Variation of the Lignan Content of Schisandra chinensis (Turcz.) Baill. and Schisandra sphenanthera Rehd. et Wils.

A rapid and convenient extraction technique followed by HPLC analysis has been developed for determination of lignans in Schisandra chinensis (Turcz.) Baill. and Schisandra sphenanthera Rehd. et Wils. Under the optimized conditions the lignans schisandrin, schisantherin, deoxyschisandrin, and γ-schisandrin were extracted from ten samples collected from different regions in China and then quantified by HPLC. The lignan content of the ten samples is different. S. sphenanthera Rehd. et Wils. is richer in deoxyschisandrin but there is less schisandrin and γ-schisandrin is not found. S. chinensis (Turcz.) Baill. is usually richer in γ-schisandrin, the maximum amount being 4.263 mg g⁻¹, although one sample contained none. This method is simple, rapid, sensitive, and repeatable, and is suitable for identification and differentiation of samples from different regions of China.     

An efficient strategy for the extraction and purification of lignans from Schisandra chinensis by a combination of supercritical fluid extraction and high‐speed counter‐current chromatography

An efficient strategy for extracting and separating five lignans from Schisandra chinensis (Turcz.) Baill has been developed using supercritical fluid extraction (SFE) and high‐speed counter‐current chromatography (HSCCC) in the present study. First, the extraction was performed by a preparative SFE system under 15 MPa of pressure at 36°C for 4 h. Then, the SFE extract was successfully separated and purified by HSCCC with a two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water (6:4:5:5, 6:4:6:4, 6:4:8:2, v/v) in a stepwise elution mode. The fractions were analyzed by HPLC, and the chemical structures of the products were identified by ESI‐MS and ¹H NMR spectroscopy. As a result, a total of 12.5 mg of schisandrin at 98.0% purity, 7.1 mg of gomisin A at 98.1% purity, 1.8 mg of schisantherin B at 93.3% purity, 4.4 mg of deoxyschisandrin at 92.9% purity, and 6.8 mg of γ‐schisandrin at 89.1% purity were obtained from 300 mg crude extract in a one‐step purification.     

Metabolic mapping of Schisandra chinensis lignans and their metabolites in rats using a metabolomic approach based on HPLC with quadrupole time‐of‐flight MS/MS spectrometry

Schisandra chinensis lignans are the main active components of the traditional Chinese medicine Schisandra chinensis in East Asia. At present, there are more and more medicines and health foods in which the total S. chinensis lignans extracts are considered as the main active components, but little research has been done on the active components of S. chinensis lignans in the blood and main target organs. In this study, the components of S. chinensis lignans in the blood, liver and brain tissues of rats at different time points after the intragastrical administration of S. chinensis lignans were determined by a metabolomic method based on high‐performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry spectrometry. Twelve Schisandra chinensis lignans and 15 metabolites in the blood, liver, and brain of rats were identified. The results showed that the main metabolic ways of S. chinensis lignans in rats were hydroxylation, demethylation, and demethylation‐hydroxylation, and some of them might undergo demethylation, dehydrogenation, epoxidation, and elimination reaction. The time‐dose characteristics of S. chinensis lignans and their metabolites in the blood and target organs were analyzed, which may be helpful to elucidate the active substances that really exert the pharmacodynamic effects of S. chinensis lignans in organisms.     

Analysis of lignans in Schisandra chinensis and rat plasma by high‐performance liquid chromatography diode‐array detection,time‐of‐flight mass spectrometry and quadrupole ion trap mass spectrometry

High‐performance liquid chromatography/diode‐array detection (HPLC/DAD), time‐of‐flight mass spectrometry (HPLC/TOFMS) and quadrupole ion trap mass spectrometry (HPLC/QIT‐MS) were used for separation, identification and structural analysis of lignans in Schisandra chinensis and rat plasma after oral administration of the herbal extract. Six lignans in Schisandra chinensis extract were identified unambiguously by comparing the retention time, their characteristic ultraviolet (UV) absorption and accurate mass measurement. A formula database of known lignans in Schisandra chinensis was established, against which the other 15 lignans were identified effectively based on the accurate extract masses and formulae acquired by HPLC/TOFMS. In order to distinguish the isomers, multi‐stage mass spectrometry (ion trap mass spectrometry, MSⁿ) was also used. The fragmentation behavior of the lignans in the ion trap mass spectrometer was studied by the six lignan standards, and their fragmentation rules in MSⁿ spectra were summarized. These deduced fragmentation rules of lignans were successfully implemented in distinguishing the three groups of isomers in Schisandra chinensis by HPLC/QIT‐MS. By using the three different analytical techniques, 21 lignans in Schisandra chinensis were identified within 30 min. After oral administration of the extract, 11 lignans in rat plasma were detected and identified by comparing their retention time, characteristic UV absorption and accurate mass measurement of peaks in HPLC/TOFMS chromatograms of the herbal extract. Finally, HPLC/TOFMS fingerprints of Schisandra chinensis in vitro and rat plasma in vivo were established. It is concluded that a rapid and effective method based on three analytical techniques for identification of chemical components was established, which is useful for rapid identification of multiple components in Schisandra chinensis in vitro and in vivo. In addition, it can provide help for further pharmacology and action mechanism study of lignans in Schisandra chinensis. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous LC–MS Quantification of 15 Lignans in Schisandra chinensis (Turcz.) Baill. Fruit

For the first time a simple, rapid, and specific liquid chromatographic–mass spectrometric (LC–MS) method for simultaneous quantification of 15 lignans from Schisandra chinensis (Turcz.) Baill. fruit has been developed and fully validated. Samples were ultrasonically extracted with methanol. Chromatographic separation was performed on a C₁₈ column with methanol–0.1% aqueous acetic acid 72:28 (v/v) as mobile phase at a flow rate of 0.8 mL min⁻¹; the run time was 40 min. The analytes were monitored by selected ion monitoring (SIM) with electrospray ionization (ESI). Fifteen regression equations revealed good linear relationships (r ² > 0.99) between peak area and concentration. Within-batch and between-batch precision of the method for the 15 lignans was <9.5% and accuracy was 93.1–107.5%. The validated method was successfully used to determine the 15 compounds in the fruits of Schisandra chinensis procured from different regions of China. The results indicated the method could be used for quality control of Schisandra chinensis fruit and might also useful for further studies of lignans.     

Simultaneous determination of nine lignans from Schisandra chinensis extract using ultra‐performance liquid chromatography with tandem mass spectrometry in rat plasma,urine, and gastrointestinal tract samples: Application to the pharmacokinetic study of Schisandra chinensis

The fruit of Schisandra chinensis is a well‐known herbal medicine and dietary supplement due to a variety of biological activities including antihepatotoxic and antihyperlipidemic activities. However, the simultaneous validation methodology and pharmacokinetic investigation of nine lignans of S. chinensis extract in biological samples have not been proved yet. Thus, the present study was undertaken to develop the proper sample preparation method and simultaneous analytical method of schisandrol A, gomisin J, schisandrol B, tigloylgomisin H, angeloylgomisin H, schisandrin A, schisandrin B, gomisin N, and schisandrin C in the hexane‐soluble extract of S. chinensis to apply for the pharmacokinetic study in rats. All intra‐ and interprecisions of nine lignans were below 13.7% and accuracies were 85.1–115% and it is enough to evaluate the pharmacokinetic parameters after both intravenous and oral administration of hexane‐soluble extract of S. chinensis to rats.     

Bioanalytical assay development and validation for simultaneous quantification of five schisandra lignans in rat primary hepatocytes based on LC‐MS/MS: application to a real‐time uptake study for Schisandra Lignan Extract

Schisandra lignans, mainly including schizandrol A, schizandrol B, schisantherin A, schizandrin A, schizandrin B, etc., are the major active ingredients of Schisandra chinensis . In the present study, a robust liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed for the simultaneous quantification of schisandra lignans in rat primary hepatocytes. Lovastatin was used as an internal standard, and chromatographic separation was achieved on a Shimadzu C₁₈ column with a gradient elution at the flow rate of 0.2 mL/min. All of the analytes were detected in multiple reaction monitoring mode with positive electrospray ionization since the sodium adduct ion [M + Na]⁺ was observed as the most intensive peak in the MS spectrum. For schizandrol A, schisantherin A and schizandrin A, the dynamic range was within 2–1000 ng/mg protein, and the linear range of schizandrol B and schizandrin B was from 5 to 1000 ng/mg protein. The intra‐ and inter‐day precision was <15% and the accuracy (relative error) ranged from −15 to 15%. No significant variation was observed in the stability tests. The validated method was then successfully applied to the time‐dependent uptake study for the Schisandra Lignan Extract in rat primary hepatocytes.     

Study on the Chromatographic Fingerprinting of Schisandra chinensis (Turcz.) Baill. by LC Coupled with Principal Component Analysis

Schisandra chinensis (Turcz.) Baill. is a rich source of lignans with diverse biological activities. Quantitative analysis and chromatographic fingerprinting of the fruitlets of the plant by liquid chromatography (LC) were performed in order to characterize and evaluate the region-related variation. Using the optimized LC method, satisfactory linearity and repeatability were obtained for all the test standards. This method was subsequently applied for the identification and quantitation of six lignans in fruitlets of Schisandra chinensis by comparing the retention time with that of authentic standards available. Principal component analysis (PCA) was also employed to assess the similarity and dissimilarity of the fingerprint profiles from different origins encompassing eleven areas from China and two samples from Japan. The work suggested that the fruitlets of Schisandra chinensis from Japan exhibited great difference with those from China as evidenced by the clear separation in the PCA scores plot as well as quantitative analysis e.g., schisandrin and deoxyschisandrin are 5.50 μg mg^?1 $ (\overline{X} ) $ and 0.90 μg mg^?1 $ (\overline{X} ) $ in the materials from China and 3.40 μg mg^?1 $ (\overline{X} ) $ and 1.90 μg mg^?1 $ (\overline{X} ) $ in the materials from Japan, respectively. Such a reliable method potentially provides an effective and convenient method for quality control of herbal medicine from different regions.     

Rapid determination and pharmacokinetics study of lignans in rat plasma after oral administration of Schisandra chinensis extract and pure deoxyschisandrin

A simple, sensitive and rapid method for analysis of six lignans in rat plasma after oral administration of Schisandra chinensis extracts, utilizing liquid chromatography tandem mass spectrometry (LC‐MS), was established and validated. Plasma samples were prepared by one‐step protein precipitation using acetonitrile and the analytes were separated on an SB‐C₁₈ column (100 mm × 3.0 mm, 3.5 µm) with the mobile phase of acetonitrile–water at a flow‐rate of 0.8 mL/min. Analytes were determined in a single‐quadrupole mass spectrometer in the selected ion monitoring (SIM) mode using electrospray source with positive mode. The method was proved to be rapid, sensitive and reproducible, and it was successfully applied to the pharmacokinetic studies of six lignans in rat plasma after oral administration of Schisandra chinensis extracts. In this research, the pharmacokinetics of deoxyschisandrin was also studied following oral administration of the pure deoxyschisandrin. It was found that most of the pharmacokinetic parameters of deoxyschisandrin in the extract were changed significantly compared with those in monomer. The content assay also revealed that the concentrations of the lignan in the extract increased in vivo compared with the pure monomer. Some ingredients in the extract may increase the dissolution of deoxyschisandrin, delay its elimination and enhance its bioavailability in rat. Copyright © 2010 John Wiley & Sons, Ltd.     

Fast quantitative determination of methylphenidate levels in rat plasma and brain ex vivo by MALDI‐MS/MS

This study presents a simple and sensitive high‐throughput matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (MALDI‐MS/MS) method for ex vivo quantification of methylphenidate (MPH) in rat plasma and brain. The common MALDI matrix alpha‐cyano‐4‐hydroxycinnamic acid was used to obtain an optimal dried droplet preparation. For method validation, standards diluted in plasma and brain homogenate prepared from untreated (control) rats were used. MPH was quantified within a concentration range of 0.1–40 ng/ml in plasma and 0.4–40 ng/ml in brain homogenate with an excellent linearity (R² ≥ 0.9997) and good precision. The intra‐day and inter‐day accuracies fulfilled the FDA's ±15/20 critera. The recovery of MPH ranged from 93.8 to 98.5% and 87.2 to 99.8% in plasma and homogenate, respectively. We show that MPH is successfully quantified in plasma and brain homogenate of rats pre‐treated with this drug using the internal standard calibration method. By means of this method, a linear correlation between plasma and brain concentration of MPH in rodents pre‐treated with MPH was detected. The simple sample preparation based on liquid‐liquid extraction and MALDI‐MS/MS measurement requires approximately 10 s per sample, and this significantly reduces analysis time compared with other analytical methods. To the best of our knowledge, this is the first MALDI‐MS/MS method for quantification of MPH in rat plasma and brain. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of three major lignans in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study after oral administration of Diphylleia sinensis extract

A sensitive and selective liquid chromatography tandem mass spectrometry was developed and validated for the simultaneous determination of three major lignans (podophyllotoxin, epipodophyllotoxin, and 4′‐demethylpodophyllotoxin) in rat plasma using diphenhydramine as the internal standard. The analytes were detected using a triple quadrupole mass spectrometer that was equipped with an electrospray ionization source in the positive ion and selected reaction monitoring modes. The linearity of the calibration curve was good, with coefficients of determination (r²) >0.9914 for all of the analytes. The developed method was successfully applied for the simultaneous determination of the three lignans in rat plasma following oral administration of Diphylleia sinensis extract to rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of calycosin‐7‐O‐β‐d‐glucoside,calycosin, formononetin,astragaloside IV and schisandrin in rat plasma by LC‐MS/MS: application to a pharmacokinetic study after oral administration of Shenqi Wuwei chewable tablets

A rapid, sensitive and reliable high‐performance liquid chromatography–mass spectrometry (LC‐MS/MS) method was developed and validated for simultaneous quantification of the five main bioactive components, calycosin, calycosin‐7‐O‐β‐d ‐glucoside, formononetin, astragaloside IV and schisandrin in rat plasma after oral administration of Shenqi Wuwei chewable tablets. Plasma samples were extracted using solid‐phase extraction separated on a CEC₁₈ column and detected by MS with an electrospray ionization interface in multiple‐reaction monitoring mode. Calibration curves offered linear ranges of two orders of magnitude with r > 0.995. The method had a lower limit of quantitation of 0.1, 0.02, 0.1, 1 and 0.1 ng/mL for calycosin, calycosin‐7‐O‐β‐d ‐glucoside, formononetin, astragaloside IV and schisandrin, respectively. Intra‐ and inter‐day precisions (relative standard deviation) for all analytes ranged from 0.97 to 7.63% and from 3.45 to 10.89%, respectively. This method was successfully applied to the pharmacokinetic study of the five compounds in rats after oral administration of Shenqi Wuwei chewable tablets. Copyright © 2014 John Wiley & Sons, Ltd.     

A simple and sensitive assay for eflornithine quantification in rat brain using pre‐column derivatization and UPLC‐MS/MS detection

Eflornithine (α‐difluoromethylornithine) has been used to treat second‐stage (or meningoencephalitic‐stage) human African trypanosomiasis and currently is under clinical development for cancer prevention. In this study, a new ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS)‐based assay was developed and validated for the quantification of eflornithine in rat brain. To improve chromatographic retention and MS detection, eflornithine was derivatized with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate for 5 min at room temperature prior to injection. Derivatized eflornithine was separated on a reverse‐phase C₁₈ UPLC column with a 6‐min gradient; elution occurred at approximately 1.5 min. Prior to derivatization, eflornithine was reproducibly extracted from rat brain homogenate by methanol protein precipitation (~70% recovery). Derivatized eflornithine was stable in the autosampler (6 °C) for at least 24 h. This new assay had acceptable intra‐ and interday accuracy and precision over a wide dynamic range (5000‐fold) and excellent sensitivity with a lower limit of quantification of 0.1 µm (18 ng/mL) using only 10 μL of rat brain homogenate. The validated eflornithine assay was applied successfully to determine eflornithine distribution in different regions of rat brain in an in situ rat brain perfusion study. Copyright © 2014 John Wiley & Sons, Ltd.     

Validated UPLC‐MS/MS method for determination of moclobemide in human brain cell supernatant and its application to bidirectional transport study

A simple and sensitive analytical method based on ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) has been developed for determination of moclobemide in human brain cell monolayer as an in vitro model of blood–brain barrier. Brucine was employed as the internal standard. Moclobemide and internal standard were extracted from cell supernatant by ethyl acetate after alkalinizing with sodium hydroxide. The UPLC separation was performed on an Acquity UPLC^TM BEH C₁₈ column (50 × 2.1 mm, 1.7 µm, Waters, USA) with a mobile phase consisting of methanol–water (29.5:70.5, v/v); the water in the mobile phase contained 0.05% ammonium acetate and 0.1% formic acid. Detection of the analytes was achieved using positive ion electrospray via multiple reaction monitoring mode. The mass transitions were m/z 269.16 → 182.01 for moclobemide and m/z 395.24 → 324.15 for brucine. The extraction recovery was 83.0–83.4% and the lower limit of quantitation (LLOQ) was 1.0 ng/mL for moclobemide. The method was validated from LLOQ to 1980 ng/mL with a coefficient of determination greater than 0.999. Intra‐ and inter‐day accuracies of the method at three concentrations ranged from 89.1 to 100.9% for moclobemide with precision of 1.1–9.6%. This validated method was successfully applied to bidirectional transport study of moclobemide blood–brain barrier permeability. Copyright © 2013 John Wiley & Sons, Ltd.     

Profiling and identification of the absorbed constituents and metabolites of schisandra lignans by ultra‐performance liquid chromatography coupled to mass spectrometry

Schisandra chinensis Baill grows wild in Russia, China, Korea and Japan, and its fruit has been found to be effective in amnesia and insomnia. It is enriched in schisandra lignans (SL) that are major components responsible for therapeutic action. However, there are no reports on the biotransformation analysis of SL. An ultra‐performance liquid chromatography/electrospray‐ionization high‐definition mass spectrometry (UPLC‐Q‐TOF‐HDMS) method was developed to investigate the metabolism of SL in vivo. MS was performed on a Waters Micromass high‐definition system with an electrospray ionization source in positive ion mode and automated MetaboLynx software analysis with excellent MS accuracy and enhanced MS data acquisition. An improved mass defect filter (MDF) method employing both drug and core structure filter templates was applied to the processing of UPLC‐Q‐TOF‐HDMS data for the detection and structural characterization of metabolites. In this study, 30 metabolites were detected and identified in vivo, and demethylation and hydroxylation were confirmed as the primacy metabolic pathway for SL in rat plasma. In conclusion, the presently developed methodology was suitable for biotransformation research of SL and will find wide use in metabolic studies for other herbal medicines. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a sensitive LC‐MS/MS method for simultaneous quantification of eleven constituents in rat serum and its application to a pharmacokinetic study of a Chinese medicine Shengmai injection

A sensitive LC‐MS/MS method was developed and validated for simultaneous quantification of 11 constituents, ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rd, Rc, ophiopogonin D, schisandrin, schisandrol B and schizandrin B, in rat serum using digoxin as the internal standard (IS). The serum samples were pretreated and extracted with a two‐step liquid–liquid extraction. Chromatographic separation was achieved on a C₁₈ analytical column with a proper gradient elution using 0.02% acetic acid aqueous solution and 0.02% acetic acid–acetonitrile as mobile phase at a flow rate of 0.5 mL/min. MS detection was performed using multiple reaction monitoring via an electrospray ionization source. Good linearity was observed in the validated concentration range for every analyte (r² ≥0.9929), and the lower limits of quantitation of the analytes were in the range of 0.044–1.190 ng/mL in rat serum. Intra‐ and inter‐day precisions were <14.2%. The accuracy expressed as recovery was within the range of 85.1–112.8%. The extraction recoveries were >75.8%.The validated method was successfully applied to a pharmacokinetic study of all analytes in rats after single intravenous administration of Shengmai injection. Copyright © 2014 John Wiley & Sons, Ltd.     

五味子中木脂素类成分的高效液相色谱-电喷雾质谱研究

采用液相色谱-电喷雾质谱联用(HPLC-ESI-MSⁿ)技术, 对北五味子与南五味子中木脂素类成分进行了系统研究. 通过HPLC-ESI-MS技术, 获得了相应化合物的保留时间、紫外光谱和分子量等信息, 利用电喷雾多级串联质谱技术(ESI-MSⁿ), 获得了相应化合物的结构信息. 研究结果表明, 北五味子与南五味子的主要木脂素成分除5个共有成分外其它成分差异较大, 并且其共有成分含量差别较大. 在此基础上, 建立了简便、快速的北五味子与南五味子药材分析鉴定的新方法.     

Qualitative and quantitative analysis of the major constituents in Acorus tatarinowii Schott by HPLC/ESI‐QTOF‐MS/MS

Acorus tatarinowii Schott (ATS) is a well‐known traditional Chinese medicine (TCM) for the treatment of epilepsy, amnesia and insomnia. In this study, a methodology utilizing high‐performance liquid chromatography (HPLC) coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐QTOF‐MS/MS) was established for the separation and structural identification of the major chemical constituents in ATS for the first time. Overall, 46 major constituents including flavonoid glycosides, phenylpropane derivatives, amides and lignans were identified or tentatively characterized. Seven major constituents, including four phenylpropane derivatives and three lignans, were further quantified as marker substances, which showed good linearity within the test ranges. These results indicated that the developed quantitative method was linear, sensitive, and precise for quality control of ATS. Copyright © 2014 John Wiley & Sons, Ltd.     

Chemical constituents isolated from stems of Schisandra chinensis and their antifeedant activity against Tribolium castaneum

Abstract

One new sesquiterpene (α-iso-cubebenol acetate, 8), together with 9 known compounds (1-7, 9, 10) were isolated from the stems of Schisandra chinensis (Turcz.) Baill. by repeated silica gel column chromatography. Based on the results of MS, NMR spectra and comparing with literature data, the six dibenzocyclooctadiene lignans were identified as schizandrin A to C (1-3), schizandrin (4), schisantherin A (5) and gomisin J (6), the two sesquiterpenes were identified as α-iso-cubebenol (7) and α-iso-cubebenol acetate (8), while the two triterpenic acids were identified as ganwuweizic acid (9) and kadsuric acid (10). The antifeedant activity of the 10 compounds against Tribolium castaneum adults was tested. Gomisin J (6) exhibited activity at 1500?ppm concentration with 40.3% antifeeding index percentages. As for the dibenzocyclooctene lignans (compounds 1–3, 6), the number of methylenedioxies and the position of hydroxyl groups were the main factors to affect their antifeedant activities.