Simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and high-performance liquid chromatography

Herba Epimedii (family Berberidaceae), Yinyanghuo in Chinese, is one of the commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a reliable pressurized liquid extraction (PLE) and HPLC method was developed for simultaneous determination of 15 flavonoids, namely hexandraside E, kaempferol-3-O-rhamnoside, hexandraside F, epimedin A, epimedin B, epimedin C, icariin, epimedoside C, baohuoside II, caohuoside C, baohuoside VII, sagittatoside A, sagittatoside B, 2'-O-rhamnosyl icariside II and baohuoside I in different species of Epimedium. The analysis was performed by using a Zorbax SB-C18 analytical column (250 mm x 4.6 mm I.D., 5 microm) at gradient elution of water and acetonitrile with diode-array detection (270 nm). All calibration curves showed good linearity (r(2)>0.9997) within test ranges. The LOD and LOQ were lower than 1.31 ng and 2.62 ng on column, respectively. The RSD for intra- and inter-day of 15 analytes was less than 3.8% at three levels, and the recoveries were 90.5-106.8%. The validated method was successfully applied for the analysis of 15 flavonoids in different species of Epimedium which had great variation on the contents of investigated flavonoids. Hierarchical clustering analysis based on the characteristics of 15 investigated compound peaks in HPLC profiles showed that 26 samples were divided into three main clusters, which were in accordance with their flavonoid contents. Four flavonoids including epimedin A, B, C and icariin were optimized as markers for quality control of the species of Epimedium used as Yinyanghuo.     

Simultaneous determination of seven flavonoids in Epimedium by liquid chromatography-tandem mass spectrometry method

In this paper, a sensitive and specific liquid chromatography-electrospray ionisation-mass spectrometry （LC-ESI-MS） method has been developed and validated for the identification and determination of seven flavonoids, namely epimedin A, epimedin B, epimedin C, icariin, sagittatoside B, 2＂-O-rhamnosyl icariside II, and baohuoside I in Epimedium from different sources.     

Rapid characterization and determination of multiple components in Bu‐Shen‐Yi‐Qi‐Fang by high‐performance liquid chromatography coupled to electrospray ionization and quadrupole time‐of‐flight mass spectrometry

In this study, a qualitative and quantitative analysis using high‐performance liquid chromatography coupled to electrospray ionization and quadrupole time‐of‐flight mass spectrometry was performed for the quality control of Bu‐Shen‐Yi‐Qi‐Fang, a traditional Chinese formula used for asthma. Thirty‐four compounds, including flavonoids, isoflavonoids, triterpenoid saponins, and iridoid glycosides were identified or tentatively characterized by comparing their retention times and mass spectra with those of authentic standards or literature data. Sixteen components were considered as the main bioactive constituents of Bu‐Shen‐Yi‐Qi‐Fang and they were chosen as the chemical markers in quantitative analysis, including catalpol, leonuride, calycosin‐7‐O‐β‐d ‐glucoside, hyperoside, acteoside, formononetin‐7‐O‐β‐d ‐glucoside, epimedin A, calycosin, icariin, epimedin B, epimedin C, formononetin, astragaloside IV, astragaloside II, baohuoside‐I, and astragaloside I. The total run time was 20 min. It was found that the calibration curves for all analytes showed good linearity (R² > 0.99) within the test ranges. The relative standard deviations for intra‐ and inter‐day precisions were below 3.9 and 11.7%, respectively. The accuracy was evaluated by the recovery test within the range of 89.20–110.71% with the relative standard deviation < 4.8%. The sample was stable for at least 48 h at 4°C. The results showed that the new approach was effective for the quality control of Bu‐Shen‐Yi‐Qi‐Fang.     

Identification of Metabolites of Epimedin A in Rats Using UPLC/Q–TOF–MS

Herba Epimedii (Epimedium) is a kind of tonic herb, widely used in China. Epimedin A is a major component of Herba Epimedii with bioactivities. Analysis of the metabolic profile in vivo plays a pivotal role in understanding how traditional Chinese medicine works. And the metabolites of epimedin A might influence the effects of Herba Epimedii. Moreover, the metabolic routes of epimedin A provide an important basis for safety evaluation. Until now, little has been known about the metabolism of epimedin A. The current study was designed to characterize the metabolic pathways of epimedin A in vivo. The metabolites in rat plasma, bile, feces, and urine were identified by UPLC/Q–TOF–MS analysis. A total of 27 metabolites from epimedin A were detected or tentatively identified. The major metabolic processes were hydrolysis, hydrogenation, hydroxylation, dehydrogenation, demethylation, and conjugation with glucuronic acid and different sugars. The present study revealed the metabolic pathways of epimedin A in rat for the first time, and epimedin A could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of epimedin A. In addition, the results of this work have shown the feasibility of the UPLC/Q–TOF–MS approach for rapid and reliable characterization of metabolites.

     

Pharmacokinetics and oral bioavailability of epimedin C after oral administration of epimedin C and Herba Epimedii extract in rats

Epimedin C, an ingredient of Herba Epimedii, has potential for treatment of cardiovascular disease and bone loss. However, there is still no sensitive analytical method to monitor epimedin C in biological samples. The goal of this study was to develop a sensitive and reliable method based on a LC‐MS/MS for evaluating the pharmacokinetics of epimedin C after administration of Herba Epimedii in rat. Electrospray ionization in positive‐ion mode and multiple reaction monitoring were used to identify and quantitate active components. Analytes were separated by a reverse‐phase C₁₈ column. Liquid–liquid extraction using ethyl acetate, evaporation and reconstitution was used to plasma sample preparation. Mass transition of precursor ion → product ion pairs were monitored at m/z 823.4 → 313.1 for epimedin C and m/z 237.1 → 178.9 for carbamazepine (internal standard). A calibration curve gave good linearity (r > 0.999) over the concentration range 2.5–500 ng/mL. Pharmacokinetic data demonstrated that there was rapid distribution and slow elimination after epimedin C administration (1 mg/kg, i.v.). Oral bioavailabilities of epimedin C in the pure compound and in the Herba Epimedii were around 0.58% and 0.13%, respectively. The result suggests that other herbal ingredients of Herba Epimedii may suppress the oral bioavailability of epimedin C. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of seven flavonoids in Epimedium using pressurized liquid extraction and capillary electrochromatography

Herba Epimedii (known as Yinyanghuo in China) is one of the commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a CEC method was developed for the simultaneous determination of seven flavonoids, including hexandraside E, kaempferol-3-O-rhamnoside, hexandraside F, icariin, epimedin A, B, and C, in Epimedium using baicalein as internal standard (IS). The influence of relevant parameters such as buffer concentration, pH, and proportion of ACN was investigated and optimized. Baseline separation was obtained using a Hypersil C18 capillary (3 microm, 100 microm/25 cm) with a mixture of 20 mM phosphate buffer (pH 4.0)/ACN (70:30 v/v) as mobile phase running at 30 kV and 25 degrees C in 20 min. All calibration curves showed good linearity (r2 >0.9992) within test ranges. The LOD and LOQ were lower than 8.6 and 42.8 microg/mL, respectively. The RSDs of intra- and interday for relative peak areas of seven analytes were less than 3.1 and 4.4%, and the recoveries were 95.2-103.3%. Samples of different Epimedium species were analyzed using the validated method, which is useful for quality control of Epimedium and its medical preparations.     

Studies on target tissue distribution of ginsenosides and epimedium flavonoids in rats after intravenous administration of Jiweiling freeze‐dried powder

A simple and rapid liquid chromatography–mass spectrometry (LC‐MS) method was developed and validated for analysis of ginsenoside Rb₁, Rb₂, Rc, Rd, Re, Rf, Rg₁, icariin and epimedin A, B, C in rat target tissues (spinal cord, brain, muscle and sciatic nerve) after intravenous administration of Jiweiling freeze‐dried powder using genistein as an internal standard (IS). The tissue samples were treated by protein precipitation with methanol prior to HPLC and chromatographic separation was performed on a C₁₈ column utilizing a gradient elution program with acetonitrile and 0.1% formic acid aqueous. Electrospray ionization (ESI) source was employed and the 11 analytes and IS were detected by multiple reaction monitoring (MRM) scanning under the negative ionization mode. Higher sensitivity was achieved and the optimized mass transition ion‐pairs (m/z) for quantitation were selected. The calibration curves were linear over the investigated concentration ranges with correlation coefficients higher than 0.995. The intra‐ and inter‐day RSDs were all less than 10% with the relative error (RE) within ±9.3%. The mean extraction recoveries for all compounds were between 93.3 and 106%. The proposed method was successfully applied to investigate the target tissue distribution of the 11 compounds in rat after intravenous administration of Jiweiling freeze‐dried powder. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of nine flavonoids and qualitative evaluation of Herba Epimedii by high performance liquid chromatography with ultraviolet detection

A simple high performance liquid chromatographic method has been developed for the determination of nine flavonoids in Herba Epimedii, including quercetin-3-O-glucoside, epimedin A, hexandraside A, epimedin B, epimedin C, icariin, icariside I, icariside II, and icaritin. The chromatographic separation was performed on a Diamonsil C(18) column (5 microm, 250x4.6 mm) with gradient elution by acetonitrile-0.5% acetic buffer (adjusted to pH 5.00 with triethylamine). Methodological validation gave acceptable linearities (r(2) >0.9992) and recoveries (ranging from 98.9 to 102.4%). The limits of detection of these flavonoids ranged from 16.3 to 83.3 ng. The results indicated that the contents of flavonoids in Herba Epimedii varied significantly from habitat to habitat with contents ranging from 0.05 to 39.0 mg/g. Twenty five Herba Epimedii samples prepared from five different botanical materials were investigated by the established method, and the results showed the influence of the botanical material and the habitat on the quality of Herba Epimedii. The proposed method is simple, effective, and suitable for the evaluation of this traditional Chinese medicine.     

Identification of Metabolites of Epimedin A in Rats Using UPLC/Q–TOF–MS

Herba Epimedii (Epimedium) is a kind of tonic herb, widely used in China. Epimedin A is a major component of Herba Epimedii with bioactivities. Analysis of the metabolic profile in vivo plays a pivotal role in understanding how traditional Chinese medicine works. And the metabolites of epimedin A might influence the effects of Herba Epimedii. Moreover, the metabolic routes of epimedin A provide an important basis for safety evaluation. Until now, little has been known about the metabolism of epimedin A. The current study was designed to characterize the metabolic pathways of epimedin A in vivo. The metabolites in rat plasma, bile, feces, and urine were identified by UPLC/Q–TOF–MS analysis. A total of 27 metabolites from epimedin A were detected or tentatively identified. The major metabolic processes were hydrolysis, hydrogenation, hydroxylation, dehydrogenation, demethylation, and conjugation with glucuronic acid and different sugars. The present study revealed the metabolic pathways of epimedin A in rat for the first time, and epimedin A could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of epimedin A. In addition, the results of this work have shown the feasibility of the UPLC/Q–TOF–MS approach for rapid and reliable characterization of metabolites.     

Metabolite profiling of four major flavonoids of Herba Epimedii in zebrafish

The zebrafish model organism was applied first in a metabolic study of icariin, baohuoside I, epimedin A and epimedin C, which are flavonoids in Herba Epimedii. Metabolites of these compounds in zebrafish after exposure for 24 h were identified by HPLC-ESI-MS, whereby the separation was performed with a Zorbax C-18 column using a gradient elution of 0.05% formic acid acetonitrile-0.05% formic acid water. The quasi-molecular ions of compounds were detected in simultaneous negative and positive ionization modes. Metabolic products of icariin and epimedin C via cleavage of glucose residue instead of rhamnose residues were found, which coincided with the results using regular metabolic analysis methods. In addition, the zebrafish model was used to predict the metabolism of the trace component epimedin A, whose metabolic mechanisms haven't been clearly elucidated with the current metabolism model. The metabolic pathway of epimedin A in zebrafish was similar to those of its homologue icariin and epimedin C. Our study demonstrated that the zebrafish model can successfully imitate the current models in elucidating metabolic pathways of model flavonoids, which has advantages of lower cost, far less amount of compound needed, easy set up and high performance. This novel model can also be applied in quickly predicting the metabolism of Chinese herb components, especially trace compounds.     

Metabolite profiles of epimedin C in rat plasma and bile by ultra‐performance liquid chromatography coupled with quadrupole‐TOF‐MS

Epimedin C is one of the major bioactive constituents of Herba Epimedii. In this study, the metabolite profiles of epimedin C in rat plasma and bile were qualitatively investigated, and the possible metabolic pathways of epimedin C were subsequently proposed. After oral administration of epimedin C at a single dose of 80 mg/kg, rat biological samples were collected and pretreated by protein precipitation. Then these pretreated samples were injected into an Acquity UPLC BEH C₁₈ column and detected by ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry. In all, 12 metabolites were identified in the biosamples. Of these, eight, two from plasma and six from bile, are, to our knowledge, reported here for the first time. The results indicated that epimedin C was metabolized via desugarization, dehydrogenation, hydrogenation, dehydroxylation, hydroxylation, demethylation and glucuronidation pathways in vivo. Thus, this study revealed the possible metabolite profiles of epimedin C in rat plasma and bile. Copyright © 2014 John Wiley & Sons, Ltd.     

A novel icariin type flavonoid from Epimedium pseudowushanense

A novel icariin type flavonoid glycoside with a malonaldehydic acid intramolecular ester and two known flavonoid glycosides were isolated from Epimedium pseudowushanense. Their structures were elucidated on the basis of spectroscopic analysis and comparison of their data to the values reported in the literatures. The anti-inflammatory activities of these compounds icariin 3′′′-O-malonaldehydic acid intramolecular 1′′′′, 2′′′ ester (1), icariin (2) and epimedin C (3) were tested. The results indicated that compounds 1, 2 and 3 showed maximal inhibitory ratio of 27.91, 44.80 and 46.61%, respectively in in vitro anti-inflammatory activity on LPS-induced TNF-α secretion in RAW264.7 cells. Compounds icariin (2) and epimedin C (3) were found to inhibit the secretion of TNF-α to a comparable degree as quercetin.     

Effect of the Peptidic Scaffold in Copper(II) Coordination and the Redox Properties of Short Histidine‐Containing Peptides

A linear decapeptide containing three His and one Asp residues and a β‐turn‐inducing dProPro unit was synthesised. A detailed potentiometric, mass spectrometric and spectroscopic study showed that at a 1:1 ratio of C_Cu/C_peptide this peptide formed a major [CuH(O_dPro?Asp)]²⁺ species (pH range 5.5–7.0), in which the Cu²⁺ ion was bound to the His and Asp residues in square‐planar or square‐pyramidal geometries. The stability constant corrected for protonated species (log K*=9.33) is almost equal to the value obtained for the parent [CuH(O?Asp)]²⁺ species (log K*_CuH(O‐Asp)=9.28), but lower than that obtained for the cyclic [CuH(C?Asp)]²⁺ complex (log K*_CuH(C‐Asp)=10.79) previously published. Thus, the replacement of the ProGly unit by the stronger β‐turn‐inducing dProPro unit did not generate a more stable copper(II) species, although the O_dPro?Asp peptide was structured in solution, as shown by circular dichroism (CD) spectroscopy. Interestingly, the calculated value of K_eff showed that this peptide behaved similarly to the O?Asp or C?Asp counterparts, depending on the pH value. The cyclic voltammetry data indicated that the most easily reducible species were [CuH(O?Asp)]²⁺ (E′⁰=262 mV versus a normal hydrogen electrode (NHE)) and [CuH(O_dPro?Asp)]²⁺ (E′⁰=294 mV versus NHE) complexes, the peptidic scaffolds of which are open. A lower value was obtained for [CuH(C?Asp)]²⁺ (E′⁰=24 mV versus NHE). A different degree of non‐reversibility was observed for the three copper(II) complexes; this could reflect a different degree of flexibility in their respective peptidic scaffolds.     

Thiomaltol‐Based Organometallic Complexes with 1‐Methylimidazole as Leaving Group: Synthesis,Stability, and Biological Behavior

Thiomaltol, a potential S,O‐coordinating molecule, has been utilized for the complexation of four different organometallic fragments, yielding the desired Ru^II, Os^II, Rh^III, and Ir^III complexes having a “piano‐stool” configuration. In addition to the synthesis of these compounds with a chlorido leaving group, the analogous 1‐methylimidazole derivatives have been prepared, giving rise to thiomaltol‐based organometallics with enhanced stability under physiological conditions. The organometallic compounds have been characterized by NMR spectroscopy, elemental analysis, and X‐ray diffraction analysis. Their behavior in aqueous solution and their interactions with certain amino acids have been studied by ESI mass spectrometry. Their pH‐dependent stability has been investigated by ¹H NMR in aqueous solution, and their cytotoxicity against three different cancer cell lines has been investigated. Furthermore, their capacity as topoisomerase IIα inhibitors as well as their effect on the cell cycle distribution and reactive oxygen species (ROS) generation have been elucidated.     

Functionalization of superparamagnetic Fe3O4@SiO2 nanoparticles with a Cu(II) binuclear Schiff base complex as an efficient and reusable nanomagnetic catalyst for N‐arylation of α‐amino acids and nitrogen‐containing heterocycles with aryl halides

Fe₃O₄@SiO₂ nanoparticles was functionalized with a binuclear Schiff base Cu(II)‐complex (Fe₃O₄@SiO₂/Schiff base‐Cu(II) NPs) and used as an effective magnetic hetereogeneous nanocatalyst for the N‐arylation of α‐amino acids and nitrogen‐containig heterocycles. The catalyst, Fe₃O₄@SiO₂/Schiff base‐Cu(II) NPs, was characterized by Fourier transform infrared (FTIR) and ultraviolet‐visible (UV‐vis) analyses step by step. Size, morphology, and size distribution of the nanocatalyst were studied by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scatterings (DLS) analyses, respectively. The structure of Fe₃O₄ nanoparticles was checked by X‐ray diffraction (XRD) technique. Furthermore, the magnetic properties of the nanocatalyst were investigated by vibrating sample magnetometer (VSM) analysis. Loading content as well as leaching amounts of copper supported by the catalyst was measured by inductive coupled plasma (ICP) analysis. Also, thermal studies of the nanocatalyst was studied by thermal gravimetric analysis (TGA) instrument. X‐ray photoelectron spectroscopy (XPS) analysis of the catalyst revealed that the copper sites are in +2 oxidation state. The Fe₃O₄@SiO₂/Schiff base‐Cu(II) complex was found to be an effective catalyst for C–N cross‐coupling reactions, which high to excellent yields were achieved for α‐amino acids as well as N‐hetereocyclic compounds. Easy recoverability of the catalyst by an external magnet, reusability up to eight runs without significant loss of activity, and its well stability during the reaction are among the other highlights of this catalyst.     

Conformational analysis of the disaccharide methyl α‐d‐mannopyranosyl‐(1→3)‐2‐O‐acetyl‐β‐d‐mannopyranoside monohydrate

The crystal structure of methyl α‐d ‐mannopyranosyl‐(1→3)‐2‐O‐acetyl‐β‐d ‐mannopyranoside monohydrate, C₁₅H₂₆O₁₂·H₂O, ( II ), has been determined and the structural parameters for its constituent α‐d ‐mannopyranosyl residue compared with those for methyl α‐d ‐mannopyranoside. Mono‐O‐acetylation appears to promote the crystallization of ( II ), inferred from the difficulty in crystallizing methyl α‐d ‐mannopyranosyl‐(1→3)‐β‐d ‐mannopyranoside despite repeated attempts. The conformational properties of the O‐acetyl side chain in ( II ) are similar to those observed in recent studies of peracetylated mannose‐containing oligosaccharides, having a preferred geometry in which the C2—H2 bond eclipses the C=O bond of the acetyl group. The C2—O2 bond in ( II ) elongates by ～0.02 Å upon O‐acetylation. The phi (?) and psi (ψ) torsion angles that dictate the conformation of the internal O‐glycosidic linkage in ( II ) are similar to those determined recently in aqueous solution by NMR spectroscopy for unacetylated ( II ) using the statistical program MA′AT, with a greater disparity found for ψ (Δ = ～16°) than for ? (Δ = ～6°).     

Efficient method for screening and identification of radical scavengers in the leaves of Olea europaea L

In this article, an efficient method was developed to screen, isolate and identify the major radical scavengers in the leaves of Olea europaea L. by DPPH‐HPLC‐DAD, HSCCC and NMR. The method of DPPH‐HPLC‐DAD was used to screen the major radical scavengers. It was found that three major constituents (A, B, C) in the extract of the leaves of O. europaea L. possessed potential antioxidant activities. In order to identify the chemical structures of those compounds, the HSCCC method with a two‐phase solvent system composed of petroleum ether–ethyl acetate–water at an optimized volume ratio of 6:600:700 (v/v/v) together with column chromatography was developed to isolate and purify the active compounds. Pure compounds A (225 mg), B (10 mg) and C (12 mg) with purities 92.6, 95.1 and 96.4%, respectively, were obtained from the crude sample (500 mg). Their structures were identified as oleuropein (A), luteolin‐7‐O‐glucoside (B) and verbascoside (C) by ¹H‐NMR and ¹³C‐NMR. Copyright © 2010 John Wiley & Sons, Ltd.     

Twist‐chair conformation of the tetraoxepane ring remains unchanged in tetraoxaspirododecane diamines

A detailed structural analysis has been performed for N,N′‐bis(4‐chlorophenyl)‐7,8,11,12‐tetraoxaspiro[5.6]dodecane‐9,10‐diamine, C₂₀H₂₂Cl₂N₂O₄, (I), N,N′‐bis(2‐fluorophenyl)‐7,8,11,12‐tetraoxaspiro[5.6]dodecane‐9,10‐diamine, C₂₀H₂₂F₂N₂O₄, (II), and N,N′‐bis(4‐fluorophenyl)‐7,8,11,12‐tetraoxaspiro[5.6]dodecane‐9,10‐diamine, C₂₀H₂₂F₂N₂O₄, (III). The seven‐membered ring with two peroxide groups adopts a twist‐chair conformation in all three compounds. The lengths of the C—N and O—O bonds are slightly shorter than the average statistical values found in the literature for azepanes and 1,2,4,5‐tetraoxepanes. The geometry analysis of compounds (I)–(III), the topological analysis of the electron density at the (3, ?1) bond critical points within Bader's quantum theory of `Atoms in molecules' (QTAIM) and NBO (natural bond orbital) analysis at the B3LYP/6‐31G(d,2p) level of theory showed that there are n_O→σ*(C—O), n_N→σ*(C—O) and n_O→σ*(C—N) stereoelectronic effects. The molecules of compounds (I) and (III) are packed in the crystals as zigzag chains due to strong N—H…O and C—H…O hydrogen‐bond interactions, whereas the molecules of compound (II) form chains in the crystals bound by N—H…O, C—H…π and C—H…O contacts. All these data show that halogen atoms and their positions have a minimal effect on the geometric parameters, stereoelectronic effects and crystal packing of compounds (I)–(III), so that the twist‐chair conformation of the tetraoxepane ring remains unchanged.     

Comparative pharmacokinetic study of nine bioactive components in osteoarthritis rat plasma using ultra-performance liquid chromatography–tandem mass spectrometry after single and combined oral administration of Epimedii Folium and Chuanxiong Rhizoma extracts

The herb pair Epimedii Folium–Chuanxiong Rhizoma (EF–CR), derived from the classical traditional Chinese medicine ‘Xian Ling Pi San’, has a distinctive compatibility therapeutic profile and is clinically safe and effective. This study aimed to investigate and compare the pharmacokinetic characteristics of nine analytes in osteoarthritis (OA) rat plasma after the oral administration of EF, CR or a combination of these two herbs. We developed an ultra-performance liquid chromatography method coupled with quadrupole linear ion-trap mass spectrometry to simultaneously quantify and assess the pharmacokinetics of icariin, epimedin A, epimedin B, epimedin C, icariside I, icariside II, ferulic acid, ligustilide and senkyunolide A of the EF–CR pair in the plasma of osteoarthritic rats. The pharmacokinetic parameters showed that the absorption of multiple components was significantly enhanced and residence time was prolonged in the EF–CR group (P < 0.05) compared to the single-herb group. These parameters revealed that the combination of EF and CR exhibited synergistic effects of the nine bioactive components, suggesting the potential application of the EF–CR combination for the treatment of OA.     

Validation of analytical procedures using HPLC‐ELSD to determine six sesquiterpene lactones in Eremanthus species

Eremanthus species display sesquiterpene lactones with therapeutic potential. We are proposing the development of a new analytical method that has been completely validated to qualify Eremanthus species and its main lactones in raw material using HPLC‐ELSD. For the sample preparation, 10.0 mg of powdered Eremanthus leaves was extracted with a 5 mL MeOH/H₂O (9:1 v/v) solution containing scopoletin at 140 µg/mL as the internal standard. For the separation of eight compounds, six of which were lactones, one internal (IS) and one secondary standard were performed utilizing monolithic columns with a nonlinear gradient. The selectivity, stability, precision and matrix effects parameters showed values of RSD of <10%. The six lactones and scopoletin (IS) were recovered with a proportion between 74 and 90% with accuracy represented by error at ?25.41%. The linear dynamic range was obtained between 10.0 and 310.0 µg/mL for all compounds with r² > 0.9987. The limits of detection and quantitation ranged from 2.00 to 6.79 µg/mL and from 6.00 to 20.40 µg/mL, respectively. Assessing the robustness study, this method can be used in inter‐laboratory studies. Using the HPLC‐ELSD method, six sesquiterpene lactones including 4β,5‐dihydro‐2′,3′‐dihydroxy‐15‐deoxy‐goyazensolide, goyazensolide, 4β,5‐dihydro‐2′,3′‐epoxy‐15‐deoxy‐goyazensolide, centratherin, 4β,5‐dihydro‐15‐deoxy‐goyazensolide and lychnofolide, were detected and quantified from distinct Eremanthus species, which were collected in different regions. Copyright © 2015 John Wiley & Sons, Ltd.