Structures and bioactivities of seven flavonoids from Osmanthus fragrans ‘Jinqiu’ essential oil extraction residues

Osmanthus fragrans are well-known for their fragrance, but it is wasteful if to discard O. fragrans flower after extracting their essential oils. In this paper, we found that O. fragrans flower residues were rich in flavonoids. Six flavonoids and one phenylethanoid glycoside were isolated from the ethanol extract of O. fragrans flower residues, identified as quercetin (1), rutin (2), verbascoside (3), genistin (4), kaempferol (5), isorhamnetin (6) and naringin (7). In bioactivity study, kaempferol (IC₅₀ = 1.43 μg/mL) showed the best anti-inflammatory activity. Isorhamnetin, quercetin, kaempferol, verbascoside and rutin (the values of IC₅₀ were 18.30, 11.05, 16.88, 20.21 and 22.76 μg/mL, respectively) showed excellent DPPH free radical scavenging activity. Verbascoside performed relatively well at inhibiting the growth of both CT26 colonic carcinoma cells (IC₅₀ = 46.87 μg/mL) and HepG2 hepatocarcinoma cells (IC₅₀ = 30.58 μg/mL). In addition, quercetin and kaempferol showed strong anti-proliferation activity against HepG2 cells.     

Molecular imprinted polymer for solid‐phase extraction of flavonol aglycones from Moringa oleifera extracts

Molecular imprinted polymer produced using quercetin as the imprinting compound was applied for the extraction of flavonol aglycones (quercetin and kaempferol) from Moringa oleifera methanolic extracts obtained using heated reflux extraction method. Identification and quantification of these flavonols in the Moringa extracts was achieved using high performance liquid chromatography with ultra violet detection. Breakthrough volume and retention capacity of molecular imprinted polymer SPE was investigated using a mixture of myricetin, quercetin and kaempferol. The calculated theoretical number of plates was found to be 14, 50 and 8 for myricetin, quercetin and kaempferol, respectively. Calculated adsorption capacities were 2.0, 3.4 and 3.7 μmol/g for myricetin, quercetin and kaempferol, respectively. No myricetin was observed in Moringa methanol extracts. Recoveries of quercetin and kaempferol from Moringa methanol extracts of leaves and flowers ranged from 77 to 85% and 75 to 86%, respectively, demonstrating the feasibility of using the developed molecularly imprinted SPE method for quantitative clean‐up of both of these flavonoids. Using heated reflux extraction combined with molecularly imprinted SPE, quercetin concentrations of 975 ± 58 and 845 ± 32 mg/kg were determined in Moringa leaves and flowers, respectively. However, the concentrations of kaempferol found in leaves and flowers were 2100 ± 176 and 2802 ± 157 mg/kg, respectively.     

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.     

Investigation of Chemiluminescence Behavior of Flavonoids with Cerium (IV)‐Rhodamine B System

Abstract

The chemiluminescence (CL) behavior of five major flavonoid types in cerium (IV)‐rhodamine B system was investigated by flow‐injection. Strong CL was observed when cerium (IV) reacted with rhodamine B in sulfuric acid medium in the presence of flavonoids. This reaction system has been established as a simple, rapid, and highly sensitive flow injection CL analysis for quercetin and kaempferol, and their detection limit (3σ) was 2.7 and 0.22 nmol/L, respectively. The relative standard deviation (n=8) was 1.2% for 1.0 µmol/L quercetin and 1.9% for 0.5 µmol/L kaempferol. This method was successfully applied to the determination of quercetin in the hydrolysate of rutin and compared well with the high performance liquid chromatography (HPLC) method. From a comparison of several related flavonoids, it was concluded that only flavonoids that contain a free 3‐hydroxyl and 2, 3‐double bond in conjugation with 4‐oxo function could produce a relatively strong CL emission.     

Simultaneous LC Determination of Quercetin, Kaempferol and Isorhamnetin in Rabbits after Intragastric Administration of an Ethanol Extract from Pollen Typhae

A simple and rapid reversed-phase LC method was developed and validated for simultaneous determination of three flavonoids, quercetin (QU), kaempferol (KA) and isorhamnetin (IS), in rabbit blood plasma. The plasma was deproteinized using 10% trichloroacetic acid and extracted by n-butanol–acetoacetate solvent prior to LC analysis. The analyte was separated on a reversed-phase column with acetonitrile and 0.1% phosphoric acid in water (27:73, v/v) as mobile phase at a flow-rate of 0.8 mL min^?1, and UV detection wavelength at 369 nm. By this developed method, the concentrations of QU, KA and IS were linearly related to their responses in the range of 0.05–2.5 μg mL^?1. The precision and accuracy for QU, KA and IS in plasma were within ±15% except for the limit of quantitation (LOQ), where they were within ±20%. The validated method has been successfully applied in the pharmacokinetic study of QU, KA and IS in rabbits after intragastric administration of an ethanol extract from traditional Chinese medicine Pollen Typhae.     

Extraction and determination of flavonoids in fruit juices and vegetables using Fe3O4/SiO2 magnetic nanoparticles modified with mixed hemi/ad‐micelle cetyltrimethylammonium bromide and high performance liquid chromatography

Extraction and determination of three flavonoids (morin, quercetin, and kaempferol) were performed by dispersive magnetic solid phase extraction based on mixed hemi/ad‐micelles and high‐performance liquid chromatography with UV detection. The Fe₃O₄/SiO₂ nanoparticles were synthesized and characterized by X‐ray diffraction, FTIR, scanning electron microscopy, and thermogravimetric analysis. Fe₃O₄/SiO₂ nanoparticles coated with mixed hemi/ad‐micelles cetyltrimethyl ammonium bromide was applied as a sorbent and used for extraction of flavonoids. Effective parameters on the extraction recovery such as amount of magnetic nano particles, volume of cetyltrimethyl ammonium bromide solution with specific concentration, pH of sample solution, adsorption equilibrium time, volume of desorption solvent, and desorption times were evaluated and optimized using fractional factorial design and central composite design. Under the optimum condition limit of detection and linearity were 0.83, 2.7–500.0 for morin, 0.18, 0.7–500.0 for quercetin and, 0.37, 1.3–500.0 µg/L for kaempferol. The extraction recovery with relative standard deviation were 97.88, 1.94 for morin, 95.77, 0.80 for quercetin, and 93.35, 1.45 for kaempferol. The proposed method was applied for simultaneous extraction and determination of flavonoids in several fruit juices and vegetable samples.     

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.     

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.     

High-Performance Liquid Chromatographic Method for the Simultaneous Determination of Four Flavonols in Food Supplements and Pharmaceutical Formulations

A method using high-performance liquid chromatography with diode array detection (HPLC-DAD) as a powerful separation technique has been developed for the simultaneous determination of the four flavonols rutin, quercetin, kaempferol and isorhamnetin in food supplements and pharmaceutical formulations. The chromatographic separation was achieved in 36?min using a Symmetry C₁₈ column (250?×?3?mm; 5?µm) as the stationary phase and a mixture of methanol, acetonitrile, and pH 2.5 aqueous acetic acid as the mobile phase in gradient elution mode. The analytical wavelengths were 256?nm for rutin, quercetin and isorhamnetin, and 368?nm for kaempferol. An ultrasound-assisted extraction protocol was performed using methanol as solvent. The detection and quantification limits were lower than 0.03?µg mL^?1 and 0.08?µg mL^?1, respectively. The inter-day and intra-day precisions were less than 4.8 and 5.1%, respectively, and the average recoveries were in the range from 96 to 107%. The method was applied for the determination of the studied flavonols in food supplements and pharmaceutical preparations. The satisfactory recovery values demonstrate the potential of the developed method for the determination of the analytes in these samples. In addition, the method is suitable for routine quality control due its ease of operation.     

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.     

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.     

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.     

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.     

Phytochemical screening,antioxidant activity,total phenolic and total flavonoid contents of seven local varieties of Rosa indica L.

Rosa indica symbol of godness and beauty known for various healing power, has astringent, sedative, anti-inflammatory and antidepressant qualities. Standard methods were used for qualitative detection of phyto-compounds, and quantitative detection of antioxidants was done using DPPH radical scavenging assay, total phenolics and total flavonoids content were expressed in mg GAE/g dry weight and mg QE/g dry weight. Results revealed phyto-compounds presence in all varieties under study however maximum % inhibition was observed by R. indica var pink perfume (94 ± 0.6) with IC50 value 0.3376 ± 0.01 mg/mL. Highest phenolic and flavonoid content was observed in the leaves extract of R. indica var cardinal red, i.e. 3.3553 ± 0.11 (ethanol) mg of Gallic acid equivalents (GAE)/g dry weight and 3.736 ± 0.001(ethanol) mg of quercetin equivalents (QE)/g dry weight, respectively, at conc. 0.125 mg/mL. Our finding provides evidence that all varieties of rose contain medicinally important bioactive compounds and justifies their use for treatment of different diseases.     

Separation and determination of flavonoids in three traditional chinese medicines by capillary electrophoresis with amperometric detection

Flavonoids are important active ingredients in many traditional Chinese medicines. In this paper, capillary electrophoresis with amperometric detection was employed to separate and detect eight flavonoids, rutin, quercetrin, quercetin, kaempferol, kaempferide, catechin, apigenin, and luteolin, in a home‐made capillary electrophoresis device. Under the separation voltage of 2000 V, the eight flavonoids could be completely separated within 33 min in 18 mM borax running buffer at pH 10.2. Good linear relationships were obtained for all analytes and the detection limits for flavonoids ranged from 0.46 to 0.85 μM. Then, the method was applied to separate and determine the flavonoids in three traditional Chinese medicines, hippophae rhamnoides, hypericum perforatum, and cacumen platycladi. Finally, rutin, kaempferol, quercetin, and quercetrin were discovered in these medicines and the concentrations ranged from 0.28 to 9.94 mg/g. The recoveries of flavonoids ranged from 84.7 to 113%, which showed the high reliability of this method.     

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

UPLC-DAD method was developed and validated for the quantitative determination of free flavonol aglycones (kaempferol, quercetin and myricetin) after acidic hydrolysis in six Lysimachia species. Quantitative analyses showed that the amounts of various flavonol aglycones were significantly different in Lysimachia vulgaris, Lysimachia nummularia, Lysimachia punctata, Lysimachia christinae, Lysimachia ciliata and Lysimachia clethroides. The L. clethroides sample was found to be the richest in kaempferol (25.77 ± 1.29 μg/mg extract) and quercetin (97.67 ± 4.61 μg/mg extract), while the L. nummularia sample contained the highest amount of myricetin (20.79 ± 1.00 μg/mg extract). The antioxidant capacity of hydrolysed extracts was evaluated using in vitro DPPH^? (2,2-diphenyl-1-picrylhydrazyl) and ABTS^?+ [2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)] decolourisation tests. The observed radical scavenging capacities of the extracts showed a relationship with the measured flavonol aglycone content and composition. The acidic treatment resulted in an increased free radical scavenging activity compared to the untreated methanol extract.     

高效液相色谱法测定野葱中黄酮类化合物

建立了测定野葱中芦丁、黄酮醇类化合物的高效液相色谱方法。80%乙醇超声提取,高效液相色谱分析野葱中芦丁和黄酮醇类化合物的含量。芦丁检测条件:V(甲醇)∶V(0.2%磷酸水)=45∶55,检测波长:360nm;槲皮素、山奈酚、异鼠李素检测条件:V(甲醇)∶V(0.2%磷酸水)=40∶60,检测波长:360 nm。结果表明,野葱中芦丁含量0.22%,槲皮素、山奈酚、异鼠李素含量依次为0.42%、0、0.23%,总黄酮醇类化合物含量为1.63%。     

高效液相色谱法测定不同产地枇杷叶中的3种黄酮类成分

建立了高效液相色谱同时测定枇杷叶中3种黄酮类成分的分析方法。该方法分析了不同产地枇杷叶中芦丁、槲皮素和山柰酚的含量差异。枇杷叶粉末用甲醇超声提取后,加盐酸回流,制备样品测试液。采用Diamonsil C18色谱柱（250 mm×4.6 mm,5 μm）,以0.4%（v/v）磷酸水溶液-乙腈为流动相,梯度洗脱。分别对7个不同产地的枇杷叶样品中的芦丁、槲皮素和山柰酚进行测定。结果表明,芦丁、槲皮素、山柰酚在各自的质量浓度范围内线性关系良好（r>0.99）,加标回收率分别为96.33%、95.81%和95.80%,RSD分别为6.48%、0.90%和3.02%。该方法操作简单、分离度好、重复性高。不同产地枇杷叶中3种黄酮类成分的含量存在差异,其中芦丁的差异最大,而山柰酚的含量最稳定且在不同产地样品中均可检出,或可用作枇杷叶药材质量控制的标志成分。     

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).