•Thirty eight constituents in ethyl acetate fraction of Gastrodia elata were identified or tentatively characterized.•Forty compounds including sixteen prototypes and twenty four metabolites were detected in dosed rat plasma sample.•The main metabolic “soft spots” was hydroxyl or carboxyl group.The chemical fingerprint and metabolic profile of traditional Chinese medicine is very complicated and has been a great challenge. In the present study, chemical fingerprint of ethyl acetate fraction of Gastrodia elata (EtAcGE) and metabolic profile of rat plasma sample after intragastric administration of EtAcGE (2.5 g/kg) were investigated using ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC/Q-TOF MS). A total of 38 chemical constituents of EtAcGE were identified by comparing their retention time, accurate molecular mass and characteristic fragment ions with those of references, or tentatively characterized by comparing molecular formula, fragment ions with that of known compound or information available in literature. And 40 compounds were detected in dosed rat plasma sample, including 16 prototypes and 24 metabolites underwent metabolic process of glucuronidation, glucosylation, sulfation, methylation, hydroxylation, dehydrogenation or mixed modes. The metabolic “soft spots” was hydroxyl or carboxy group. This is the first research for chemical fingerprint and metabolic profile of EtAcGE, which lay a foundation for the further investigation of EtAcGE.

Estradiol is a major drug used clinically to alleviate osteoporosis, partly through inhibition of the activity of osteoclasts, which play a crucial role in bone resorption. So far, little is known about the effects of estradiol on osteoclast metabolism. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC/MS)-based metabolomics strategy was used to investigate the metabolite response to 17β-estradiol in mouse osteoclast RAW264.7, a commonly used cell model for studying osteoporosis. Our results showed that the application of estradiol altered the levels of 27 intracellular metabolites, including lysophosphatidylcholines (LysoPCs), other lipids and amino acid derivants. The changes of all the 27 metabolites were observed in the study of estradiol induced osteoclast proliferation inhibition (1 μM estradiol applied), while the changes of only 18 metabolites were observed in the study of differentiation inhibition (0.1 μM estradiol applied). Further pathway impact analysis determined glycerophospholipid metabolism as the main potential target pathway of estradiol, which was further confirmed by LCAT (phosphatidylcholine-sterol acyltransferase) activity changes and lipid peroxidative product (MDA, methane dicarboxylic aldehyde) changes caused by estradiol. Additionally, we found that estradiol significantly decreased intracellular oxidative stress during cell proliferation but not during cell differentiation. Our study suggested that estradiol generated a highly condition-dependent influence on osteoclast metabolism.

An applicable affinity capture-based method was developed for screening of enzyme inhibitors from complex plant extracts directly. By elimination of false positives, three non-competitive α-glucosidase inhibitors were fished out from 400 μL green tea extract rapidly, using affinity capture of immobilized α-glucosidase combined with UHPLC-QTOF MS analysis.

•Ethanol–ammonium sulfate–water aqueous two-phase system for separation of nucleosides on multilayer coil HSCCC.•The polarity of ethanol–ammonium sulfate–water system was between that of 1-butanol–water and PEG–ammonium sulfate–water system.•The retention of stationary phase on multilayer coil HSCCC was 26–71% for four elution modes.Hydrophilic organic/salt-containing aqueous two-phase system composing of ethanol, water and ammonium sulfate for separation polar compounds was investigated on multilayer coil associated with J-type HSCCC devices. Compared to the classical polar solvent system based on 1-butanol–water or PEG1000–ammonium sulfate–water, the water content of upper phase in ethanol–ammonium sulfate–water systems was from 53.7% to 32.8% (wt%), closed to PEG1000–ammonium sulfate–water aqueous two-phase systems and higher than 1-butanol–water (22.0%, wt%). Therefore, the polarity of ethanol–ammonium sulfate–water is in the middle of 1-butanol–water and PEG–ammonium sulfate–water system, which is quite good for separating polar compounds like phenols, nucleosides and amino acids with low partition coefficient in 1-octanol–water system. The retention of stationary phase in four elution mode on type-J counter-current chromatography devices with multilayer coil column changed from 26% to 71%. Hydrodynamic trend possess both intermediate and hydrophilic solvent system property, which closely related to the composition of solvent system. The applicability of this system was demonstrated by successful separation of adenosine, uridine guanosine and cytidine.

•A new preparative recycle HPLC system with a multifunctional trap was built up.•The system was designed for separating a low abundant compound from a complex mixture.•Absorption capability of the trap can be modified by online mobile phase adjusting.•The system could work against the problems of volume overloading and solvent effect.•The system could speed up the drying procedure of the final product.A novel recycle preparative HPLC system was developed to repeatedly enrich and purify low-abundance compounds from complex samples. The recycle separation involves injecting a large volume of the sample through a trap column, releasing the absorbed sample for preparative HPLC separation, and then capturing the target component in the same trap column for the next separation. After several cycles, the final purified target compound absorbed in the trap column is eluted with a small volume of solvent. The capture and recycle procedure was realized by adjusting the strength of the mobile phase using an auxiliary pump and switching the two valves connecting the trap and preparative columns. Compared with standard HPLC or existing recycle chromatography, this system not only reduces the solvent side-effects and sample volume overloading during injection but also avoids peak–broadening and sensitivity loss during the recycle runs. Moreover, the final purified products can be easily concentrated. With the aid of this system, a 10% polyphenol in extracts from Saussurea involucrata cultured cells reached >95% purity with more than 95% recovery after three recycle purifications, and a 1% unstable epimer impurity in the raw material of the drug silybin reached >95% purity with 75% recovery after a six-cycle separation.

•Two new phenolic acids were obtained from cultured cells of Saussurea involucrata.•The new compounds belong to caffeoyl quinic acids with an additional maloyl group.•The new phenolic acids showed strong radical scavenging abilities.Two new phenolic acids, 1, 5-O-dicaffeoyl-3-O-(4-maloyl)-quinic acid (1) and 3, 5-di-O-caffeoyl-1-O-(2-O-caffeoyl-4-maloyl)-quinic acid (2), were isolated from cultured cells of Saussurea involucrata. Their structures were elucidated using 2D NMR spectroscopy and MS. Further in vitro bioactive investigations demonstrated that 3, 5-di-O-caffeoyl-1-O-(2-O-caffeoyl-4-maloyl)-quinic acid (2) had significant scavenging activities against radicals 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) and 2, 2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS).

•A simple, rapid and sensitivity UHPLC-FLD method was developed.•Low LOD and high recovery achieved with good precision and accuracy.•The method has been successfully applied to study parishin pharmacokinetics.A simple, rapid and sensitive ultra high performance liquid chromatography with fluorescence detection (UHPLC-FLD) method was developed and validated for quantification of parishin and its metabolites in rats. Plasma samples were prepared by protein precipitation and then analyzed using UHPLC-FLD system. Repeated optimization showed that parishin and its metabolites, including gastrodin, p-hydroxybenzyl alcohol, parishin B and parishin C, could be sensitively detected based on the autofluorescence when excitation and emission wavelengths were set at 225 nm and 295 nm, respectively. The limit of detections (LODs) of GAS, HBA, PB, PC and PA reached 0.6, 0.8, 1, 1 and 1 ng/mL, respectively. The linearity for all targets was within the range 2.5–5000 ng/mL and the correlation coefficient (r2) was larger than 0.999. Importantly, our method was almost free from matrix effects and the recoveries were higher than 80%. Additionally, our method also had high precision and accuracy for all analytes, presenting RSDs and REs within ±6% and ±14%, respectively. Finally, the validated UHPLC-FLD method was successfully applied for studying the pharmacokinetics of parishin following intragastrically administration in rats.

A rapid and sensitive method for simultaneous quantitative determination of terpenoids in Tripterygium wilfordii Hook F (TwHF) was developed by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). In this method, five major terpenoids, including triptolide, triptonide, demethylzelastral, tripterine and wilforlide A, with large content range in TwHF, were determined simultaneously in a UHPLC run of 4.5 minutes. The intra-day and inter-day precisions of five terpenoids were less than 4.58% and 4.82%, respectively, and the mean recovery rates obtained were in the range of 97.2% to 104.5%. All calibration curves showed good linearity (r2 > 0.996) from 20 ng mL−1 and 20000 ng mL−1. The limit of detection (LOD) and limit of quantitation (LOQ) were less than 5.7 ng mL−1 and 19.0 ng mL−1. Compared with previous studies, this method could be employed for accurate quantification of five terpenoids in different parts and products of TwHF in a very short time after simple ultrasonic extraction. The results indicated that this method would provide a rapid and sensitive way for evaluating the quality of the TwHF and its products.

Bioassay-directed phytochemical study of the Jerusalem artichoke (Helianthus tuberosus L.) leaves led to the isolation of a new sesquiterpene lactone of 3-Hydroxy-8β-tigloyloxy-1,10-dehydroariglovin (1), ten known sesquiterpene lactones (2–11) and two known flavones (12–13). Their chemical structures were elucidated on the basis of NMR (1D and 2D) and mass spectroscopic analysis. The cytotoxic activities of those compounds were subsequently tested against the MCF-7, A549 and HeLa cancer cells lines. The results indicated that sesquiterpene lactones 1–11 exhibited consistent cytotoxicity against all three cancer cell lines, while flavones 12 and 13 showed selective inhibitory activity against HeLa cell lines. Among them, compound 3 exhibited significant growth inhibitory activity against all three cell lines. The IC50 values of compound 3 against MCF-7, A549 and HeLa were 1.97 ± 0.04, 7.79 ± 0.44, 9.87 ± 0.20 μg/ml, respectively. In addition, some structure–activity relationships of these sesquiterpene lactones for cytotoxicity were explored and summarized in this study.Graphical abstractA new sesquiterpene lactone, 3-Hydroxy-8β-tigloyloxy-1,10-dehydroariglovin (1) along with ten known sesquiterpene lactones (2–11) and two flavones (12–13) were isolated from the leaves of H. tuberosus. Compounds 1–11 showed potent cytotoxicity against selected human cancer cell lines.Highlights► The leaves of Jerusalem artichoke possess strong anticancer activities. ► A new sesquiterpene lactone was isolated and characterized. ► Sesquiterpene lactones are the major active compounds. ► Structure–activity relationships of sesquiterpene lactones were discussed.

•Seven strategies were reviewed to improve identification accuracy.•Construction of special database of MS and RI might be advisable.•Feature mining of MS and RI data in database benefits structure elucidation.•Mass spectral transfer improves the accuracy of identifying stereoisomers.In this review, we summarize seven strategies for structure elucidation of small molecules based on several aspects: discovery of spectral characteristics; construction of a special database; and elimination of retention-index variation and mass spectral differences.We also discuss trends and future perspectives of the structure elucidation of small molecules using data from gas chromatography coupled to mass spectrometry.

The total phenolic content and radical scavenging activities of Jerusalem artichoke (Helianthus tuberosus L.) leaves were investigated. Results indicated that the ethyl acetate fraction contained the highest total phenolic content (266.69 ± 2.51 mg GAE/g dry extract) accompanied with strongest free radical scavenging abilities. Following an in vitro radical scavenging activity-guide fractionation procedure, six phenolic compounds which strongly quenched free radicals were separated from ethyl acetate fraction. Among them, 3-O-caffeoylquinic acid and 1,5-dicaffeoylquinic acid played a dominant role due to their strong free radical scavenging abilities and their high contents. The content of 3-O-caffeoylquinic acid in n-butanol fraction was 74.58 ± 1.05 mg/g, while 1,5-dicaffeoylquinic acid in ethyl acetate fraction was 104.51 ± 2.86 mg/g. The results imply that the leaves of Jerusalem artichoke might be a potential source of natural antioxidants.Highlights► The leaves of Jerusalem artichoke possess strong radical scavenging activities. ► The high antiradical activities ascribe to the abounding phenolic compounds. ► Chlorogenic acid and 1,5-dicaffeoylquinic acids are the major active compounds.

Metabolite profiles and biomarker discovery benefit clinical diagnosis of type 2 diabetes mellitus (T2DM) and reflection of metabolite variations in pathophysiology. Fatty acids play an important role in the occurrence and development of diabetes mellitus. The fasting plasma glucose (FPG) values of T2DM patients are partially adjusted to normal range (FPG < 7.0 mmol/L) after the intervention of drug or other treatments. However, whether these patients return to normal is still unknown. In this study, graphical index of separation (GIOS) was employed to discover the potential biomarkers of fatty acids for T2DM and subsequently investigate whether the fatty acid differences exist between T2DM patients with low and high FPG values (low fasting plasma glucose, FPG < 7.0 mmol/L and high fasting plasma glucose, FPG ≥ 7.0 mmol/L). As a result, C16:0, C20:2, C20:5, C22:6 and FPG were selected as potential biomarkers, which could significantly decline the misdiagnosis rate of diabetes (from 35/98 to 0/98). Additionally, the partial least squares-discriminant analysis (PLS-DA) was also employed to identify fatty acids responsible for T2DM and subsequently build a discriminant model using significant variables. After Monte Carlo cross validation, the prediction rate of PLS-DA reaches to 89.1%. Moreover, the biomarkers selected by GIOS were compared with those selected by PLS-DA model. The almost accordant results indicate that GIOS is a simple but comprehensible method to screen biomarkers.Highlights► A strategy of plasma fatty acid profiles was employed to investigate T2DM. ► GIOS-PCA was applied to select biomarkers of T2DM patients and healthy control. ► PLS-DA was used to build discriminate model of selected variables for T2DM patients. ► C22:6, C20:5, 16:0 and C20:2 were biomarkers for assisting FPG in T2DM diagnosis. ► T2DM patients with different FPG values possess no significant metabolic difference.

The anti-tumor action of Taxol was investigated in the changes of amino-acids involved in tumor cell survival. By tracing the intracellular amino-acid profiles of HeLa cells treated with non-conditioned and three conditioned media (Taxol, l-alanine, and Taxol + l-alanine), it was observed that an alteration of amino-acid metabolism participates in Taxol-induced death of HeLa cells. The contents of 18 out of 21 detected amino-acids are 5–95% and the ones of lysine and methionine are 158 and 117% of the corresponding contents in the control after treatment with Taxol for 24 h, respectively. Addition of l-alanine inhibited cell apoptosis upon Taxol treatment by partially blocking the increase of lysine and methionine and reversing decrease trend of alanine, glycine, and glutamic acid. These results suggest that interference of amino-acid metabolism might be an important mechanism of Taxol cytotoxicity.

A selective and sensitive liquid chromatography coupled with triple stage quadruple tandem mass spectrometry (HPLC/TSQ-MS/MS) was developed and validated for simultaneous quantification of calycosin-7-O-β-d-glycoside (CCSG), formononetin-7-O-β-d-glycoside (Ononin) and (6R,10R)-9,10-dimethoxypterocarpan-3-O-β-d-glycoside (DPG) in rabbit plasma. Plasma samples were extracted with solid-phase extraction (SPE), separated on an Inertsil ODS-3 column and detected by tandem mass spectrometry with electrospray ionization (ESI) interface in positive selective reaction monitoring (SRM) mode. 3,7,8-Trimethoxy-xanthone-1-O-primaverose was used as internal standard (IS) for quantitative measurement. For each analyte, one major product ion was chosen and used for screening of it. Calibration curves were generated over the range of 2–1000 ng mL−1 with the correlation coefficients greater than 0.99 by using a weighted (1/χ) least squares linear regression. The method had the lower limit quantification of 0.15, 0.21 and 0.19 for CCSG, Ononin and DPG, respectively, with precision less than 20%. The intra- and inter-day precisions ranged from 2.48 to 6.38% and 4.81 to 11.78% (R.S.D.%), respectively. This assay is suitable for determining the above three trace glycosides in rabbit plasma simultaneously and thus investigating the pharmacokinetics of glycosides from Astragalus mongholicus extract in rabbits.

Ethnopharmacological relevanceEcliptae herba, also known as “Mo-Han-Lian”, has long been used in China to nourish Kidney and thereafter strengthen bones. Accumulating evidence indicates that extracts of Ecliptae herba have antiosteoporotic effect. However, the effective compounds and cellular mode of action are still unclear. To investigate the effect of ethyl acetate extract of Ecliptae herba (EAE) and its component wedelolactone on proliferation and differentiation of preosteoclastic RAW264.7 cells as well as proliferation of bone marrow stromal cells (BMSC).Materials and methodsRAW264.7 and BMSC were examined for proliferation by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Tartrate-resistant acid phosphatase (TRAP) activity of RAW264.7 was measured by using p-nitrophenyl sodium phosphate (pNPP) assay after the cells were treated with 30 ng/ml receptor activator for nuclear factor-κ B ligand (RANKL) plus various concentrations of EAE, wedelolactone or alendronate. The formation of multinucleated TRAP-positive RAW264.7 cells was observed by using a TRAP-staining kit.ResultsTreatment of RAW264.7 cells with EAE at high doses (20 µg/ml and 40 µg/ml) or wedelolactone at 10 µg/ml resulted in a decrease in proliferation of RAW264.7 cells. Low doses of EAE (5, 10 µg/ml) and wedelolactone (2.5 µg/ml) inhibited RANKL-induced TRAP activity by 20.3%, 37.9%, and 48.3%. The inhibitory effect of wedelolactone is more potent than that of alendronate, an anti-resorptive drug. Morphological changes revealed that 5 µg/ml EAE and 2.5 µg/ml wedelolactone reduced the number of multinucleated osteoclast-like cells. At the high doses, EAE (20 µg/ml) and wedelolactone (10 µg/ml) inhibited the growth of BMSC.ConclusionsEAE and its component wedelolactone inhibited osteoclast RAW264.7 proliferation and differentiation at the low doses, but at the high doses, showed cytotoxic effect on BMSC. These results indicated that EAE and wedelolatone might be potential alternative therapy for osteoporosis.Download high-res image (174KB)Download full-size image

Increasing evidence shows the therapeutic superiority of herbal extracts in comparison to isolated single constituents. One of the reasons may be attributed to the synergy effect of compound combinations. Flavonoids from Herba Epimedii have been shown to have therapeutic effect against bone loss. Our previous study showed that Icariside II inhibited pre-osteoclast RAW264.7 growth. The aim of this study was to investigate whether the activity of Icariside II is synergized by other components of Herba Epimedii. The inhibitory activity of Icariside II was significantly enhanced in the presence of the extract of Herba Epimedii (EHE) at the ratio of 1:1, 1:5 and 1:10. Icaritin, another flavonoid constituent, was shown here to inhibit RAW264.7 growth in a dose-dependent manner. Further, we found that Icariside II, together with Icaritin, synergistically inhibited RAW264.7 growth. The synergistic effect is significant when the ratio of Icariside II and Icaritin was 10:1, 5:1, 1:1, 1:2, and 1:5, respectively. In conclusion, Icaritin were an active component. The inhibitory activity of Icariside II on pre-osteoclast RAW264.7 growth was synergized by Icaritin, which maybe contribute to the efficiency of Herba Epimedii extract on curing bone-related diseases, such as osteoporosisDownload high-res image (121KB)Download full-size image

•Metabolomics method was used to study time-selected combination of LWDH and JKSQ.•GIOS was taken as a tool to select kidney deficiency and aging related biomarkers.•PCP was employed to evaluate the influence of TCM on endogenous metabolites.•Metabolic pathway function analysis was applied to analyzed biomarkers.•This study provides a clue for the study of time-selected combination of herbs.Jinkui Shenqi pill (JKSQ) and Liuwei Dihuang (LWDH) pills are ancient traditional Chinese medicines (TCMs), which are usually used for the treatment of kidney deficiency for thousands of years in China. Time-selected combination of LWDH and JKSQ pills in treating kidney deficiency and aging is one of the features of traditional Chinese medicines (TCMs). However, the molecular mechanisms of time-selected combination remain unclear. In this work, UHPLC–QTOF/MS based metabolomics research was conducted to evaluate the therapeutic effect of LWDH, JKSQ pills and their combinations on kidney deficiency in Sprague–Dawley rats induced by d-galactose and Dexamethasone. Based on peak areas of serum extracts, analysis of variance (ANOVA) and graphical index of separation (GIOS) were employed to select the significant variables for kidney deficiency and aging and principal component projection (PCP) was subsequently applied to evaluate the influence of drugs on endogenous metabolites. 10 endogenous metabolites from 22 important ions were identified via database search. The score plot of PCA shows that nourishing Yang–nourishing Yin group shows the strongest rehabilitation for metabolic disorder induced by kidney deficiency and aging, which is consistent with the classic theories of traditional Chinese medicine. Moreover, metabolic pathway function analysis indicates that kidney deficiency and aging might possess closed relationships with lipid metabolism and energy metabolism. In this work, the change trends of potential biomarkers after administration provide molecular evidence for combined administration of Jinkui Shenqi pill in the morning and Liuwei Dihuang pill at night for the patients with kidney deficiency. The method proposed in this study may provide inspiration for evaluation of the therapeutic effect of Chinese medicines by comparing the rehabilitation of potential biomarkers.

A rapid and sensitive method for simultaneous quantitative determination of terpenoids in Tripterygium wilfordii Hook F (TwHF) was developed by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). In this method, five major terpenoids, including triptolide, triptonide, demethylzelastral, tripterine and wilforlide A, with large content range in TwHF, were determined simultaneously in a UHPLC run of 4.5 minutes. The intra-day and inter-day precisions of five terpenoids were less than 4.58% and 4.82%, respectively, and the mean recovery rates obtained were in the range of 97.2% to 104.5%. All calibration curves showed good linearity (r2 > 0.996) from 20 ng mL−1 and 20000 ng mL−1. The limit of detection (LOD) and limit of quantitation (LOQ) were less than 5.7 ng mL−1 and 19.0 ng mL−1. Compared with previous studies, this method could be employed for accurate quantification of five terpenoids in different parts and products of TwHF in a very short time after simple ultrasonic extraction. The results indicated that this method would provide a rapid and sensitive way for evaluating the quality of the TwHF and its products.

An applicable affinity capture-based method was developed for screening of enzyme inhibitors from complex plant extracts directly. By elimination of false positives, three non-competitive α-glucosidase inhibitors were fished out from 400 μL green tea extract rapidly, using affinity capture of immobilized α-glucosidase combined with UHPLC-QTOF MS analysis.