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.     

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.     

Identification of complex,naturally occurring flavonoid glycosides in kale (Brassica oleracea var. sabellica) by high‐performance liquid chromatography diode‐array detection/electrospray ionization multi‐stage mass spectrometry

Kale is a member of the Brassicaceae family and has a complex profile of flavonoid glycosides. Therefore, kale is a suitable matrix to discuss in a comprehensive study the different fragmentation patterns of flavonoid glycosides. The wide variety of glycosylation and acylation patterns determines the health‐promoting effects of these glycosides. The aim of this study is to investigate the naturally occurring flavonoids in kale. A total of 71 flavonoid glycosides of quercetin, kaempferol and isorhamnetin were identified using a high‐performance liquid chromatography diode‐array detection/electrospray ionization multi‐stage mass spectrometry (HPLC‐DAD/ESI‐MSⁿ) method. Of these 71 flavonol glycosides, 27 were non‐acylated, 30 were monoacylated and 14 were diacylated. Non‐acylated flavonol glycosides were present as mono‐, di‐, tri‐ and tetraglycosides. This is the first time that the occurrence of four different fragmentation patterns of non‐acylated flavonol triglycosides has been reported in one matrix simultaneously. In addition, 44 flavonol glycosides were acylated with p‐coumaric, caffeic, ferulic, hydroxyferulic or sinapic acid. While monoacylated glycosides existed as di‐, tri‐ and tetraglycosides, diacylated glycosides occurred as tetra‐ and pentaglycosides. To the best of our knowledge, 28 compounds in kale are reported here for the first time. These include three acylated isorhamnetin glycosides (isorhamnetin‐3‐O‐sinapoyl‐sophoroside‐7‐O‐D‐glucoside, isorhamnetin‐3‐O‐feruloyl‐sophoroside‐7‐O‐diglucoside and isorhamnetin‐3‐O‐disinapoyl‐triglucoside‐7‐O‐diglucoside) and seven non‐acylated isorhamnetin glycosides. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatography–tandem mass spectrometry reveals the widespread occurrence of flavonoid glycosides in honey,and their potential as floral origin markers

HPLC-MS–MS analysis of unifloral honey extracts has shown the occurrence of flavonoid glycosides in most of the analyzed samples. These compounds are not present in large amounts, but can reach up to 600 μg/100 g honey in canola and rapeseed honeys. Rhamnosyl-hexosides (tentatively rutinosides and neohesperidosides) and dihexosides (hexosyl(1→2)hexosides and hexosyl(1→6)hexosides) of flavonols such as quercetin, kaempferol, isorhamnetin and 8-methoxykaempferol, are the main flavonoid glycosides found in honey. However, flavonoid triglycosides and monoglycosides are also detected in some floral origins. Eucalyptus and orange blossom nectars were collected and analyzed showing that nectar flavonoid glucosides, as is the case of eucalyptus flavonoids, can be readily hydrolyzed by the bee saliva enzymes, while flavonoid rhamnosyl-glucosides, as is the case of citrus nectar flavonoids, are not hydrolyzed, and because of these reasons the flavonoid glycoside content of citrus honey is higher than that of eucalyptus honey that contains mainly aglycones. The flavonoid glycoside profiles detected in honeys suggest that this could be related to their floral origin and the results show that the HPLC-MSn ion trap analysis of flavonoid glycosides in honey is a promising analytical method to help in the objective determination of the floral origin of unifloral honeys.     

Palynological origin, phenolic content, and antioxidant properties of honeybee-collected pollen from Bahia, Brazil

The aim of this study was to determine the palynological origin, phenolic and flavonoid content, and antioxidant properties of twenty-five samples of bee pollen harvested during a nine-month period (February-November) from the Canavieiras municipality (northeastern Brazil). Of the 25 samples analyzed, only two (February 01 and 02) were heterofloral. The predominant pollens in the samples analyzed during that month were: Cecropia, Eucalyptus, Elaeis, Mimosa pudica, Eupatorium, and Scoparia. Ethyl acetate fractions were analyzed by HPLC-DAD. The flavonoids isoquercetin, myricetin, tricetin, quercetin, luteolin, selagin, kaempferol, and isorhamnetin were detected. The flavonoid present in all 22 samples was isolated and identified as isorhamnetin 3-O-b-neohesperidoside. The total phenolic contents determined using the Folin-Ciocalteu reagent ranged from 41.5 to 213.2 mg GAE/g. Antioxidant activities based on the 1,1-diphenyl-2-picryl hydrazyl (DPPH), 2,2-azinobis 3-ethylbenzothiozoline-6-sulfonic acid (ABTS), and Fe2+ ion chelating activity assays were observed for all extracts, and correlated with the total phenolic content.     

High-performance liquid chromatographic fingerprint analysis for different origins of sea buckthorn berries

Using high-performance liquid chromatography (HPLC), a chemical fingerprint method was developed for investigating and demonstrating the variance of flavonoids among different origins of sea buckthorn berries. Thirty-four samples were analyzed including 15 RS (Hippophae rhamnoides ssp. sinensis) samples, 7 RY (H. rhamnoindes ssp. yunnanensis) samples, 5 RW (H. rhamnoides ssp. wolongensis) samples, 4 NS (H. neurocarpa ssp. stellatopilosa) samples and 3 TI (H. tibetana) samples. In the HPLC chromatograms, 12 compounds were identified as flavonoids, including quercetin 3-O-sophoroside-7-rhamnoside, kaempferol 3-O-sophoroside-7-O-rhamnoside, isorhamnetin 3-O-sophoroside-7-O-rhamnoside, isorhamnetin 3-O-glucoside-7-O-rhamnoside, quercetin 3-O-rutinoside, quercetin 3-O-glucoside, isorhamnetin 3-O-rutinoside, isorhamnetin 3-O-glucoside, quercetin, kaempferol 7-O-rhamnoside, kaempferol and isorhamnetin. Both correlation coefficient of similarity in chromatograms and relative peak areas of characteristic compounds were calculated for quantitative expression of the HPLC fingerprints. Our results revealed that the chromatographic fingerprint combining similarity evaluation could efficiently identify and distinguish sea buckthorn berries from different species. However, no obvious difference between RS and RY suggested that the two subspecies might have very close relationship in terms of chemotaxonomy. The established method was considered to be suitable for fingerprint analysis to check the genuine origin and control the quality of sea buckthorn berries and extracts.     

Identification of flavonoids and their glycosides by high-performance liquid chromatography with electrospray ionization mass spectrometry and with diode array ultraviolet detection

Identification of flavonoids and flavonoid glycosides was carried out on Psidium guajava Linn leaves by means of high-performance liquid chromatography ultraviolet (HPLC-UV) analysis and HPLC mass spectrometry. By using HPLC-UV, two known phenolics (gallic acid and quercetin) and five newly reported ones (procatechuic acid, chlorogenic acid, caffeic acid, kaempferol and ferulic acid) were identified in alcohol guava leaf extract. Structural information about the compounds was obtained from the retention times, the UV spectra and mass spectra without the need to isolate the individual compounds. Two flavonoids (quercetin and kaempferol) and four flavonoid glycosides (three known components, quercetin 3-O-alpha-L-arabinoside, quercetin 3-O-beta-D-glucoside and quercetin 3-O-beta-D-galactoside, along with one novel compound, kaempferol-glycoside) and three other unknown compounds have been identified in the fractions.     

Study on the PEG‐based microwave‐assisted extraction of flavonoid compounds from persimmon leaves

A method for PEG‐based microwave‐assisted extraction (MAE) of flavonoid compounds from persimmon leaves has been successfully developed. The extraction efficiency of total flavonoid content was evaluated by the chromatographic peak areas of quercetin and kaempferol, which are two bioactive components typically found in persimmon leaves. The best combination of extraction parameters was obtained with response surface methodology. A microwave power of 525 W, liquid to solid ratio of 17:1 mL/g, and PEG aqueous solution concentration of 60% w/w were identified as the optimum parameters. Extraction dynamics analysis indicated that the quercetin, kaempferol, and total flavonoid contents were rising with increasing extraction time up to 20–25 min, from which point onwards they all decreased. Under the optimum conditions, quercetin, kaempferol, and total flavonoid contents obtained from the sample were 1.20 ± 0.05, 0.64 ± 0.11, and 16.90 ± 0.06 mg/g, respectively. Compared with ethanol‐based MAE, and ethanol‐based and PEG‐based ultrasonic‐assisted extractions, PEG‐based MAE had higher efficiency for the extraction of flavonoid compounds from persimmon leaves. Overall, PEG‐based MAE represents an efficient choice for the extraction of bioactive substances from traditional Chinese medicines.     

Phytochemical study of Centaurea cyanus

Flavonoid aglycons (quercetin, kaempferol, isorhamnetin, apigenin, luteolin, hispidulin) and their glycosides, and caffeic, chlorogenic, neochlorogenic, and isochlorogenic acids have been isolated from the epigeal part of theCentaurea cyanus L. and have been identified, and ten amino acids have also been identified.     

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.     

Analysis of flavonoids in honey by HPLC coupled with coulometric electrode array detection and electrospray ionization mass spectrometry

The analysis of flavonoids in unifloral honeys by high-performance liquid chromatography (HPLC) coupled with coulometric electrode array detection (CEAD) is described. The compounds were extracted by a nonionic polymeric resin (Amberlite XAD-2) and then separated on a reversed phase column using gradient elution. Quercetin, naringenin, hesperetin, luteolin, kaempferol, isorhamnetin, and galangin were detected in a coulometric electrode array detection system between +300 and +800 mV against palladium reference electrodes, and their presence was additionally confirmed by HPLC coupled with electrospray ionization mass spectrometry. The method was applied to analysis of 19 honeys of different varieties and origin. The limits of detection and quantitation ranged between 1.6 and 8.3 μg/kg and 3.9 and 27.4 μg/kg, respectively. The recoveries were above 96% in fluid and above 89% in creamy honeys. Some of these honeys (melon, pumpkin, cherry blossom, dandelion, maple, and pine tree honey) were investigated for their flavonoid content and profile for the first time. Differences between honeys were observed both in flavonoid concentrations and in the flavonoid profiles. The flavonoid concentrations ranged from 0.015 to 3.4 mg/kg honey. Galangin, kaempferol, quercetin, isorhamnetin, and luteolin were detected in all investigated honeys, whereas hesperetin occurred only in lemon and orange honeys and naringenin in lemon, orange, rhododendron, rosemary, and cherry blossom honeys.     

Development and validation of a gas chromatographic-mass spectrometric method for simultaneous identification and quantification of marker compounds including bilobalide,ginkgolides and flavonoids in Ginkgo biloba L. extract and pharmaceutical preparations

A gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the simultaneous determination of seven major chemical markers (bilobalide, ginkgolides A, B, C, kaempferol, quercetin and isorhamnetin) in phytopharmaceuticals of Ginkgo biloba L. The intra-day relative standard deviations (RSD) and inter-day RSD's were based on the analysis of the standardized Ginkgo biloba L. extract on the same day and on the following 3 consecutive days. The intra-day RSD's ranged from 1.21% (bilobalide) to 6.20% (kaempferol). The inter-day RSD's ranged from 2.10% (bilobalide) to 10.42% (isorhamnetin). Mean recoveries ranged from a low of 63.0 +/- 5.3% (isorhamnetin) to a maximum of 103.5 +/- 6.0% (ginkgolide A). Calibration curves were linear in ranges between 2.73 and 36.36 microg/ml for the markers. Limits of detection ranged from a low of 0.5 microg/ml (bilobalide) to a high of 2.5 microg/ml (quercetin). The limits of quantitation were a low of 1.1 microg/ml (gingkolides A, B, C) to a high of 7.5 microg/ml (kaempferol). The method was applied to a standard extract (>6% total terpenoids and >24% total flavonoids) and six ginkgo capsule phytopharmaceuticals.     

NMR spectral analysis of flavonoids from <Emphasis Type="Italic">Chrysanthemum coronarium</Emphasis>

Naringenin 5-O-glucoside, apigenin 7-O-glucoside, luteolin 7-O-glucoside, kaempferol 3-O-glucoside, quercetin 3-O-glucoside, apigenin, luteolin, kaempferol, and quercetin, nine flavonoid derivatives, were isolated for the first time from the aqueous methanolic extract of the aerial parts of Chrysanthemum coronarium. Their structures were elucidated on the basis of chemical and spectroscopic (UV, ¹H, ¹³C NMR) analyses. 1-and 2-dimensional NMR spectroscopy of the rare naringenin 5-O-glucoside have been recorded and assigned for the first time. The flavonoid glucosides from Chrysanthemum coronarium showed week activity against Poliovirus I and Adenovirus type 7. Published in Khimiya Prirodnykh Soedinenii, No. 6, pp. 546–548, November–December, 2007.     

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.     

Simultaneous determination of six herbal components in intestinal perfusate by high‐performance liquid chromatography

An effective, accurate and reliable HPLC with UV detection method was developed and validated for quantitation of six components: baicalin, berberine hydrochloride, quercetin, kaempferol, isorhamnetin and baicalein in intestinal perfusate using rotundin as an internal standard. The chromatographic separation was performed on a Welchrom‐C₁₈ column (250 × 4.6 mm i.d. with 5.0 µm particle size) with a mobile phase consisting of acetonitrile, water, phosphoric acid and triethylamine (30:70:0.2:0.1,v/v) at a flow rate of 1.0 mL/min and a UV detection at 270 nm. The method had a chromatographic run time of 30 min and excellent linear behavior over the investigated concentration ranges observed with the values of r higher than 0.99 for all the analytes. The lower limit of quantification of the analytical method was 0.09 µg/mL for berberine hydrochloride, quercetin, kaempferol and baicalein and 0.18 µg/mL for baicalin and isorhamnetin. The intra‐ and inter‐day precisions measured at three concentration levels were all less than 10% for all analytes. The bias ranged from ?6.91 to 4.33%. The validated method has been successfully applied to investigate the rat intestine absorption profiles of baicalin, berberine hydrochloride, quercetin, kaempferol, isorhamnetin and baicalein. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Phytochemical study of Centaurea cyanus

Flavonoid aglycons (quercetin, kaempferol, isorhamnetin, apigenin, luteolin, hispidulin) and their glycosides, and caffeic, chlorogenic, neochlorogenic, and isochlorogenic acids have been isolated from the epigeal part of theCentaurea cyanus L. and have been identified, and ten amino acids have also been identified.Kursk State Medical Institute. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 792–795, November–December, 1988.     

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.     

Ultra-high-performance liquid chromatography-Quadrupole-Exactive-Orbitrap-tandem mass spectrometry-based putative identification for Eucommiae Folium (Duzhongye) and its quality-marker candidate for pharmacopeia

Eucommiae Folium (Duzhongye) is a traditional Chinese medicine with a long history of use in China. However, its quality-marker in Chinese Pharmacopoeia is poorly defined nowadays. The study, therefore, conducted an ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap tandem mass spectrometry analysis to obtain accurate data. The obtained data were then compared with the authentic standards library using Xcalibur 4.1 software package and TraceFinder General Quan. Through the comparison, the study has putatively identified 26 bioactive compounds, which include 17 flavonoid derivatives (catechin, quercetin 3-gentiobioside, quercetin 3-O-β-D-glucose-7-O-β-D-gentiobioside, taxifolin, myricetin 3-O-galactoside, myricitrin, hyperoside, rutin, isoquercitrin, quercetin 3-O-β-xylopyranoside, quercitrin, isorhamnetin 3-O-β-D-glucoside, quercetin, kaempferol, S-eriodictyol, S-naringenin, and phloridzin), four caffeoylquinic acids (neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C), two alkaloids (vincamine and jervine), one lignan (pinoresinol), one xanthone (cowaxanthone B), and one steroid (cholesteryl acetate). Of these, flavonoid isoquercitrin is recommended as the new and additional pharmacopeia quality-marker candidate, which can not only overcome the unreliability of old quality-marker but also recognize the possible counterfeit.     

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.