside (3), 6-methoxyluteolin (4), patuletin 7-O-glucoside (5), and hispidulin 7-O-glucoside (6), were isolated from a n-butanolic fraction of Centaurea microcarpa Coss et Dur. flowers. This work describes for the first time the phytochemical composition of this endemic Algerian plant.