Quality evaluation of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins by high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors

A method, high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC-DAD-ELSD), was developed to evaluate the quality of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins. The wavelength at 280 nm was chosen to determine six isoflavonoids: calycosin-7-O-beta-D-glucoside (1), ononin (2), (6alphaR, 11alphaR)-9,10-dimethoxypterocarpan-3-O-beta-D-glucoside (3), (3R)-2'-hydroxy-3',4'-dimethoxyisoflavan-7-O-beta-D-glucoside (4), calycosin (5), and formononetin (6); and ELSD connected after DAD was employed to determine four saponins: astragaloside IV (7), astragaloside II (8), astragaloside I (9), and acetylastragaloside I (10). This assay was fully validated with respect to precision, repeatability and accuracy. The proposed method was successfully applied to quantify the ten components in eleven samples from different localities in China; significant variations were demonstrated in the content of these compounds in the samples from different areas. This simple, rapid, low-cost and reliable HPLC-DAD-ELSD method is suitable for routine quantitative analysis and quality control of traditional Chinese medicines (TCMs) consisting of bioactive multi-components with different structures such as Radix Astragali.     

Determination and identification of isoflavonoids in Radix astragali by matrix solid-phase dispersion extraction and high-performance liquid chromatography with photodiode array and mass spectrometric detection

The isoflavonoids in Radix astragali were determined and identified by HPLC-photodiode array detection-MS after extraction employing matrix solid-phase dispersion (MSPD). As a new sample preparation method for R. astragali, the MSPD procedure was optimized, validated and compared with conventional methods including ultrasonic and Soxhlet extraction. The amounts of two major components in this herb, formononetin (6) and ononin (2), were determined based on their authentic standards. Four major isoflavonoids, formononetin (6), ononin (2), calycosin (5) and its glycoside (1), and three minor isoflavonoids, (6aR,11aR)-3-hydroxy-9, 10-dimethoxypterocarpan (7), its glycoside (3), and (3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavone-7-O-beta-D-glycoside (4), were identified based on their characteristic two-band UV spectra and [M + H], [aglycone + H]+ and [A1 + H]+ ions, etc. The combined MSPD and HPLC-DAD-MS method was suitable for quantitative and qualitative determination of the isoflavonoids in R. astragali.     

Separation and sweeping of flavonoids by microemulsion electrokinetic chromatography using mixed anionic and cationic surfactants

In this report, a novel means for the separation and sweeping of flavonoids (quercetin, rutin, calycosin, ononin and calycosin-7-O-β-d-glucoside) by microemulsion electrokinetic chromatography using mixed anionic and cationic surfactants as modified pseudostationary phase was presented. The optimized background electrolyte consisted of 0.5% (w/v) ethyl acetate, 2.0% (w/v) SDS, 9 mM DTAC, 4.0% (w/v) 1-butanol and 10 mM sodium borate or 25 mM phosphoric acid. We systematically investigated the separation and preconcentration conditions, including the concentrations of surfactant, types of sweeping, sample matrix, the effect of high salt or acetonitrile, and sample injection volume. It was found that the use of mixed surfactants significantly enhanced the separation efficiency through the change of the efficient electrophoretic mobility of analytes. Compared with normal sample injection, 185-508-fold sensitivity enhancement in terms of limit of detection was achieved through effective sweeping of large sample volume at 50 mbar pressure (up to 45% capillary length). At last, the proposed method was suitable for the determination of Radix Astragali sample.     

Determination of nine active components in Radix Hedysari and Radix Astragali using capillary HPLC with diode array detection and MS detection

A capillary HPLC (cHPLC) coupled with diode array detection (DAD) and MS method was developed for the simultaneously qualitative and quantitative determination of nine components, namely vanillic acid, calycosin-7-O-beta-D-glucoside, (6alphaR,11alphaR)-9,10-dimethoxypterocarpan-3-O-beta-D-glucoside, ononin, calycosin, (3R)-2'-hydroxy-3',4'-dimethoxyisoflavan-7-O-beta-D-glucoside, isoliquiritigenin, formononetin, (3R)-8,2'-dihydroxy-7,4'-dimethoxyisoflavan, in Radix Hedysari (Hongqi) and Radix Astragali (Huangqi). Simultaneous separation of these nine compounds was achieved on a Zorbax C18 microcolumn (5 microm, 150 x 0.3 mm). The mobile phase consisted of (A) 0.3% aqueous formic acid and (B) ACN with a gradient elution. The identification of nine compounds in both Hongqi and Huangqi was confirmed by TOF-MS. All calibration curves showed good linearity (R(2) >0.998) within test ranges. This method showed good repeatability for the quantification of these nine components in Hongqi and Huangqi with intra- and inter-day variations of less than 1.89 and 3.13%, respectively. The validated method was successfully applied to quantify nine investigated components in eighteen samples of Hongqi and Huangqi. Hierarchical cluster analysis of 18 samples was performed using the peak area of nine analytes on cHPLC chromatograms. The result showed that Hongqi and Huangqi are significantly different, though the two species of Astragalus are very similar.     

Liquid chromatography-electrospray ionization mass spectrometry study of the flavonoids of the roots of Astragalus mongholicus and A. membranaceus

High-performance liquid chromatography-electrospray ionization mass spectrometry has been applied to analyze the flavonoids of Huangqi, the roots of Astragalus mongholicus and A. membranaceus. Eight flavonoids were identified as calycosin-7-O-beta-D-glucoside, calycosin-7-O-beta-D-glucoside-6"-O-malonate (2), ononin, (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan-3-O-bet a-D-glucoside, calycosin, (3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavan-7-O-beta-D-glucoside, formononetin-7-O-beta-D-glucoside-6"-O-malonate and formononetin by direct comparison with the isolated standards from Huangqi. The existence of (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan, (3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavan, astrapterocarpanglucoside-6'-O-malonate and astraisoflavanglucoside-6'-O-malonate was detected. This is the first report of flavonoid glycoside malonates in these two Astragalus species, and malonate 2 is a structurally completely identified new compound.     

Determination of paeoniflorin,calycosin‐7‐O‐β‐d‐glucoside,ononin, calycosin and formononetin in rat plasma after oral administration of Buyang Huanwu decoction for their pharmacokinetic study by liquid chromatography–mass spectrometry

The purpose of this study was to simultaneously investigate the pharmacokinetics of five bioactive compounds in rat plasma after oral administration of Buyang Huanwu decoction (BYHWD) using high‐performance liquid chromatography coupled with mass spectrometry (HPLC‐MS). The separations were performed on a Thermo Hypersil Gold C₁₈ analytical column (50 × 2.1 mm, 3 µm) with the column temperature kept at 30°C. The quantitative analysis was performed using a quadrupole mass spectrometer detector operated under selected ion monitoring mode. A linear gradient elution of A (0.1% formic acid solution) and B (100% acetonitrile) was used at a flow rate of 0.2 mL/min. The method was validated within the concentration ranges 1.8–450, 6.0–1500, 2.0–500, 1.2–300 and 1.2–150 ng/mL for paeoniflorin, calycosin‐7‐O‐β‐d ‐glucoside, ononin, calycosin and formononetin, respectively. The calibration curves were linear with correlation coefficients > 0.99. The lower limits of quantitations were < 6.0 ng/mL. The method was further applied to assess the pharmacokinetics of the five bioactive constituents of BYHWD in rat plasma. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis and retention behavior of isoflavone glycosides and aglycones in Radix Astragali by HPLC with hydroxypropyl‐β‐cyclodextrin as a mobile phase additive

In order to determine isoflavone glycosides (calycosin‐7‐O‐β‐d ‐glucoside and formononetin‐7‐O‐β‐d ‐glucoside) and aglycones (calycosin and formononetin), a simple HPLC method with isocratic elution employing hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as a mobile phase additive was developed. Various factors affecting the retention of isoflavone glycosides and aglycones in the C₁₈ reversed‐phase column, such as the nature of cyclodextrins, HP‐β‐CD concentration, and methanol concentration, were systematically studied. The results show that HP‐β‐CD, as a very effective mobile phase additive, can markedly reduce the retention of isoflavone glycosides and aglycones, and the decrease magnitudes of isoflavone aglycones are more than those of their glycosides. The role of HP‐β‐CD in the developed HPLC method is attributed to the formation of the inclusion complexes between isoflavone glycosides (or aglycones) and HP‐β‐CD. So, the apparent formation constants of the isoflavone glycosides (or aglycones)/HP‐β‐CD inclusion complexes also were investigated. Isoflavone glycosides (and aglycones) form the 1:1 inclusion complexes with HP‐β‐CD, and the isoflavone aglycones/HP‐β‐CD complexes are more stable than the isoflavone glycosides/HP‐β‐CD complexes. Finally, the optimized method was successfully applied for the determination of isoflavone glycosides and aglycones in Radix Astragali samples.     

Capillary liquid chromatography-microcoil 1H nuclear magnetic resonance spectroscopy and liquid chromatography-ion trap mass spectrometry for on-line structure elucidation of isoflavones in Radix astragali

Miniaturization and hyphenation of chromatographic separation techniques to nuclear magnetic resonance spectroscopy is being increasingly demanded in the field of biomedical, drug metabolite and natural product analysis. Herein, capillary liquid chromatography was coupled on-line to microcoil 1H nuclear magnetic resonance spectroscopy (capLC-NMR) equipped with a 1.5 microL solenoidal probe for structure elucidation of isoflavones in Radix astragali. The extract was screened by HPLC-UV-MS as the preliminary step and four major peaks were identified tentatively by ion trap mass spectrometry molecular weights and characteristic fragments. Then, stopped-flow capLC-UV-NMR was performed using 33 microg extract injected on-column. The four peaks were parked manually in the micro probe one by one and corresponding 1H NMR spectra were recorded with good resolutions under the applied capLC-NMR conditions (120 and 220 ng injected on-column for peaks 2 and 4, respectively). All aromatic regions of 1H NMR spectra correlated well to the characteristic signals of isoflavone aglycone protons. And the signal corresponding to the anomeric proton of the glucopyranoside of isoflavone glycoside was also obtained for peak 1. Therefore, these four peaks are determined as calycosin-7-O-beta-D-glucopyranoside (1), ononin (2), calycosin (3) and formononetin (4) unambiguously. The capLC-NMR results indicate that this hyphenated technique could be used for the determination of a great variety of natural products from small sample amounts, e.g., only 5 g R. astragali in this study.     

Fast separation of flavonoids by supercritical fluid chromatography using a column packed with a sub‐2 μm particle stationary phase

The purpose of this study was to compare the effects of different chromatographic columns for the separation of seven flavonoids. Four different stationary phases are available, including bridged ethyl hybrid, BEH and the same hybrid phase modified with 2‐ethylpyridine, CSH fluorophenyl, and HSS C₁₈ SB. The analytes included calycosin, genistein, medicarpin, calycosin‐7‐O‐β‐d ‐glucoside, formononetin, formononetin‐7‐O‐β‐d ‐glucoside, and liquiritigenin. The CSH fluorophenyl column was determined to be the most suitable and provided the fastest separation within 17 min using gradient elution with carbon dioxide as the mobile phase and methanol as the co‐solvent. Good peak shapes were obtained, and the values of the peak asymmetry were close to 1.0 for all of the flavonoids. The resolution was more than 1.41 for all of the separated peaks. Baseline separation on the optimal columns was achieved by changing the co‐solvent type and adjusting the temperature and pressure. Quantitative performance was evaluated under optimized conditions, and method validation was accomplished. The validation parameters, such as linearity, sensitivity, precision, and accuracy, were satisfactory. Good repeatability of both peak area (relative standard deviation <1.02%) and retention time (relative standard deviation <0.88%) was observed. The optimized chromatographic methods were successfully used for the determination of seven flavonoids in Radix astragali . The sensitivity was sufficient for the analysis of real samples.     

Concordance between antioxidant activities in vitro and chemical components of Radix Astragali (Huangqi)

Five Radix Astragali (RA) extracts were prepared and their antioxidant activities were measured in vitro using ABTS [2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid diammonium salt)], DPPH?·?(1,1-diphenyl-2-picrylhydrazyl radical), reducing power, ?O?? and ?OH assays. Their chemical contents were then determined, including total phenolics, total flavonoids, total saponins and total sugars. The 1/IC?? values of the various antioxidant assays were used to evaluate the level of antioxidant activity of the RA extracts (plot showing 1/IC?? values vs. chemical contents), and the average R values (correlation coefficients) of total phenolics and total flavonoids were 0.762 and 0.638. In contrast, the average R values of the total saponins and total sugars were -0.0386 and -0.132, respectively. This large difference clearly demonstrates that the antioxidant effects of RA in vitro might be generally considered to be a result of the presence of phenolic compounds (including flavonoids) but not astragalosides and polysaccharides.     

Octadecylamine and glucose-coderived hydrophobic carbon dots-modified porous silica for chromatographic separation

A hydrophobic carbon dots (Glc-OCDs) derived from octadecylamine and glucose were successfully synthesized for the first time and then grafted onto the porous silica surface by the “Nano-on-Micro” strategy, which was served as a new stationary phase (Sil-Glc-OCDs) for reversed-phase liquid chromatography. The structure of this stationary phase was carefully verified by laser scanning confocal microscope, Fourier transform infrared spectrometry, elemental analysis, contact angle measurement, etc. Several analytes including seven polycyclic aromatic hydrocarbons, eight alkylbenzenes, eight phenols and seven sulfonamides can be well separated on this stationary phase. Better separation performance for certain analytes over commercial C₁₈ column was obtained. Interestingly, this stationary phase exhibited excellent chromatographic selectivity in the separation of the isomers of tert‑butylbenzene, sec‑butylbenzene, isobutylbenzene and n-butylbenzene. In addition, this new Sil-Glc-OCDs column was also applied for detection of calycosin-7-glucoside, ononin, calycosin, formononetin, genistein and isorhamnetin in the extract of Radix Astragali, which were found that the concentration was 0.15 g/L, 0.088 g/L, 0.14 g/L, 0.086 g/L, 0.18 g/L and 0.29 g/L, respectively. We believe that this CDs-grafted silica materials are promising for chromatographic separation.     

Quality assessment of Astragali Radix based on pseudo-targeted metabolomics and chemometric approach

Astragali Radix is widely used because of its dual use in medicine and food, and its quality evaluation is of great importance. In this study, a pseudo-targeted metabolomics approach based on scheduled multiple reaction monitoring was developed, and a total of 114 compounds with good linearity, sensitivity, and reproducibility were selected for relative quantification, and the chemical differences between Astragali Radix of different growth patterns were further compared by chemometric analysis. With the help of multivariate and univariate analysis, 26 differential compounds between wild/semi-wild Astragali Radix and cultivated Astragali Radix were determined. Then five marker compounds were screened out by lasso regression, and further verified by systematic clustering, random forest, support vector machine, and logistic regression. In addition, malonyl-substituted flavonoids showed relatively higher content in wild/semi-wild Astragali Radix. Thus, the malonyl substitution was characteristic for flavonoids in wild/semi-wild Astragali Radix. In conclusion, the application of pseudo-targeted metabolomics and various statistical methods could offer multi-dimensional information for the holistic quality evaluation of Astragali Radix.     

An ultra high performance liquid chromatography with tandem mass spectrometry method for simultaneous determination of thirteen components extracted from Radix Puerariae in rat plasma and tissues: Application to pharmacokinetic and tissue distribution study

A rapid and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was established and validated for simultaneous determination of thirteen bioactive components (gallic acid, protocatechuic acid, puerarin, p‐hydroxycinnamic acid, daidzin, ononin, daidzein, naringenin, genistein, apigenin, formononetin, biochanin A, and β‐sitosterol) of Radix Puerariae extract in rat plasma and tissues. The plasma and tissues samples were pretreated by protein precipitation extraction, and umbelliferone and rutin were used as internal standards. Sample separation was performed on a ZORBAX RRHD Eclipse plus C₁₈ column (2.1 mm × 50 mm, 1.8 µm, Agilent) with a mobile phase consisting of methanol–water (containing 0.1% formic acid). The mass spectrometry analysis was conducted in positive and negative ionization modes with multiple reaction monitoring. The lower limit of quantitation range for the 13 analytes was 0.2?35 ng/mL. The intra‐ and inter‐day precision of all the analytes were less than 10.92%, with an accuracy ranging from ?13.10 to 11.96%. Both the recovery and matrix effect were within acceptable limits. This method was successfully applied to pharmacokinetic and tissue distribution study of the 13 bioactive components in rats after oral administration of R. Puerariae extract.     

Simultaneous quantification of multiple components in rat plasma by UPLC‐MS/MS and pharmacokinetic study after oral administration of Huangqi decoction

A rapid, sensitive and accurate UPLC‐MS/MS method was developed for the simultaneous quantification of components of Huangqi decoction (HQD), such as calycosin‐7‐O‐β‐d ‐glucoside, calycosin‐glucuronide, liquiritin, formononetin‐glucuronide, isoliquiritin, liquiritigenin, ononin, calycosin, isoliquiritigenin, formononetin, glycyrrhizic acid, astragaloside IV, cycloastragenol, and glycyrrhetinic acid, in rat plasma. After plasma samples were extracted by protein precipitation, chromatographic separation was performed with a C₁₈ column, using a gradient of methanol and 0.05% acetic acid containing 4mm ammonium acetate as the mobile phase. Multiple reaction monitoring scanning was performed to quantify the analytes, and the electrospray ion source polarity was switched between positive and negative modes in a single run of 10 min. Method validation showed that specificity, linearity, accuracy, precision, extraction recovery, matrix effect and stability for 14 components met the requirements for their quantitation in biological samples. The established method was successfully applied to the pharmacokinetic study of multiple components in rats after intragastric administration of HQD. The results clarified the pharmacokinetic characteristics of multiple components found in HQD. This research provides useful information for understanding the relation between the chemical components of HQD and their therapeutic effects.     

Phenolics and flavonoids compounds, phenylanine ammonia lyase and antioxidant activity responses to elevated CO₂ in Labisia pumila (Myrisinaceae)

A split plot 3 × 3 experiment was designed to examine the impact of three concentrations of CO? (400, 800 and 1,200 μmol·mol?1) on the phenolic and flavonoid compound profiles, phenylalanine ammonia lyase (PAL) and antioxidant activity in three varieties of Labisia pumila Benth. (var. alata, pumila and lanceolata) after 15 weeks of exposure. HPLC analysis revealed a strong influence of increased CO? concentration on the modification of phenolic and flavonoid profiles, whose intensity depended on the interaction between CO? levels and L. pumila varieties. Gallic acid and quercetin were the most abundant phenolics and flavonoids commonly present in all the varieties. With elevated CO? (1,200 μmol·mol?1) exposure, gallic acid increased tremendously, especially in var. alata and pumila (101-111%), whilst a large quercetin increase was noted in var. lanceolata (260%), followed closely by alata (201%). Kaempferol, although detected under ambient CO? conditions, was undetected in all varieties after exposure. Instead, caffeic acid was enhanced tremendously in var. alata (338~1,100%) and pumila (298~433%). Meanwhile, pyragallol and rutin were only seen in var. alata (810 μg·g?1 DW) and pumila (25 μg·g?1 DW), respectively, under ambient conditions; but the former compound went undetected in all varieties while rutin continued to increase by 262% after CO? enrichment. Interestingly, naringenin that was present in all varieties under ambient conditions went undetected under enrichment, except for var. pumila where it was enhanced by 1,100%. PAL activity, DPPH and FRAP also increased with increasing CO? levels implying the possible improvement of health-promoting quality of Malaysian L. pumila under high CO? enrichment conditions.     

高速逆流色谱法分离纯化黄芪中的芒柄花素和毛蕊异黄酮

采用高速逆流色谱法(HSCCC),以正己烷-氯仿-甲醇-水组成二相系统作为固定相与流动相,对黄芪的乙酸乙酯粗提 物进行了分离纯化。 结果发现:以正己烷-氯仿-甲醇-水(体积比为1.5∶3∶3∶2)组成的系统可以从黄芪的乙酸乙酯粗 提物中分离出毛蕊异黄酮,纯度可达95％以上,并可以初步纯化芒柄花素;接着用正己烷-氯仿-甲醇-水(体积比为4∶4∶5 ∶4)组成的系统进一步纯化芒柄花素,其纯度达95％以上。利用该方法,可以对中药黄芪中的异黄酮进行快速的分离和纯 化。     

Simultaneous Separation of Four Flavonoids and Two Astragalosides from Radix Astragali by Semi-Preparative LC

An effective method for simultaneous separation of four flavonoids and two astragalosides was developed by semi-preparative high-performance liquid chromatography in this work. A crude sample was firstly cleaned-up from ethanol aqueous extract of Radix Astragali by LS-300B resin-based column chromatography and was further isolated by semi-preparative LC with a gradient elution of water–methanol. Six compounds including calycosin (3.5 mg), formononetin (2.6 mg), (6aR,11aR)-10-hydroxy-3,9-dimethoxypterocarpan (2.6 mg), (3R)-7,2′-dihydroxy-3′,4′-dimethoxyisoflavan) (2.0 mg), astragaloside I (5.4 mg) and astragaloside II (9.8 mg) were obtained with a recovery of 67.8, 77.2, 88.4, 85.3, 62.1 and 57.9%, respectively. Their chemical structures were confirmed by MS and NMR analysis. This study developed an effective and rapid method for simultaneous separation of multiple components from Radix astragali.     

Screening anti‐allergic components of Astragali Radix using LAD2 cell membrane chromatography coupled online with UHPLC‐ESI‐MS/MS method

Huangqi (Astragali Radix), a traditional Chinese herb, is widely used in clinical therapy in China. In addition, an anti‐allergic effect of constituents in Huangqi has been reported in the scientific literature. In the present study, cell membrane chromatography coupled online with UHPLC‐ESI‐MS/MS method was developed to screen, analyze and identify the anti‐allergic components of Huangqi. The Laboratory of Allergic Disease 2 (LAD2) cell was used to establish cell membrane chromatography, which was combined with UHPLC‐ESI‐MS/MS. The coupled system was then used to screen anti‐allergic components from Huangqi. Effects of active components were verified by histamine release assay. A component retained on the LAD2 cell membrane chromatography was identified as formononetin. Bioactivity of formononetin was investigated by histamine release assay in LAD2 cells, and it was found that formononetin could inhibit histamine release in a dose‐dependent manner from 1 to 100 μm . The LAD2 cell membrane chromatography online with UHPLC‐ESI‐MS/MS method is an effective technique for screening the anti‐allergic components of Huangqi.     

毛细管电泳用于中药白鲜皮中生物碱的分析

建立了毛细管电泳同时分离白鲜皮中3种生物碱(胡芦巴碱、白鲜碱和胆碱)的的定量分析方法.以缓冲液H3BO3—Na2B4O7(pH=8.4,5 mmol/L)、添加剂3 mmol/L SDS和0.1%triton X-100为电泳运行液,24 kV为分离电压,分离在5 min内就可以快速完成.在230 nm检测波长处,3种组分的线性范围为:胡芦巴碱5～100μg/mL、白鲜碱5～75μg/mL、胆碱50～300μg/mL,检测限分别为1.4、1.4和16.0μg/mL.方法用于白鲜皮实际样品的测定,回收率范围在92.0%～101.0%之间。