Evaluation of phenyldimethylethoxysilane treated high-performance thin-layer chromatographic plates. Application to analysis of flavonoids inScutellariae radix

Summary The retention and selectivity of flavonoids (baicalin, baicalein, wogonin, oroxylin A) inScutellariae radix have been studies by high-performance thin-layer chromatography on phenyldimethylethoxysilane-treated silica plates. The silica plates treated with phenyl groups were used for physical and chemical analysis. From elemental carbon analysis, the maximum number of bonded phenyl surface groups per gram was calculated to be 0.467×10²¹ (Oginal silica plate: Merck Art. 15109, Silica gel 100 F₂₅₄). With methanol-1/15 M phosphate buffer (pH 6.2) mixtures as mobile phase, baicalin, baicalein, wogonin, and oroxylin A inScutellariae radix were separated. It has been shown that phenyl-treated plates are more suitable for selective separation of baicalin, baicalein, wogonin, and oroxylin A than octadecyl-treated plates.     

Isolation and purification of baicalein, wogonin and oroxylin A from the medicinal plant Scutellaria baicalensis by high-speed counter-current chromatography

The medicinal plant Scutellaria baicalensis Georgi has been used widely in traditional Chinese medicine for anti-inflammation, anticancer, antiviral and antibacterial infections, reducing the total cholesterol level and decreasing blood pressures. A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of three bioactive flavonoids, namely, baicalein, wogonin and oroxylin A, from S. baicalensis Georgi. Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-n-butanol-water (1:1:8:10, v/v/v/v) was successfully performed by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml min(-1) after 4 h. The components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 144.8 mg of baicalein at 95.7% purity, 50.2 mg of wogonin at 98.5% purity, and 12.4 mg of oroxylin A at 93.2% purity from 500 mg of the crude extract in a one-step separation. The recoveries of baicalein, wogonin and oroxylin A were 92.7%, 91.6% and 92.5%, respectively.     

Study of major flavonoids in crude Scutellariae Radix by micellar electrokinetic capillary chromatography

A method of micellar electrokinetic capillary electrophoresis for determining the six flavonoids in Scutellariae Radix (i.e., baicalin, baicalein, wogonin 7-O-glucuronide, wogonin, oroxylin A 7-O-glucuronide, and oroxylin A) has been developed. The buffer solution (pH 7.24) composed of 20 mM sodium dodecyl sulfate (SDS), 20 mM sodium dihydrogen phosphate, and 25 mM sodium borate was found to be the most suitable electrolyte for the separation. The contents of the six flavonoids in crude Scutellariae Radix could easily be determined within about 15 min. On-column UV (254 nm) monitoring allowed the quantitative determination of baicalin. The effects of pH, surfactant concentration, and applied voltage on the migration behavior of the solutes were studied.     

Development and validation of a UHPLC–MS/MS method for the simultaneous determination of five bioactive flavonoids in rat plasma and comparative pharmacokinetic study after oral administration of Xiaochaihu Tang and three compatibilities

An accurate, rapid, and reliable ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of baicalin, wogonoside, baicalein, wogonin, and oroxylin A in rat plasma. Then, the stability of baicalin and baicalein in the preparation of plasma sample was systematically investigated. The Waters BEH C₁₈ column was used with a gradient mobile phase system of acetonitrile and water containing 0.1% formic acid. The analytes were detected in the multiple reaction monitoring mode with positive electrospray ionization. 100 μL fresh plasma was added with 50 μL antioxidant reagent (1 mol/L HCl containing 0.5% Vitamin C), and liquid–liquid extraction with ethyl acetate was used to extract the analytes from plasma. Lower limits of quantification of baicalin, wogonoside, baicalein, wogonin, and oroxylin A were 21.9, 4.80, 1.20, 0.848, and 0.800 ng/mL, respectively. The mean extract recoveries of five flavonoids were 69.1∼89.2%, and the precision and accuracy were within the acceptable limits. This method was further successfully applied to the comparative pharmacokinetic study of these five flavonoids in rats after oral administration of Xiaochaihutang and three compatibilities. The obtained results may be helpful to reveal the mechanism of Xiaochaihutang formula compatibility.     

Flavones of the roots of Scutellaria baicalensis

Summary 1. A qualitative analysis of the flavonoid aglycones in the roots ofScutellaria baicalensis Georg. has been performed, and more than 20 substances have been detected in them.2. Six individual flavonoid aglycones have been obtained, and on the basis of chemical and spectral characteristics they have been identified as baicalein, 7-O-methylbaicalein, oroxylin, norwogonin, 7-O-methylnorwogonin, and wogonin.Khar'kov Scientific-Research Institute of Pharmaceutical Chemistry. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 729–733, November–December, 1973.     

Simultaneous LC–MS Analysis and of Wogonin and Oroxylin A in Rat Plasma,and Pharmacokinetic Studies After Administration of the Active Fraction from Xiao-Xu-Ming Decoction

A rapid and specific high-performance liquid chromatographic method coupled with electrospray ionization mass spectrometric detection has been developed and validated for identification and quantification of wogonin and oroxylin A in rat plasma. Wogonin, oroxylin A, and diazepam (internal standard) were extracted from plasma samples by liquid–liquid extraction with ethyl acetate. Chromatographic separation was achieved on a C₁₈ column with acetonitrile–0.6% aqueous formic acid 35:65 (v/v) as mobile phase at a flow rate of 0.2 mL min⁻¹. Detection was performed with a single-quadrupole mass spectrometer in selected-ion-monitoring (SIM) mode. Linearity was good within the concentration range 14.4–360 ng mL⁻¹ for wogonin and 10.8–271 ng mL⁻¹ for oroxylin A; the correlation coefficients (r ²) were 0.9999. The intra-day and inter-day precision, as RSD, was below 12.4%, and accuracy ranged from 81.1 to 111.9%. The lower limit of quantification was 14.4 ng mL⁻¹ for wogonin and 10.8 ng mL⁻¹ for oroxylin A. This method was successfully used in the first pharmacokinetic study of wogonin and oroxylin A in rat plasma after oral administration of the active fraction from Xiao-xu-ming decoction.

     

Simultaneous LC–MS Analysis and of Wogonin and Oroxylin A in Rat Plasma, and Pharmacokinetic Studies After Administration of the Active Fraction from Xiao-Xu-Ming Decoction

A rapid and specific high-performance liquid chromatographic method coupled with electrospray ionization mass spectrometric detection has been developed and validated for identification and quantification of wogonin and oroxylin A in rat plasma. Wogonin, oroxylin A, and diazepam (internal standard) were extracted from plasma samples by liquid–liquid extraction with ethyl acetate. Chromatographic separation was achieved on a C₁₈ column with acetonitrile–0.6% aqueous formic acid 35:65 (v/v) as mobile phase at a flow rate of 0.2 mL min^?1. Detection was performed with a single-quadrupole mass spectrometer in selected-ion-monitoring (SIM) mode. Linearity was good within the concentration range 14.4–360 ng mL^?1 for wogonin and 10.8–271 ng mL^?1 for oroxylin A; the correlation coefficients (r ²) were 0.9999. The intra-day and inter-day precision, as RSD, was below 12.4%, and accuracy ranged from 81.1 to 111.9%. The lower limit of quantification was 14.4 ng mL^?1 for wogonin and 10.8 ng mL^?1 for oroxylin A. This method was successfully used in the first pharmacokinetic study of wogonin and oroxylin A in rat plasma after oral administration of the active fraction from Xiao-xu-ming decoction.     

Optimization of Extraction Conditions for Flavonoid Composition and Antioxidant Activity of Radix Scutellariae

Microwave, ultrasonic, and reflux extraction were compared to provide an optimized method for flavonoids from Radix Scutellariae including the antioxidant activity of the extracts. The antioxidant activities of the extracts were evaluated by 2,2-diphenyl-1-picrylhydrazyl and oxygen radical absorbance capacity assays. Baicalin, wogonoside, baicalein, wogonin, and oroxylin A were quantified using ultra-high performance liquid chromatography. For microwave extraction, with a ten minutes treatment, the extraction efficiencies of the analytes were higher than treatments by refluxing for 180 minutes and sonication for sixty minutes. In addition, the antioxidant activity of the microwave extracts was slightly higher than the extracts obtained by the other methods. Microwave extraction conditions were further optimized by a single-factor experiment and the optimum conditions were 50 percent ethanol–water (volume by volume), an extraction temperature of 100 degrees celsius, an extraction time of ten minutes, and a sample to solvent ratio of 1:50. This study combines extraction, phytochemistry, and bioactivity to screen the most efficient extraction procedure for flavonoids from Radix Scutellariae.     

Application of LC/MS and ICP/MS for establishing the fingerprint spectrum of the traditional Chinese medicinal preparation Gan-Lu-Yin

We developed a method to analyze the fingerprint spectrum qualitatively and quantitatively for the traditional Chinese herbal medicinal preparation Gan-Lu-Yin with HPLC combined with photodiode array detection, and MS, and to identify the preparation's 14 main components including baicalin, baicalein, oroxylin A-7-O-glucuronide, wogonin-7-O-glucuronide, wogonin, and oroxylin A in Radix Scutellariae; naringin and neohesperidin in Aurantii fructus; liquiritigenin, liquiritin, and glycyrrhizic acid in Radix Glycyrrhizae. In LC/UV assay, a Cosmosil 5C18-MS-II column was used as the stationary phase, and a gradient of potassium dihydrogen phosphate, ACN, and water as the elute solution. The UV detection wavelengths were 250 and 280 nm. In LC/MS assay, a gradient of phosphoric acid, ACN, and water was used as the elute solution, and electrospray positive ion mode ((+)-ESI) as the analytic mode. In order to explore the distribution of trace metal elements effectively in Gan-Lu-Yin, a microwave digestion method was used for sample treatment, and an inductively coupled plasma MS assay was used to analyze fingerprint spectra of the inorganic metals in Gan-Lu-Yin. Combined with fingerprint spectra of organic compounds by LC/UV and LC/MS, it was expected to provide effective quality control in the production of Gan-Lu-Yin.     

Two new alkaloids from Capparis himalayensis

Two new alkaloids, Capparin A (1) and B (2), along with seven known compounds 6-methoxyindoline-2,3-dione (3), wogonin (4), oroxylin A (5), kaempferol (6), apigenin (7), quercetin (8) and luteolin (9), were isolated from the whole plant of Capparis himalayensis. Their structures have been established on the basis of spectral methods and the structure of 1 was confirmed by X-ray crystallographic analysis.     

Biliary excretion of metabolites of baicalin and baicalein in rats

Biliary excretion of metabolites of baicalin, present in Scutellariae Radix, was investigated using rats. The bile of rats administered baicalin orally was shown to contain five major metabolites, which were identified as baicalein 6-O-beta-glucopyranuronoside (M1), 6-O-methyl-baicalein 7-O-beta-glucopyranuronoside (oroxylin A 7-O-beta-glucuronide (M2], baicalein 7-O-beta-glucopyranuronoside (M3), 6-O-beta-glucopyranuronosyl- baicalein 7-O-sulfate (M4), and baicalein 6,7-di-O-beta-glucopyranuronoside (M5) on the basis of chemical and spectroscopic evidence. The bile of rats treated with baicalein also contained the above metabolites. Slower biliary excretion of the metabolites after baicalin administration suggested that it was absorbed as baicalein after hydrolysis in the gastrointestinal tract. The total cumulative amounts of the five metabolites excreted in the bile during 30 h after oral administration of baicalin and that of baicalein were approximately 54% and 40% of the doses, respectively. In addition the bilary metabolites of both drugs were shown to be mainly composed of M5 and M4, which have high polarity and large molecular weight.     

An efficient strategy based on two-stage chromatography and in vitro evaluation for rapid screening and isolation of acetylcholinesterase inhibitors from Scutellaria baicalensis Georgi

The extraction of Scutellaria baicalensis Georgi was investigated using the response surface methodology-genetic algorithm mathematical regression model, and the extraction variables were optimized to maximize the flavonoid yield. Furthermore, a simple and efficient ultrafiltration–liquid chromatography–mass spectrometry and molecular docking methods were developed for the rapid screening and identification of acetylcholinesterase inhibitors present in Scutellaria baicalensis Georgi. Subsequently, four major chemical constituents, namely baicalein, norwogonin, wogonin, and oroxylin A, were identified as potent acetylcholinesterase inhibitors. This novel approach, involving the use of ultrafiltration–liquid chromatography–mass spectrometry and molecular docking methods combined with stepwise flow rate counter-current chromatography and semi-preparative high-performance liquid chromatography, could potentially provide a powerful tool for the screening and extraction of acetylcholinesterase inhibitors from complex matrices and be a useful platform for the production of bioactive and nutraceutical ingredients.     

Simultaneous determination of ten flavonoids of crude and wine‐processed Radix Scutellariae aqueous extracts in rat plasma by UPLC‐ESI‐MS/MS and its application to a comparative pharmacokinetic study

Radix Scutellariae (RS) is a herbal medicine with various pharmacological activities to treat inflammation, respiratory and gastrointestinal infections, etc. In this study, a rapid, sensitive and selective UPLC‐ESI‐MS/MS method was developed for simultaneous determination of 10 flavonoids – scutellarin, scutellarein, chrysin, wogonin, baicalein, apigenin, wogonoside, oroxylin A‐7‐O‐glucuronide, oroxylin A and baicalin – from RS aqueous extracts in rat plasma with propyl paraben as internal standard (IS). Chromatographic separation was achieved on a C₁₈ column using gradient elution with the mobile phase consisting of methanol and water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min. The detection was performed in multiple reaction monitoring mode using electrospray ionization in negative mode. The validated method showed good linearity over a wide concentration range (r >0.9935). The intra‐ and interday assay variabilities were <9.5% and <12.4% for all analytes, respectively. The extraction recovery ranged from 71.2 to 89.7% for each analyte and IS. This method was successfully applied to pharmacokinetic comparision after oral administration of crude and wine‐processed RS aqueous extracts. There were significant differences in some pharmacokinetic parameters of most analytes between crude and wine‐processed RS. This suggested that wine‐processing exerted effects absorption of most flavonoids. Copyright © 2014 John Wiley & Sons, Ltd.     

An improved cell membrane chromatography method for the simultaneous screening of two epidermal growth factor receptor antagonists from radix scutellariae

A high‐expression epidermal growth factor receptor cell membrane chromatography using the silica gel with the average particle size of 3 μm as the stationary phase carrier coupled with high‐performance liquid chromatography and mass spectrometry was established for the online screening of epidermal growth factor receptor antagonists from Radix Scutellariae (Huang Qin in Chinese), a traditional Chinese medicine. In this study, the growth factor receptor cell membrane chromatography model using the smaller particle size carrier showed a higher efficiency for simultaneous screening baicalein, another one of the potential epidermal growth factor receptor antagonists from Radix Scutellariae extract besides wogonin, which was found in our previous work. The molecular docking result showed the occupancy site and binding mode of baicalein and wogonin with epidermal growth factor receptor tyrosine kinase were similar to gefitinib. The result of the assay for the in vitro inhibitory activity showed that baicalein and wogonin inhibited the growth of the high‐expression epidermal growth factor receptor cell in a dose‐dependent manner and even achieved a better inhibition effect than gifitinib in the low‐dosage range.     

HPLC Determination of Flavonoids in Hairy-Root Cultures of <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi

A simple and sensitive liquid chromatographic method has been established for determination of the main flavonoid components of Scutellaria baicalensis Georgi (Lamiaceae) roots and hairy-root cultures. Wogon, the dried root of the plant, is used in traditional Chinese medicine for treatment of bronchitis, hepatitis, tumors, and inflammatory diseases. Lyophilized hairy roots were extracted with methanol. The crude extracts were purified by SPE on Supelco LC-8 cartridges. HPLC separations were performed on a Eurospher 100-C₈ reversed-phase column. The mobile phase was a gradient prepared from mixtures of acetonitrile and 0.1% trifluoroacetic acid. Peaks were identified by addition of standards and/or by diode-array detection. Baicalein 7-O-glucuronide (baicalin), wogonin 7-O-glucuronide (wogonoside), baicalein, wogonin, and acteoside were determined by the external standard method at 280 nm. We found that the aglycon (baicalein and wogonin) content of the transformed roots was consistently higher than that of the intact root from Siberia.     

Simultaneous determination of the bioactive components in rat plasma by UPLC‐MS/MS and application in pharmacokinetic studies after oral administration of radix Scutellariae extract

A highly sensitive and rapid ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for simultaneous quantification of the four main bioactive compounds, i.e. baicalin, baicalein, wogonoside and wogonin, in rat plasma after oral administration of Radix Scutellariae extract. Clarithromycin was used as an internal standard (IS). Plasma samples were processed by protein precipitation with methanol. The separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm, 1.7 μm) at a flow rate of 0.4 mL/min, using 0.1% formic acid–acetonitrile as mobile phase. The MS/MS ion transit ions monitored were 447.5 → 270.1 for baicalin, 270.1 → 168.1 for baicalein, 461.2 → 284.0 for wogonoside, 284.2 → 168.1 for wogonin and 748.5 → 158.1 for IS. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantification (LLOQ) achieved was 1.13 ng/mL for baicalin, 1.23 ng/mL for baicalein, 0.82 ng/mL for wogonoside and 0.36 ng/mL for wogonin. The calibration curves obtained were linear (r > 0.99) over the concentration range ~ 1–1000 ng/mL. The intra‐ and inter‐day precision was <15% and the accuracy was within ±14.7%. After validation, this method was successfully applied to a pharmacokinetic study of Radix Scutellariae extract.     

Simultaneous Determination of Baicalin, Baicalein, Wogonin, Oxysophocarpine, Oxymatrine and Matrine in the Chinese Herbal Preparation of Sanwu-Huangqin-Tang by Ion-Paired HPLC

A sensitive and reliable ion-paired high-performance liquid chromatographic method has been established for the simultaneous quantification of six major active ingredients, namely baicalin, baicalein, wogonin, oxysophocarpine, oxymatrine and matrine in the Chinese herbal preparation, Sanwu-Huangqin-Tang. HPLC analyses were performed on a Phenomenex luna C₁₈ column with mobile phase of methanol–acetonitrile–aqueous phosphoric acid at a flow rate of 0.9 mL min⁻¹. The complete separation was achieved within 35 min for the six target constituents. A good linear regression relationship between peak-areas and concentrations was obtained over the range of 12.10–242.0 μg*mL⁻¹ for baicalin, 5.05–101.0 μg*mL⁻¹ for baicalein, 0.95–19.0 μg*mL⁻¹ for wogonin, 2.75–55.0 μg*mL⁻¹ for oxysophocarpin, 2.75–55.0 μg*mL⁻¹ for oxymatrine and 4.90–98.0 μg*mL⁻¹ for matrine, respectively. The repeatability was evaluated by intra- and inter-day assays with relative standard deviation (RSD) being less than 5.1%. The recoveries, measured at three concentration levels, varied from 93.8 to 102.1%. The assay was successfully applied for determination of six bioactive compounds in Sanwu-Huangqin-Tang. The interaction of chemical constituents was observed when the herbs were used in compatibility. The results indicated that the developed assay method was rapid, accurate and could be readily utilized as a quality control method for Sanwu-Huangqin-Tang.     

On-line continuous flow ultrasonic extraction coupled with high performance liquid chromatographic separation for determination of the flavonoids from root of Scutellaria baicalensis Georgi

An on-line method, based on coupling dynamic ultrasonic extraction (DUE), continuously sampling the suspension of sample and solvent, high performance liquid chromatographic separation with diode array detection, has been developed for the determination of the flavonoids, including baicalin, baicalein and wogonin, from the root of Scutellaria baicalensis Georgi. Variables influencing the DUE were evaluated by orthogonal test. The extraction yields of baicalin, baicalein and wogonin in the roots of S. baicalensis Georgi obtained from five different cultivated areas are 73.8–131.5 μg mg⁻¹ (RSD ≤ 6.24%), 6.8–15.9 μg mg⁻¹ (RSD ≤ 5.36%) and 4.4–14.3 μg mg⁻¹ (RSD ≤ 5.30%), respectively. The limits of detection for baicalin, baicalein and wogonin are 0.30, 0.37 and 0.41 μg mL⁻¹, respectively. Linearity is from 0.55 to 109 μg mL⁻¹ for baicalin, from 0.51 to 105 μg mL⁻¹ for baicalein and from 0.53 to 102 μg mL⁻¹ for wogonin. Compared with off-line continuous flow-DUE, the proposed method would be more convenient for the determination of the analytes and the rapid optimization of the extraction process. The extraction yields of flavonoids obtained by the proposed method are comparable with those obtained by dynamic microwave assisted extraction, static ultrasonic extraction and reflux extraction. The result indicated that the proposed method is suitable to determine the active components in Chinese herbal medicine.