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。     

On-line coupling of capillary isotachophoresis and capillary zone electrophoresis for the determination of flavonoids in methanolic extracts of Hypericum perforatum leaves or flowers

Five flavonoids (hyperoside, isoquercitrin, quercitrin, quercetin and rutin) were separated and determined in extracts of Hypericum perforatum leaves or flowers by capillary zone electrophoresis (CZE) with isotachophoretic (ITP) sample pre-treatment using on-line column coupling configuration. The background electrolyte (BGE) used in the CZE step was different from the leading and terminating ITP electrolytes but all the electrolytes contained 20% (v/v) of methanol. The optimal leading electrolyte was 10 mM HCl of pH* approximately 7.2 (adjusted with Tris) and the terminating electrolyte was 50 mM H3BO3 of pH* approximately 8.2 (adjusted with barium hydroxide). This operational system allowed to concentrate and pre-separate selectively the flavonoid fraction from other plant constituents before the introduction of the flavonoids into the CZE capillary. The BGE for the CZE step was 50 mM Tris buffer of pH* approximately 8.75 containing 25 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid as co-ion and 55 mM H3BO3 as complex-forming agent. The ITP-CZE method with spectrophotometric detection at 254 nm was suitable for the quantitation of the flavonoids in real natural samples; kaempferol was used as internal standard. The limit of detection for quercetin-3-O-glycosides was 100 ng ml(-1) and calibration curves were rectilinear in the range 1-10 microg ml (-1) for most of the analytes. The RSD values ranged between 0.9 and 2.7% (n=3) when determining approximately 0.07-1.2% of the individual flavonoids in dried medicinal plants.     

液相色谱法同时测定维药蜀葵花中芦丁、槲皮素和山柰酚

维药是祖国医药学不可分割的组成部分。维药现代化,即利用现代技术研究维药的有效成分,是维药科学化、标准化、规范化、商品化和产业化的必经之路。本文建立了维药蜀葵花中有效成分芦丁、槲皮素和山柰酚的选择性提取方法,优化了高效液相色谱法(HPLC)同时测定这3种有效成分的分析条件。采用HC-C₁₈色谱柱(250 mm×4.6 mm, 5 μm)和甲醇-0.4%磷酸(50:50, v/v)流动相,在柱温30 ℃和流速1.00 mL/min的条件下实现了3种物质之间以及和干扰物之间的基线分离。维药蜀葵花中芦丁、槲皮素及山柰酚的线性范围分别为12.5~150 μg/mL (r=0.9998), 12.5~125 μg/mL (r=0.9999)及12.5~125 μg/mL (r=0.9988),加标回收率(n=5)分别为100.3%(RSD=1.1%)、97.60%(RSD=0.47%)、97.75%(RSD=0.71%)。该方法实现了同时测定维药蜀葵花中芦丁、槲皮素及山柰酚,为其他黄酮类物质的开发应用提供了科学依据,同时也可为其他维药分析提供借鉴。     

Separation and determination of flavonoids in Agrimonia pilosa Ledeb. by capillary electrophoresis with electrochemical detection

Five flavonoids (catechin, hyperoside, quercitrin, quercetin, and rutin) were separated and determined by capillary electrophoresis with electrochemical detection. Effects of several important factors, such as the pH and concentration of running buffer, separation voltage, injection time, and detection potential were investigated to determine the optimum conditions. The five flavonoids were baseline separated within 20 min in a 60 cm length capillary at a separation voltage of 19.5 kV with a running buffer consisting of 60 mmoL/L Na2B4O7 - 120 mmoL/L NaH2PO4 (pH = 8.8). The relationship between peak current and analyte concentration was linear over about two orders of magnitude with detection limits (S/N = 3) ranging from 0.02 to 0.05 microg/mL for all compounds. This method was successfully used to determine the above five flavonoids in Agrimonia pilosa Ledeb. with relatively simple extraction procedures, and the assay results were satisfactory.     

中药菟丝子中生物活性成分的毛细管电泳-电化学检测

采用毛细管电泳-电化学检测法(CE-ECD)同时测定了菟丝子中芦丁、金丝桃甙、山柰酚、对香豆酸和槲皮素等5种主要生物活性成分的含量,考察了运行缓冲液酸度和浓度、分离电压、氧化电位和进样时间等实验参数对分离检测的影响。在最佳实验条件下,以直径300 μm的碳圆盘电极为工作电极,检测电位为+950 mV(vs. 参比电极),以50 mmol/L的硼砂缓冲溶液(pH 9.0)为运行缓冲液,上述各组分在19 min内能完全分离。芦丁、金丝桃甙、山柰酚、对香豆酸和槲皮素在两个数量级的范围内呈良好线性关系,检测下限(按S/N=3计) 分别为1.93×10-5,3.55×10-4,3.65×10-5,1.73×10-5和1.46×10-4 g/L。该法已成功地应用于菟丝子中活性成分的分离检测,结果令人满意。     

Flavonoid separation by capillary electrophoresis. Effect of temperature and pH

Summary The use of capillary electrophoresis for the analysis of selected flavonols present in fruit juices and wines (kaempferol-3- rutinoside, rutin, avicularin, quercitrin, isoquercitrin, isorhamnetin, kaempferol and quercetin) was explored, and the effect of pH and temperature on the separation studied. The method had good reproducibility and analyses were carried out in less than 10 minutes.     

Flavonoid separation by capillary electrophoresis. Effect of temperature and pH

Summary The use of capillary electrophoresis for the analysis of selected flavonols present in fruit juices and wines (kaempferol-3-rutinoside, rutin, avicularin, quercitrin, isoquercitrin, isorhamnetin, kaempferol and quercetin) was explored, and the effect of pH and temperature on the separation studied. The method had good reproducibility and analyses were carried out in less than 10 minutes.     

Application of hot platinum microelectrodes for determination of flavonoids in flow injection analysis and capillary electrophoresis

The determination of quercetin and rutin by flow injection analysis (FIA) and capillary electrophoresis (CE) using electrochemical detection was described. These flavonoids were determined at normal (unheated) and hot platinum microelectrodes using cyclic voltammetry. When quercetin or rutin is reaching the platinum electrode, a change of the current in the region of the platinum oxide formation is observed. Integration of the current changes in this in this region creates analytical signals in the form of peaks. An increase of temperature to about 76 ?C in a small zone adjacent to the microelectrode causes an increase of the analytical signal by more than 6 times under FIA conditions. This method enables the use of hot microelectrodes as detectors in HPLC or CE. In CE the improvement of the analytical signal at hot microelectrodes is smaller than in FIA and increase only 1.3–3.4 times. Heated microelectrodes were used for analysis of the flavonoids in natural samples of the plant (extract of sea buckthorn) and a pharmaceutical preparation (Cerutin).     

Simultaneous determination of phytoestrogens in different medicinal parts of Sophora japonica L. by capillary electrophoresis with electrochemical detection

A high-performance capillary electrophoresis with electrochemical detection (CE-ED) method has been developed for the determination of phytoestrogens from the pericarps and seeds of Sophora japonica L. in this work. Genistin, genistein, rutin, kaempferol and quercetin are important bioactive constituents in these plants. The effects of several factors such as the acidity and concentration of running buffer, the separation voltage, the applied potential and the injection time on the CE-ED procedure were investigated. Under the optimum conditions, the five analytes could be well separated within 18 min in a 75 cm length capillary (i.d. 25 microm) at the separation voltage of 16 kV in a 50 mmol L(-1) borax running buffer (pH 9.0). A 300 microm diameter carbon disk electrode was used as the working electrode positioned carefully opposite the outlet of the capillary in a wall-jet configuration at the potential of +950 mV (vs SCE). Detection limits (S/N = 3) ranged from 1.1 x 10(-7) to 2.8 x 10(-7) g mL(-1) for all fi ve analytes. This method was successfully used to analyse dried Flos sophorae immaturus, pericarps and seeds of dried Fructus sophorae after a relatively simple extraction procedure, and the assay results were satisfactory.     

毛细管电泳法分离测定芦丁、槲皮素和连翘苷

用毛细管电泳紫外检测法同时测定了芦丁、槲皮素和连翘苷，研究了各种条件的影响，得到了优化的实验条件，在20mmol/L的Na2B4O7(H3B03调节至pH8．40)-30mmol/L十二烷基硫酸钠-10％乙腈(1 9)的缓冲溶液中，分离电压为12kV时，芦丁、槲皮素和连翘苷在10min内得到了良好的分离，检测波长为254nm，芦丁、槲皮素和连翘苷分别在0．01-1．0mg/mL，0．01-1．0mg／mL和0．05-1．0mg/mL质量浓度范围内与电泳峰高呈现良好线性关系，检测下限分别为0．005mg/mL，0．005mg/mL和0．01mg／mL，应用于实际样品的测定。     

Quantitative analysis of aglycone quercetin in mulberry leaves (Morus alba L.) by capillary zone electrophoresis.

A capillary zone electrophoresis method was established for analysis of aglycone quercetin in mulberry leaves (Morus alba L.). The influence of, e.g., background electrolyte concentrations and pH, surfactant concentrations, organic solvents, temperature, and voltage on the separation of aglycone quercetin, rutin, quercitrin, kaempferol, catechin, and gallic acid was systematically investigated. The optimum condition providing baseline separation of all compounds within 16.5 min was obtained in 150 mM boric acid (pH 10.0) using a fused-silica capillary with an effective length of 42.5 cm (50 microm inner diameter), temperature of 32 degrees C, and voltage of 15 kV. Method assessment was performed by standard addition method using rutin as an internal standard. Linearity of the method was excellent (r(2) > 0.999) over the concentration tested (40-160 microg/mL). The relative standard deviations (%RSDs) from injection, intraday, and interday precision were less than 2.5%. Recoveries were good (asymptotically equal to 100.0%,%RSD = 0.8%) with a limit of detection (LOD) and limit of quantitation (LOQ) of 0.86 and 3.16 microg/mL (%RSD = 1.8%), respectively. The aglycone quercetin found in the mulberry leaves was 0.452 g/100 g (%RSD = 0.6%) on dry weight.     

基于漂浮有机液滴凝固液相微萃取的黄酮类化合物分配系数及含量测定

以漂浮有机液滴凝固液相微萃取（SFODLPME）对中药中黄酮类化合物进行富集,建立SFODLPME-HPLC快速测定中药材样品中低丰度黄酮类化学成分含量的方法. 并讨论了黄酮类化合物富集倍数与浓度之间的关系、富集倍数与供相体积之间的关系,定义和阐述了EF1μg?mL-1及其物理意义,计算了漂浮有机液滴凝固相体积及黄酮类化合物的表观分配系数. 在SFODLPME的优化条件下结合高效液相色谱测得8种黄酮类成分的线性范围均为0.01~10.00μg/ml;检测限在1.0~0.01ng/mL;精密度RSD<7.0%;药材中分析物的回收率在96.0% ~104.2%. 当加入萃取溶剂体积为40μl 时,形成漂浮有机液滴凝固相体积为52±14μl;8种黄酮类成分的表观分配系数在44.8~243.9之间.     

Precision improvement for the analysis of flavonoids in selected Thai plants by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method for the analyses of kaempferol in Centella asiatica and Rosa hybrids and rutin in Chromolaena odorata was developed. The optimization was performed on analyses of flavonoids (e.g., rutin, kaempferol, quercetin, myricetin, and apigenin) and organic carboxylic acids (e.g., ethacrynic acid and xanthene-9-carboxylic acid) by investigation of the effects of types and amounts of organic modifiers, background electrolyte concentrations, temperature, and voltage. Baseline separation (R(s) = 2.83) of the compounds was achieved within 10 min in 20 mM NaH2PO4 - Na2HPO4 (pH 8.0) containing 10% v/v ACN and 6% v/v MeOH using a voltage of 25 kV, a temperature of 30 degrees C, and a detection wavelength set at 220 nm. The application of the corrected migration time (t(c)), using ethacrynic acid as the single marker, was efficient to improve the precision of flavonoid identification (% relative standard deviation (RSD) = 0.65%). The method linearity was excellent (r2 > 0.999) over 50-150 microg/mL. Precision (%RSD < 1.66%) and recoveries were good (> 96% and %RSDs < 1.70%) with detection and quantitation limits of 2.23 and 7.14 microg/mL, respectively. Kaempferol in C. asiatica and R. hybrids was 0.014 g/100 g (%RSD = 0.59%) and 0.044 g/100 g (%RSD = 1.04%), respectively, and rutin in C. odorata was 0.088 g/100 g (%RSD = 0.06%).     

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.     

Flavonoids and biological activities of Jussiaea repens

A new acylated avicularin, namely avicularin 2'-(4'-O-n-pentanoyl)-gallate (1) along with 12 metabolites have been isolated from the ethyl acetate extract of the aerial parts of Jussiaea repens L. (Onagraceae). Their structures were established as trifolin 2'-O-gallate (2), quercetrin (3), guaijaverin (4), reynoutrin (5), juglanin (6), avicularin (7), hyperin (8), trifolin (9), hyperin 2'-O-gallate (10), rutin (11), kaempferol (12) and quercetin (13) on the basis of their chromatographic properties, chemical and spectroscopic evidences. The investigated ethyl acetate extract was found to be non-toxic (LD(50) up to the maximum soluble dose 4 g kg(-1) body wt.) and had significant antioxidant, hepatoprotective, anti-inflammatory and antidiabetic activities. Also some of the isolated flavonoids showed cytotoxic activity against Ehrlich ascitis carcinoma cells.     

Screening of tyrosinase inhibitors by capillary electrophoresis with immobilized enzyme microreactor and molecular docking

A new method for screening tyrosinase inhibitors from traditional Chinese medicines (TCMs) was successfully developed by capillary electrophoresis with reliable online immobilized enzyme microreactor (IMER). In addition, molecular docking study has been used for supporting inhibition interaction between enzyme and inhibitors. The IMER of tyrosinase was constructed at the outlet of the capillary by using glutaraldehyde as cross‐linker. The parameters including enzyme reaction, separation of the substrate and product, and the performance of immobilized tyrosinase were investigated systematically. Because of using short‐end injection procedure, the product and substrate were effectively separated within 2 min. The immobilized tyrosinase could remain 80% active for 30 days at 4°C. The Michaelis–Menten constant of tyrosinase was determined as 1.78 mM. Kojic acid, a known tyrosinase inhibitor, was used as a model compound for the validation of the inhibitors screening method. The half‐maximal inhibitory concentration of kojic acid was 5.55 μM. The method was successfully applied for screening tyrosinase inhibitors from 15 compounds of TCM. Four compounds including quercetin, kaempferol, bavachinin, and bakuchiol were found having inhibitory potentials. The results obtained in this work were supported by molecular docking study.     

Determination of flavonoids by high-performance liquid chromatography and capillary electrophoresis

The compounds of flavonoid, an important group in nature, can prevent coronary heart disease and anticancer by virtue of the characteristics of antioxidation. Nine flavonoids most often seen in grape wine, namely apigenin, baicalein, naringenin, luteolin, hesperetin, galangin, kaempferol, quercetin, and myricetine, were determined by means of high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) in this work. A successful resolution was obtained from an unusual additive of tetrahydrofuran in mobile phase by HPLC. One notable thing is that the mixture of luteolin and quercetin could be separated for the first time by HPLC. In addition, the better detection limit was still attainable even with the use of tetrahydrofuran. The detection limits of CZE performed in borate buffer were hundreds-fold better than in previous reports. Furthermore, the retention and migration behavior of the analytes studied were discussed. As the result of this study, the elution order of flavone and flavonone was reversed to the contention proposed by Wulf et al. It was predictable from the interaction with tetrahydrofuran. Consequently, the extracts from grape wine with solid-phase extraction were analyzed by developing methods of HPLC and CZE. The obtained recoveries ranged from 90 to 107% and the relative standard deviations were under 6.3%.     

Simultaneous determination of flavonoids in chrysanthemum by capillary zone electrophoresis with running buffer modifiers

Despite the separation efficiency of capillary electrophoresis (CE) is much higher than other chromatographic methods, it is sometimes difficult to perfectly separate the complex ingredients in biological samples. One possible and simple way to develop the separation effect in CE is to add some modifiers in the running buffer. In this paper, the suitable running buffer modifiers were explored to simultaneously separate and detect six typical flavonoids (apigenin, luteolin, kaempferol, quercetin, (+)-catechin and (−)-epicatechin) which are the main active ingredients in chrysanthemum by capillary zone electrophoresis with amperometric detection (CZE-AD). It was found that when β-cyclodextrin (β-CD) and the mixture of methanol and ethanol were used as running buffer modifiers, a baseline separation of the six analytes could be accomplished in less than 20 min and the detection limits were as low as 10⁻⁷ or 10⁻⁸ g ml⁻¹. Other factors affecting the CZE separation, such as working potential, pH value and ionic strength of running buffer, separation voltage and sample injection time were extensively investigated. Under the optimum conditions, a successful practical application on the determination of chrysanthemum samples confirmed the validity and practicability of this method.     

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.     

Analysis of flavonoids in <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L. and its phytopharmaceuticals by capillary electrophoresis with electrochemical detection

A high-performance capillary electrophoretic (CE) method with electrochemical detection (ED) has been developed for determination of the pharmacologically active flavonoids in Ginkgo biloba L. and phytopharmaceuticals containing its extract. Epicatechin, catechin, rutin, apigenin, luteolin, and quercetin are important flavonoids in this plant. Operated in a wall-jet configuration, a 300 micro m diameter carbon-disk electrode was used as working electrode with good response to the six analytes at +1000 mV (relative to the SCE). Under the optimum conditions, the analytes were separated within 22 min in a borax buffer (pH 9.0). Excellent linearity was obtained over two orders of magnitude and detection limits (S/N=3) ranged from 1.4 x 10(-7) to 5.0 x 10(-7) g mL(-1) for all six analytes. The method was successfully used for assay of Ginkgo biloba L. and its phytopharmaceuticals after a relatively simple extraction procedure; the results obtained were satisfactory.