LC Analysis of Lignans from Schisandra sphenanthera Rehd. et Wils.

Schisandra sphenanthera Rehd. et Wils. is widely used in traditional Chinese medicine. A rapid and convenient method to separate and quantify four lignans (schisandrin, schisantherin A, deoxyschizandrin, and γ-schizandrin) was established by reversed-phase liquid chromatographic. On a Shimadzu C₁₈ column (Phenomenex, 150 × 4.6 mm; 5 μm particle size), an isocratic flow elution program and a simplified sample pretreatment approach were used in the experiment. Samples from different parts of S. sphenanthera were extracted by chloroform and then separated with methanol and deionized water (70:30 v/v) at a flow rate of 0.8 mL min^?1. The detection wavelength was set at 280 nm. The content of lignans in fruits is the highest, and the quantities of schisantherin A, deoxyschizandrin, and γ-schizandrin from fruits are 0.56, 0.54 and 0.30%, respectively. Schisandrin is not detected in all the plant extracts. This research forms a basic framework for the better use of S. sphenanthera in medicine.     

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.     

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.     

Validated method for bioactive lignans in Schisandra chinensis in vitro cultures using a solid phase extraction and a monolithic column application

A simple and rapid method for determination of six lignans found in plant cell cultures of Schisandra chinensis was developed and validated. The lignans were extracted from plant samples with methanol and the extracts were effectively cleaned by solid‐phase extraction using Strata C18‐E (Phenomenex) cartridges. Chromatographic separation was carried out on a Chromolith Performance RP‐18e monolithic column (100 × 4.6 mm, Merck) using an isocratic mobile phase of acetonitrile and water in a 50:50 (v/v) ratio. The eluent was monitored at 220 nm. The baseline separation of schizandrin, gomisin A, deoxyschizandrin, γ‐schizandrin, gomisin N and wuweizisu C was achieved in a relatively short time period (20 min), which was made possible by the relatively high flow rate of the mobile phase (2 mL/min). The lower limit of quantitation was 0.1 mg/L for schizandrin and gomisin A, 0.3 mg/L for deoxyschizandrin, γ‐schizandrin, and gomisin N and 1 mg/L for wuweizisu C. The analysis of spiked samples containing six lignans provided absolute recoveries between 93 and 101% in all cases. The validated method was successfully applied to the determination of lignans in embryogenic plant cell cultures of Schisandra chinensis. Copyright © 2010 John Wiley & Sons, Ltd.     

Discrimination of the Traditional Chinese Medicine from Schisandra Fruits by Flash Evaporation-Gas Chromatography/Mass Spectrometry and Fingerprint Analysis

Flash evaporation-gas chromatography/mass spectrometry (FE-GC/MS) with 0.3 mg sample powder in a vertical microfurnace pyrolyzer at 300 °C was applied to analyze Schisandra fruits without any tedious pretreatment. In total, 80 compounds, 74 compounds of which were identified, were observed, including low-molecular-weight compounds, essential oils (especially terpenoids), fatty acids and esters, and lignans, with the relative standard deviations (RSDs) of the relative percent of peak areas less than 7.79 % (n = 5). 32 compounds of terpenoids and lignans were selected as fingerprint components, since they are the main bioactive constituents in Schisandra fruits. The standard characteristic fingerprints of S. chinensis fruits and S. sphenantherae fruits were established, based on the 32 fingerprint components of 11 genuine S. chinensis fruit samples and 9 genuine S. sphenantherae fruit samples. The discrimination of samples from different growing places was achieved by principle component analysis and hierarchical cluster analysis. Furthermore, a similarity evaluation method was developed to evaluate the quality of each Schisandra fruit sample on the basis of the 32 fingerprint components. The results proved that the FE-GC fingerprint combined with a chemometric approach is a simple, rapid, and effective method for the origin discrimination and quality control of Schisandra fruits.

     

Development of a quality evaluation method for Fructus schisandrae by pressurized capillary electrochromatography

A pressurized CEC (pCEC) method with postcolumn detection cell had been developed for quantifying the lignans from Fructus schisandrae extracts. The effects of different experimental conditions, such as the ACN content of the mobile phase, the concentration and pH of the buffer, the applied voltage, and the supplementary pressure were studied. Five lignans (schisandrin, gomisin A, schisantherin C, deoxyschizandrin, schisandrin B) were baseline separated using a mobile phase of ACN-phosphate buffer (pH 5.4; 5 mM) (40:60 v/v) under -4 kV applied voltage. The method showed the satisfactory retention time and peak area repeatability. The calibration curves were linear in the range 50.0-1000.0 microg/mL for schisandrin, 20.0-500.0 microg/mL for gomisin A, 10.0-200.0 microg/mL for schisantherin C, 20.0-500.0 microg/mL for deoxyschizandrin, and 20.0-500.0 microg/mL for schisandrin B. The correlation coefficients were between 0.9978 and 0.9989. With this pCEC system, fingerprints of F. schisandrae were preliminarily established to distinguish two members S. chinensis (Turcz.) Baill. and S. sphenanthera Rehd. Et Wils. of F. schisandrae by characteristic peaks, and evaluate the quality of various sources of raw materials by determining the contents of the five lignans.     

UFLC–MS/MS method for simultaneous determination of six lignans of Schisandra chinensis (Turcz.) Baill. in normal and insomniac rats brain microdialysates and homogenate samples: towards an in‐depth study for its sedative‐hypnotic activity

Schisandra chinensis (Turcz.) Baill., a traditional Chinese medicine, has been clinically used for the treatment of insomnia for centuries. The insomnia mechanism and the possible active ingredients of S. chinensis remain largely unknown. The objective of this study was to develop a method to detect its components which could pass through the blood brain barrier (BBB) by determining the brain microdialysate and brain tissue homogenate samples and then obtain the pharmacokinetic profile in brain for comprehensive understanding of its hypnotic clinical efficacy. Therefore, an efficient, sensitive and selective ultra fast liquid chromatography/tandem mass spectrometry method for the simultaneous determination of six sedative and hypnotic lignans (schisandrin, schisandrol B, schisantherin A, deoxyshisandrin, γ‐schisandrin and gomisin N) of Schisandra chinensis (Turcz.) Baill. in rat brain tissue homogenate and brain microdialysates has been developed and validated. The analysis was performed on a Shim‐pack XR‐ODS column (75 mm × 3.0 mm, 2.2 µm) using gradient elution with the mobile phase consisting of acetonitrile and 0.1% formic acid water. The method was validated in brain homogenate and microdialysate samples, which all showed good linearity over a wide concentration range (r² > 0.99), and the obtained lower limit of quantification was 0.1 ng · ml^?1 for the analytes in brain microdialysate samples. The intra‐ and inter‐day assay variability was less than 15% for all analytes. The study proved the six lignans, as sedative and hypnotic ingredients, could pass through the BBB with brain targeting, distributed mainly in the hypothalamus and possessed complete pharmacokinetics process in brain. The results also indicated that significant difference in pharmacokinetic parameters of the analytes was observed between two groups, while absorptions of these analytes in insomniac group were significantly better than those in normal group. Copyright © 2013 John Wiley & Sons, Ltd.     

Enrichment and purification of deoxyschizandrin and γ-schizandrin from the extract of Schisandra chinensis fruit by macroporous resins

In present study, the performance and separation characteristics of 21 macroporous resins for the enrichment and purification of deoxyschizandrin and γ-schizandrin, the two major lignans from Schisandra chinensis extracts, were evaluated. According to our results, HPD5000, which adsorbs by the molecular tiers model, was the best macroporous resin, offering higher adsorption and desorption capacities and higher adsorption speed for deoxyschizandrin and γ-schizandrin than other resins. Columns packed with HPD5000 resin were used to perform dynamic adsorption and desorption tests to optimize the technical parameters of the separation process. The results showed that the best adsorption time is 4 h, the rate of adsorption is 0.85 mL/min (4 BV/h) and the rate of desorption is 0.43 mL/min (2 BV/h). After elution with 90% ethanol, the purity of deoxy-schizandrin increased 12.62-fold from 0.37% to 4.67%, the purity of γ-schizandrin increased 15.8-fold from 0.65% to 10.27%, and the recovery rate was more than 80%.     

Complete assignments of 1H and 13C NMR data for new dibenzocyclooctadiene lignans from Kadsura oblongifolia

Two new dibenzocyclooctadiene lignans, named kadoblongifolins A (1) and B (2), and one new natural product dibenzocyclooctadiene lignan, named kadoblongifolin C (3), were isolated from the stems of Kadsura oblongifolia (K. oblongifolia), together with five known ones, schizanrin F (4), propinquanin C (5), schisantherin G (6), heteroclitin Q (7), kadsurarin (8). The structures of these new lignans were elucidated by a combination of high‐resolution electron ionization mass spectrometry (HR‐EI‐MS), ¹H NMR, ¹³C NMR, HMQC, HMBC, and NOESY spectra. Copyright © 2009 John Wiley & Sons, Ltd.     

Inhibitory effects of schisantherin F from Schisandra propinqua subsp. sinensis on human melanoma A375 cells through ROS-induced mitochondrial dysfunction and mitochondria-mediated apoptosis

Abstract

Schisandra propinqua subsp. sinensis is a traditional medicinal plant used in Chinese folk medicine. Melanoma is the most dangerous form of skin cancer. To discover bioactive phytochemicals for preventing human melanoma, we have investigated the inhibitory effects of schisantherin F in Schisandra propinqua subsp. sinensis on human melanoma A375 cells and relevant mechanisms. The results showed that schisantherin F can inhibit A375 cells through inducing apoptosis. Further investigations have demonstrated schisantherin F attenuated the overproduction of ROS, depolarization of MMP, and mPTP opening. Meanwhile, schisantherin F inhibited the activity of Caspase-3 and up-stream Caspase-9, down-regulated Bcl-2 and up-regulated Bax. These findings propose the inhibitory mechanisms of schisantherin F in A375 cells include induction of mitochondrial dysfunction and mitochondria-mediated apoptosis.     

A simple and sensitive HPLC method for the simultaneous determination of eight bioactive components and fingerprint analysis of Schisandra sphenanthera

A simple and sensitive high performance liquid chromatography method with photodiode array detection (HPLC-DAD) was developed for simultaneous determination of eight bioactive constituents (schisandrin, schisandrol B, schisantherin A, schisanhenol, anwulignan, deoxyshisandrin, schisandrin B and schisandrin C) in the ripe fruit of Schisandra sphenanthera and its traditional Chinese herbal preparations Wuzhi-capsule by optimizing the extraction, separation and analytical conditions of HPLC-DAD. The chemical fingerprint of S. sphenanthera was established using raw materials of 15 different origins in China. The chromatographic separations were obtained by an Agilent Eclipse XDB-C18 reserved-phase column (250 mm × 4.6 mm i.d., 5 μm) using gradient elution with water-formic acid (100:0.1, v/v) and acetonitrile, at a flow rate of 1.0 mL min⁻¹, an operating temperature of 35 °C, and a wavelength of 230 nm. The constituents were confirmed by (+) electrospray ionization LC-MS. The new method was validated and was successfully applied to simultaneous determination of components in 13 batches of Wuzhi-capsule. The results indicate that this multi-component determination method in combination with chromatographic fingerprint analysis is suitable for quantitative analysis and quality control of S. sphenanthera.     

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.     

Quantitative Determination of Lignan Constituents from Schisandra chinensis by Liquid Chromatography

The fruits of Schisandra chinensis (Schisandraceae/Magnoliaceae) are a traditional oriental medicine possessing diverse biological activities. A simple and specific analytical method for the quantitative determination of eight lignan constituents from the methanolic extract of the fruits of Schisandra chinensis was developed. The lignan constituents present in the fruits of Schisandra chinensis were separated with an acetonitrile-water-reagent alcohol gradient at a flow rate of 1.0 mL per minute. The HPLC separation was performed on a Phenomenex Luna C18 (2) (150 × 4.6 mm I.D., particle size 5 μm) reversed phase column with detection at 215 nm. The limit of detection was in the range from 0.2 to1.5 μg mL^?1. The relative standard deviation (RSD) values for the determination of lignan constituents in fruits of Schisandra chinensis were less than 2.0%. The method was successfully used to analyze different products available in the market containing Schisandra chinensis and also to study the percentage compositions of various lignans present in Schisandra chinensis procured from different regions in S. Korea.     

Determination of Monobromobimane-Labeled Phytochelatins by High-Performance Liquid Chromatography

A new method for phytochelatins by high-performance liquid chromatography (HPLC) was developed based on a condensation reaction with monobromobimane to produce fluorescent derivatives. Glutathione, H-(γ-glutamic acid-cysteine)₂-glycine-OH, H-(γ-glutamic acid-cysteine)₃-glycine-OH, H-(γ-glutamic acid-cysteine)₄-glycine-OH, H-(γ-glutamic acid-cysteine)₅-glycine-OH, and H-(γ-glutamic acid-cysteine)₆-glycine-OH were well separated, with retention times between 14.68 and 22.0 min. The HPLC method had good linearity (r < 0.9991) between 0.1 mg L^?1 and 100 mg L^?1. The limits of quantification for the analytes (S/N = 3) were 0.08, 0.3, 0.05, 0.3, 0.5, and 0.8 mg L^?1, respectively. The recoveries were between 83.0% and 101.33% with relative standard deviations less than 2%. The reported method is simple, accurate, and suitable for the determination of phytochelatins.     

The Use of Boron‐Doped Diamond Electrodes for the Amperometric Determination of Flavonoids in a Flow Injection System

Abstract

This paper describes the use of boron‐doped diamond electrode for the amperometric detection and quantification of flavonoids in tea samples using a flow injection system. Flavonoids are phenol derivates widely distributed in fruits, nuts, seeds, flowers, vegetable peels, and beverages; they are known to exhibit high antioxidative activities. After optimization, the experiments were performed at a fixed potential of 0.42 V vs Ag/AgCl with a flow rate of 2.5 ml min^?1 in a Britton‐Robinson buffer at pH 5.0. The results revealed that the peak current increases linearly with rutin concentration in the interval 0.1–2.5×10^?4 mol L^?1 and the detection limit was 7.7×10^?6 mol L^?1. The method was used for the determination of rutin in three different green tea (Camellia sinensis) infusions by the direct addition of 150 µL of untreated sample. The repeatability of current responses for injections of 150 µL rutin was evaluated to be 2.1% (n=20) and the analytical frequency was 100 Hz h^?1. The results were compared with the results obtained by high‐performance liquid chromatrography‐diode ray detector.     

Rapid Determination of Creatinine in Human Urine by Microchip Electrophoresis with LED Induced Fluorescence Detection

In clinical medicine, urine creatinine concentration is an important marker in the evaluation of renal function and muscular dysfunctions. Herein, we reported a novel method for rapid determination of creatinine in urine by microchip electrophoresis with light-emitting diode induced fluorescence detection. Creatinine was derivatized by fluorescein isothiocyanate, and then quantitatively detected by the developed microchip LED induced fluorescence detection system. The excitation and emission wavelengths were 490 and 523 nm, respectively. The urine samples were analyzed after centrifuge and filtration. A baseline separation was obtained in <30 s using 10 mM borate buffer (pH 9.0, containing 45 mM sodium dodecylsulfate), with separation voltage of 1.5 kV. Good linearity was obtained (r ² = 0.9978) in the concentration range of 10.0–2.00 × 10³ μM, and the limit of detection was 2.87 μM (S/N = 3). The recovery was 96.0–107 %, and the interday precision was <4.5 % (n = 6). To validate assay results, we compared the present method with the Jaffe’s colorimetric assay by measuring real urine samples. The method was reliable, sensitive, high-speed, low-cost and suitable for the routine analysis of creatinine in biofluids.

     

Simultaneous HPLC–DAD Determination of Retinol and Eight Vitamin E Isomers in Human Serum

An efficient, high-performance liquid-chromatographic method with diode-array detection (HPLC–DAD) has been established for simultaneous determination of retinol, α, (β + γ), and δ-tocopherols, and α, β, γ, and δ-tocotrienols in human serum. After deproteinization, the target vitamins in serum were extracted with n-hexane and the extract was evaporated under weak nitrogen flow. The residue was redissolved in methanol and the resulting solution was used for HPLC analysis. Retinol acetate and α-tocopherol acetate were used as internal standards. The internal standard calibration curves were linear over the range of 0.010–50.0 µg mL⁻¹, with correlation coefficients >0.999. Mean recoveries of the method were 86.3–110 %, with intra-day and inter-day relative standard deviations less than 12.2 and 14.9 %, respectively. The detection limits of the method ranged from 0.001 to 0. 002 µg mL⁻¹, and the quantification limits ranged from 0.002 to 0.008 µg mL⁻¹. The method was successfully applied to analysis of the target vitamins in 50 human serum samples; all the analytes were detected at concentrations ranging from <0.002–23.0 µg mL⁻¹.

     

Vibrational circular dichroism study for natural bioactive schizandrin and reassignment of its absolute configuration

Bioactive natural product (+)-schizandrin was assigned as (7S,8S) using NMR. Recently, we obtained (+)-schizandrin from TCM Schisandra sphenanthera Rehd. et Wils. Its planar structure was well established using NMR and HR-MS including the reported references. Its absolute configuration is assigned using vibrational circular dichroism (VCD). By careful VCD investigation of (7S,8S) and (7S,8R) using B3LYP/6-311+G(d) methods, absolute configuration of (+)-schizandrin is assigned as (7S,8R). Electronic circular dichroism (ECD) was used for the discussion too and it gave the same conclusion.     

Survismeter Unit for Surface Tension,Viscosity, and Dipole Moment Determination for Polystyrene Interactions in Benzene

Novel instrumental set up is developed for surface tension (γ, N m^?1), viscosity (η, N s m^?2)) and dipole moment (μ/Debye) measurements. A new instrument is economic, viable and pollution free in use, the γ, η and μ from viscosities (η, N s m^?2) and surface tension (γ, N m^?1) for 0.005 to 0.125 g% polystyrene at an interval of 0.0005 g% in benzene are measured at 288.15, 293.15, and 298.15 Kelvin temperatures. The γ, η, and μ are reported stronger frictional and cohesive forces due to stronger interactions of polystyrene with benzene. The survismeter is highly accurate and mechanically operated for pulling up liquid from reservoir bulb B1 to operational bulbs B2, B3, and B4. Densities are higher than those of benzene and also infer stronger polystyrene–benzene interactions, and hydrodynamic volume (HV) calculated with intrinsic viscosity [η] is noted in 293.15 (HV) >298.15 (HV) >288.15 (HV) order with higher values at 293.15 K. Surface tension (γ) values are slightly higher than of benzene and fall in a range of 33.47 to 33.35 N m^?1.     

An Efficient Separation Method of Polysaccharides: Preparation of an Antitumor Polysaccharide APS-2 from Auricularia polytricha by Radial Flow Chromatography

Auricularia polytricha is an edible mushroom, quite popular in China. Modern pharmacology research indicate that A. polytricha polysaccharides possess antitumor and immunomodulatory activities. In this paper, an antitumor Auricularia polytricha polysaccharide (APS)-2 was purified by radial flow chromatography (RFC) in a column of 500 mL bed-volume (40 cm i.d × 15 cm) packed with DEAE52-cellulose anion ion-exchange resin. The optimal separation conditions were: sample flow-velocity 15–20 mL min⁻¹, sample volume 160–200 mL, and elution with distilled water and 0.2 M NaCl at a velocity of 35–45 mL min⁻¹. The yield and content of APS-2 obtained under these conditions were 182 mg g⁻¹ total polysaccharides and 98.35 %, respectively. The study provides guidelines for the separation and purification of polysaccharides using a radial flow chromatography column.