•An integral strategy was developed for systemic screening and identifying of metabolites from multiple biological samples.•The data processing method of combination of MMDF and tool Progenesis QI based on UPLC-LTQ-Orbitrap MS was applied.•The workload of metabolites identification with the new approach decreased 46%.•67 metabolites of hirsutine in rat multiple biological samples were identified and an in-house library was built.•Comprehensive metabolic profile of hirsutine was proposed for the first time.Drug metabolites identification and construction of metabolic profile are meaningful work for the drug discovery and development. The great challenge during this process is the work of the structural clarification of possible metabolites in the complicated biological matrix, which often resulting in a huge amount data sets, especially in multi-samples in vivo. Analyzing these complex data manually is time-consuming and laborious. The object of this study was to develop a practical strategy for screening and identifying of metabolites from multiple biological samples efficiently. Using hirsutine (HTI), an active components of Uncaria rhynchophylla (Gouteng in Chinese) as a model and its plasma, urine, bile, feces and various tissues were analyzed with data processing software (Metwork), data mining tool (Progenesis QI), and HR-MSn data by ultra-high performance liquid chromatography/linear ion trap-Orbitrap mass spectrometry (U-HPLC/LTQ-Orbitrap-MS). A total of 67 metabolites of HTI in rat biological samples were tentatively identified with established library, and to our knowledge most of which were reported for the first time. The possible metabolic pathways were subsequently proposed, hydroxylation, dehydrogenation, oxidation, N-oxidation, hydrolysis, reduction and glucuronide conjugation were mainly involved according to metabolic profile. The result proved application of this improved strategy was efficient, rapid, and reliable for metabolic profiling of components in multiple biological samples and could significantly expand our understanding of metabolic situation of TCM in vivo.

•SFC was used to separate and isolate two pairs of 7-epimers of SOAs.•Acetonitrile stabilized two pairs of epimers and was used as modifier in SFC.•Two achiral UPC2 methods were established on the Torus 1-AA and Diol columns.•Preparative SFC enabled isolation of four SOA compounds with the purity >95%.•SFC provides a solution to preparation of high-purity reference standards.Increasing challenge arising from configurational interconversion in aqueous solvent renders it rather difficult to isolate high-purity tautomeric reference standards and thus largely hinders the holistic quality control of traditional Chinese medicine (TCM). Spiro oxindole alkaloids (SOAs), as the markers for the medicinal Uncaria herbs, can easily isomerize in polar or aqueous solvent via a retro-Mannich reaction. In the present study, supercritical fluid chromatography (SFC) is utilized to separate and isolate two pairs of 7-epimeric SOAs, including rhynchophylline (R) and isorhynchophylline (IR), corynoxine (C) and corynoxine B (CB), from Uncaria macrophylla. Initially, the solvent that can stabilize SOA epimers was systematically screened, and acetonitrile was used to dissolve and as the modifier in SFC. Then, key parameters of ultra-high performance SFC (ultra-performance convergence chromatography, UPC2), comprising stationary phase, additive in modifier, column temperature, ABPR pressure, and flow rate, were optimized in sequence. Two isocratic UPC2 methods were developed on the achiral Torus 1-AA and Torus Diol columns, suitable for UV and MS detection, respectively. MCI gel column chromatography fractionated the U. macrophylla extract into two mixtures (R/IR and C/CB). Preparative SFC, using a Viridis Prep Silica 2-EP OBD column and acetonitrile-0.2% diethylamine in CO2 as the mobile phase, was finally employed for compound purification. As a result, the purity of four SOA compounds was all higher than 95%. Different from reversed-phase HPLC, SFC, by use of water-free mobile phase (inert CO2 and aprotic modifier), provides a solution to rapid analysis and isolation of tautomeric reference standards for quality control of TCM.

•Two new depsides (1–2) were isolated from Salvia miltiorrhiza Bge, and the absolute configuration of 1 was determined by comparison of the experimental and calculated ECD spectra.•The radical-scavenging capacity and protective effects against H2O2-induced H9c2 cells of compounds 1–15 were evaluated.•The negative mode collision-induced dissociations (CID) of the new depsides (1-2) on an LTQ-Orbitrap Velos Pro hybrid mass spectrometer were investigated.Phytochemical investigation of the roots of Salvia miltiorrhiza led to isolations of two new depsides (1–2), along with thirteen known compounds (3–15). Their structures and relative stereochemistry were elucidated by NMR spectral (1H and 13C NMR, HSQC, HMBC), CD and HR-ESIMS data analyses. The absolute configuration of 1 was determined by comparison of the experimental and calculated ECD spectra. All the depsides (1 − 10) were found to exhibit stronger free radical scavenging capacity (ranging from 2.62 to 22.05 μM) than diterpenoids (11–15, IC50 > 100 μM), among which Salvianolic acid A and Salvianolic acid B were the most potent compounds. Additionally, most of depsides (1, 3, 5, 7, 8, 9, 10) possessed significant protective effects against H2O2-induced H9c2 apoptosis in low concentrations. The negative mode collision-induced dissociations of compound 1 and 2 were featured by the α-cleavage and β-cleavage to lose danshensu (198 Da) and caffeic acid (180 Da), respectively, while α,β-dihydroxybenzenes depsides (8-hydroxy) showed characteristic neutral elimination of H2O.Download high-res image (63KB)Download full-size image

AbstractEuphorbia kansui is a commonly used traditional Chinese medicine for the treatment of edema, pleural effusion, and asthma, etc. According to the previous researches, terpenoids in E. kansui possess various biological activities, e.g., anti-virus, anti-allergy, antitumor effects. In this work, twenty five terpenoids were isolated from E. kansui, including thirteen ingenane- and eight jatrophane-type diterpenoids (with two new compounds, kansuinin P and Q) and four triterpenoids. Eighteen of them were analyzed by MTS assay for in vitro anticancer activity in five human cancer cell lines. Structure-activity relationship for 12 ingenane-type diterpenoids in colorectal cancer Colo205 cells were preliminary studied. Significant anti-proliferation activities were observed in human melanoma cells breast cancer MDA-MB-435 cells and Colo205 cells. More than half of the isolated ingenane-type diterpenoids showed inhibitory activities in MDA-MB-435 cells. Eight ingenane- and one jatrophane-type diterpenoids possessed much lower IC50 values in MDA-MB-435 cells than positive control staurosporine. Preliminary structure-activity relationship analysis showed that substituent on position 20 was important for the activity of ingenane-type diterpenoids in Colo205 cells and substituent on position 3 contributed more significant biological activity of the compounds than that on position 5 in both MDA-MB-435 and Colo205 cells.

Aconiti Lateralis Radix Praeparata (Fuzi) is a commonly used traditional Chinese medicine in clinic for its potency in restoring yang and rescuing from collapse. Aconiti alkaloids, mainly including monoester-diterpenoidaconitines (MDAs) and diester-diterpenoidaconitines (DDAs), are considered to act as both bioactive and toxic constituents. In the present study, a feasible, economical, and accurate HPLC method for simultaneous determination of six alkaloid markers using the Single Standard for Determination of Multi-Components (SSDMC) method was developed and fully validated. Benzoylmesaconine was used as the unique reference standard. This method was proven as accurate (recovery varying between 97.5%−101.8%, RSD < 3%), precise (RSD 0.63%−2.05%), and linear (R > 0.999 9) over the concentration ranges, and subsequently applied to quantitative evaluation of 62 batches of samples, among which 45 batches were from good manufacturing practice (GMP) facilities and 17 batches from the drug market. The contents were then analyzed by principal component analysis (PCA) and homogeneity test. The present study provided valuable information for improving the quality standard of Aconiti Lateralis Radix Praeparata. The developed method also has the potential in analysis of other Aconitum species, such as Aconitum carmichaelii (prepared parent root) and Aconitum kusnezoffii (prepared root).

Fatty acids conjugates (FACs) are ubiquitous but found in trace amounts in the natural world. They are composed of multiple unknown substructures and side chains. Thus, FACs are difficult to be analyzed by traditional mass spectrometric methods. In this study, an integrated strategy was developed to global profiling and targeted structure annotation of FACs in complex matrix by LTQ Orbitrap. Dicarboxylic acid conjugated bufotoxins (DACBs) in Venenum bufonis (VB) were used as model compounds. The new strategy (abbreviated as HPNA) combined higher-energy C-trap dissociation (HCD) with product ion- (PI), neutral loss- (NL) based MSn (n ≥ 3) acquisition in both positive-ion mode and negative-ion mode. Several advantages are presented. First, various side chains were found under HCD in negative-ion mode, which included both known and unknown side chains. Second, DACBs with multiple side chains were simultaneously detected in one run. Compared with traditional quadrupole-based mass method, it greatly increased analysis throughput. Third, the fragment ions of side chain and steroids substructure could be obtained by PI- and NL-based MSn acquisition, respectively, which greatly increased the accuracy of the structure annotation of DACBs. In all, 78 DACBs have been discovered, of which 68 were new compounds; 25 types of substructure formulas and seven dicarboxylic acid side chains were found, especially five new side chains, including two saturated dicarboxylic acids [(azelaic acid (C9) and sebacic acid (C10)] and three unsaturated dicarboxylic acids (u-C8, u-C9, and u-C10). All these results greatly enriched the structures of DACBs in VB.

•A dereplication strategy is proposed and used to discover new alkaloids.•It combines MDF-oriented novelty classification and PIL-triggered target detection.•In-house MDF more exactly screens the target components and classifies the novelty.•A novel PIL-DE-CID/MS2-HCD/MS3 scan method is developed for target detection.•New indole alkaloids with unexpected structures are discovered.Discovery of new natural compounds is becoming increasingly challenging because of the interference from those known and abundant components. The aim of this study is to report a dereplication strategy, by integrating mass defect filtering (MDF)-oriented novelty classification and precursor ions list (PIL)-triggered high-resolution mass spectrometry analysis, and to validate it by discovering new indole alkaloids from the medicinal herb Uncaria sinensis. Rapid chromatographic separation was achieved on a Kinetex® EVO C18 column (<16 min). An in-house MDF algorithm, developed based on the informed phytochemistry information and molecular design, could more exactly screen the target alkaloids and divide them into three novelty levels: Known (KN), Unknown-but-Predicted (UP), and Unexpected (UN). A hybrid data acquisition method, namely PIL-triggered collision-induced dissociation-MS2 and high-energy C-trap dissociation-MS3 with dynamic exclusion on a linear ion trap/Orbitrap mass spectrometer, facilitated the acquisition of diverse product ions sufficient for the structural elucidation of both indole alkaloids and the N-oxides. Ultimately, 158 potentially new alkaloids, including 10 UP and 108 UN, were rapidly characterized from the stem, leaf, and flower of U. sinensis. Two new alkaloid compounds thereof were successfully isolated and identified by 1D and 2D NMR analyses. The varied ring E and novel alkaloid-acylquinic acid conjugates were first reported from the whole Uncaria genus. Conclusively, it is a practical chemical dereplication strategy that can enhance the efficiency and has the potential to be a routine approach for the discovery of new natural compounds.

•An integrated strategy was presented for authentication of herbal medicines.•“Commercial-homophyletic” comparison was used to obtain robust biomarkers.•Automatic metabolites annotation was achieved by UNIFI™.•Eleven robust biomarkers were deduced to differentiate four parts of P. ginseng.•ANN models enabled exact authentication of P. ginseng including adulteration.A key segment in authentication of herbal medicines is the establishment of robust biomarkers that embody the intrinsic metabolites difference independent of the growing environment or processing technics. We present a strategy by nontargeted metabolomics and “Commercial-homophyletic” comparison-induced biomarkers verification with new bioinformatic vehicles, to improve the efficiency and reliability in authentication of herbal medicines. The chemical differentiation of five different parts (root, leaf, flower bud, berry, and seed) of Panax ginseng was illustrated as a case study. First, an optimized ultra-performance liquid chromatography/quadrupole time-of-flight-MSE (UPLC/QTOF-MSE) approach was established for global metabolites profiling. Second, UNIFI™ combined with search of an in-house library was employed to automatically characterize the metabolites. Third, pattern recognition multivariate statistical analysis of the MSE data of different parts of commercial and homophyletic samples were separately performed to explore potential biomarkers. Fourth, potential biomarkers deduced from commercial and homophyletic root and leaf samples were cross-compared to infer robust biomarkers. Fifth, discriminating models by artificial neutral network (ANN) were established to identify different parts of P. ginseng. Consequently, 164 compounds were characterized, and 11 robust biomarkers enabling the differentiation among root, leaf, flower bud, and berry, were discovered by removing those structurally unstable and possibly processing-related ones. The ANN models using the robust biomarkers managed to exactly discriminate four different parts and root adulterant with leaf as well. Conclusively, biomarkers verification using homophyletic samples conduces to the discovery of robust biomarkers. The integrated strategy facilitates authentication of herbal medicines in a more efficient and more intelligent manner.

Authentication of Chinese patent medicine (CPM) is more challenging than that of a single herbal medicine due to the increasing complexity in the chemical matrix. A strategy from systematic multi-component characterization to selective ion monitoring of chemical markers (SMC-SIM) is presented and applied to authenticate Shuxiong tablet (SXT) as a case study. First, an ultra-performance liquid chromatography/quadrupole time-of-flight-Fast DDA (UPLC/QTOF-Fast DDA) approach was developed to comprehensively profile and characterize multi-components in SXT. Second, chemical markers were established using a home-made SXT sample. The SIM method for detection of these markers was initially established on an advanced QTOF mass spectrometer, and then transferred onto an easily accessible single quadrupole QDa detector. Third, the UPLC/QDa-SIM method was used to authenticate commercial SXT samples. Consequently, 250 compounds were characterized, and 73 of them were identified by comparison with reference standards. Two UPLC-SIM methods enabling the monitoring of eleven chemical markers (including five saponins for Notoginseng Radix et Rhizoma, two quinochalcone C-glycosides and a flavonoid O-glycoside for Carthami Flos, one phenolic acid and two phthalides for Chuanxiong Rhizoma), were developed on QTOF and QDa, respectively. Twelve batches of commercial SXT samples were identified by the UPLC/QDa-SIM method. Despite the lower sensitivity, the QDa-SIM method by simple sample preparation achieved similar authentication results, and was more easily generalized in practice than the QTOF-SIM method. In conclusion, targeted monitoring of multiple chemical markers was proven as feasible in comprehensive identification of CPM, and the SMC-SIM strategy enabled establishment of practical quality control methods for CPM that contains a complex chemical matrix.

Quinochalcone C-glycosides (QCGs) are a series of pharmacologically bioactive components chemotaxonomic for Carthamus tinctorius L. The low abundance and ubiquitous interference from flavonoid O-glycosides (FOGs) frequently hinder the systematic exposure and characterization of these QCG homologs. We herein present an offline comprehensive two-dimensional liquid chromatography/linear ion-trap quadrupole/Orbitrap mass spectrometry (2D LC/LTQ-Orbitrap MS) approach coupled with versatile data mining strategies, to systematically characterize the QCGs from C. tinctorius. Initially, an offline 2D LC system, with an orthogonality of 71% and a theoretical peak capacity of 7654, was established by combining an Acchrom XAmide column and a BEH Shield RP-18 column. Subsequently, the water extract of C. tinctorius was separated by first dimensional hydrophilic interaction liquid chromatography (HILIC) yielding twelve fractions, which were further analyzed by reversed-phase ultra-high performance liquid chromatography/LTQ-Orbitrap MS using high energy C-trap dissociation (HCD) and collision-induced dissociation (CID) in the negative ion mode. The characteristic product ion filtering of m/z 119.05 (C8H7O−) in the HCD spectra, ring double bond equivalent (RDB 10–30), characteristic UV absorption around 405 nm, preferred 0,2X0 cleavage for C-glycosides, and diagnostic product ions analysis, were simultaneously employed for the structural elucidation of QCGs. Ultimately, 163 QCQ homologs were putatively characterized, and 149 are potential new ones. Particularly, nine dimers of QCG-FOG have not been previously reported. The obtained results have greatly expanded the knowledge on the structural diversity of QCGs, demonstrating the potency of the offline comprehensive 2D LC/LTQ-Orbitrap MS approach in separation and characterization of minor herbal components.

•A “monomethod-heterotrait matrix” strategy was proposed for identification of CPMs.•27 QCGs were characterized by PIF and analysis of the MSE and Fast DDA data.•Six QCG markers were selected as the biomarkers of safflower.•Identification of safflower from 11 CPMs was achieved by a UHPLC/QTOF-SIM method.•UHPLC/QTOF-SIM enabled a practical and efficient identification of CPMs.Current China Pharmacopoeia standards for the Chinese patent medicines (CPMs) that contain one or several the same drug(s) employ case-dependent TLC or HPLC approaches to achieve qualitative identification. A qualitative “monomethod-heterotrait matrix” (MHM) strategy is thus proposed, by selective monitoring of multi-biomarkers, to achieve the identification of different CPMs. Carthamus tinctorius L. (safflower) is a reputable gynecological herbal medicine containing characteristic quinochalcone C-glycosides (QCGs) as the major bioactive components. Qualitative identification of safflower in diverse CPMs by selective monitoring of QCG markers was performed by use of the MHM strategy. Initially, 27 QCG analogs (involving 16 potentially new ones) were selectively characterized by product ion filtering (m/z 119.05) and integrated analysis of the negative mode MSE and Fast DDA data obtained on a UHPLC/QTOF mass spectrometer. Subsequently, by fingerprint analysis of 20 batches of safflower samples followed by a thermostable test, six QCGs (hydroxysafflor yellow A and its two isomers, anhydrosafflor yellow B, safflomin C, and isosafflomin C) were selected as the biomarkers for safflower. Then, a highly specific selective ion monitoring (SIM) method by recording centroided data was developed and applied to selectively profile six QCG biomarkers from 28 batches of CPM samples collected from versatile vendors. By reference to a standard SIM spectrum established using a home-made safflower reference extract, simultaneous identification of safflower in eleven different CPMs was accomplished with the unified sample preparation and a single UHPLC/QTOF-SIM method. The qualitative MHM strategy represents the novel methodology that facilitates the quality control of CPMs more efficiently.

•Offline 2D LC was used to separate and characterize minor saponins from XSTI.•The offline 2D LC system integrated HILIC × RPLC for orthogonal separation.•High-accuracy data were acquired by QTOF MS using Fast-DDA in negative ion mode.•143 minor saponins were characterized by combined MS data interpretation methods.•Offline 2D LC is powerful and easily accessible for characterizing TCM components.Xueshuantong Injection (XSTI), derived from Notoginseng total saponins, is a popular traditional Chinese medicine injection for the treatment of thrombus-resultant diseases. Current knowledge on its therapeutic basis is limited to five major saponins, whereas those minor ones are rarely investigated. We herein develop an offline two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry-fast data directed analysis (offline 2D LC/QTOF-Fast DDA) approach to systematically characterize the saponins contained in XSTI. Key parameters affecting chromatographic separation in 2D LC (including stationary phase, mobile phase, column temperature, and gradient elution program) and the detection by QTOF MS (involving spray voltage, cone voltage, and ramp collision energy) were optimized in sequence. The configured offline 2D LC system showed an orthogonality of 0.84 and a theoretical peak capacity of 8976. Total saponins in XSTI were fractionated into eleven samples by the first-dimensional hydrophilic interaction chromatography, which were further analyzed by reversed-phase UHPLC/QTOF-Fast DDA in negative ion mode. The fragmentation features evidenced from 36 saponin reference standards, high-accuracy MS and Fast-DDA-MS2 data, elemental composition (C < 80, H < 120, O < 50), double-bond equivalent (DBE 5–15), and searching an in-house library of Panax notoginseng, were simultaneously utilized for structural elucidation. Ultimately, 148 saponins were separated and characterized, and 80 have not been isolated from P. notoginseng. An in-depth depiction of the chemical composition of XSTI was achieved. The results obtained would benefit better understanding of the therapeutic basis and significant promotion on the quality standard of XSTI as well as other homologous products.

•Untargeted metabolomics was used to investigate the mechanism of acute toxicity.•The contents of six different sections of intestine were collected as samples.•Three identified biomarkers were used to assess the different processing method.Kansui radix (KR) is a poisonous Chinese herbal medicine recorded in the Chinese Pharmacopoeia, and the acute toxicity obstructs its clinical applications. To explore its acute toxicity mechanism to enhance clinical safety, a metabolomics study based on UPLC-ESI-QTOF-MSE was performed. Wistar rats were exposed for 4 h to the aqueous and ethyl acetate extracts prepared from KR at a high dose (25 g/kg). The contents of six different sections of rat intestine, including the duodenum, jejunum, ileum, cecum, colon, and rectum were collected as samples for the first time, as well as the rat plasma. The interesting results showed that only those rats exposed to the ethyl acetate extract showed a watery diarrhea, similar to the observed acute human toxicity. The identified biomarkers found in the plasma, such as phenol sulfate, indoxyl sulfate, and p-cresol sulfate were significantly perturbed in the rats. These biomarkers are known as colon-derived uremic compounds, which were first reported with respect to KR. The three essential amino acids which produced these biomarkers were only found in the contents of colon and rectum. A hypothesis was proposed that only the colon-derived uremic compounds induced by KR might be responsible for the acute toxicity. Three traditional process methods to reduce the toxicity of KR were compared based on these biomarkers, and different levels of toxicity modulation were observed. These results may be helpful to further understand the mechanism of acute toxicity, and the relevance of the traditional process methods to ameliorate the adverse effects of KR.

⿢The method of UPLC-LTQ-Orbitrap MS coupled to data post-processing techniques was developed.⿢47 metabolites of isorhynchophyllin in rat urine, plasma, liver and rat liver microsomes were identified.⿢The relative quantification of metabolite and main metabolite types of isorhynchophyllin were determined.⿢Comprehensive metabolic profile of isorhynchophyllin was proposed for the first time.The searching of potentially bioactive metabolites in the biological body is an interesting and meaningful work for the drug study. However, the structural clarification of possible metabolites is one of the most challenging tasks in drug metabolism studies because of the variety of metabolic reactions and complexity of metabolites in vivo. Here, an ultra high performance liquid chromatography/linear ion trap-Orbitrap mass spectrometry (U-HPLC/LTQ-Orbitrap-MS) with combination of data post-processing techniques, including extracted ion chromatogram (EIC) and multiple mass defect filters (MMDF), was established for profiling and identification of metabolites of isorhynchophylline (IR) in vivo and in vitro, and the possible metabolic pathways were subsequently proposed after the oral dose of 20 mg/kg; A total of 47 metabolites of IR were tentatively identified, including 47, 21, 18, and 25 metabolites in rat urine, plasma, liver and rat liver microsomes (RLM) samples, respectively. To our knowledge, most of them were reported for the first time. Seven metabolic pathways, including dehydrogenation, oxidation, hydrolysis, reduction, demethylation, hydroxylation and glucuronide conjugation were involved in the metabolism. Among them, dehydrogenation, hydrolysis, hydroxylation and oxidation were considered as the main metabolic pathway of metabolism according to metabolic profile of in vivo and in vitro. The relative percentage of each metabolite and main metabolite types were also determined to better understand the metabolic behavior of IR in rats. The newly discovered IR metabolites significantly expanded our understanding and were going to be greatly helpful for the further pharmacokinetic study of IR in vivo.

•A green protocol was established for efficient discovery of new natural compounds.•Offline comprehensive 2D LC, LTQ-Orbitrap-MS, and NMR analysis were applied.•A total of 427 potentially new ginsenosides were tentatively characterized.•Ginsenosides showed distinct sapogenin diversity and molecular isomerism.•The integrated strategy was proven as efficient and environmentally friendly.Exploration of new natural compounds is of vital significance for drug discovery and development. The conventional approaches by systematic phytochemical isolation are low-efficiency and consume masses of organic solvent. This study presents an integrated strategy that combines offline comprehensive two-dimensional liquid chromatography, hybrid linear ion-trap/Orbitrap mass spectrometry, and NMR analysis (2D LC/LTQ-Orbitrap-MS/NMR), aimed to establish a green protocol for the efficient discovery of new natural molecules. A comprehensive chemical analysis of the total ginsenosides of stems and leaves of Panax ginseng (SLP), a cardiovascular disease medicine, was performed following this strategy. An offline 2D LC system was constructed with an orthogonality of 0.79 and a practical peak capacity of 11,000. The much greener UHPLC separation and LTQ-Orbitrap-MS detection by data-dependent high-energy C-trap dissociation (HCD)/dynamic exclusion were employed for separation and characterization of ginsenosides from thirteen fractionated SLP samples. Consequently, a total of 646 ginsenosides were characterized, and 427 have not been isolated from the genus of Panax L. The ginsenosides identified from SLP exhibited distinct sapogenin diversity and molecular isomerism. NMR analysis was finally employed to verify and offer complementary structural information to MS-oriented characterization. The established 2D LC/LTQ-Orbitrap-MS/NMR approach outperforms the conventional approaches in respect of significantly improved efficiency, much less use of drug materials and organic solvent. The integrated strategy enables a deep investigation on the therapeutic basis of an herbal medicine, and facilitates new compounds discovery in an efficient and environmentally friendly manner as well.

•A “monomethod-heterotrait matrix” strategy was proposed for TCM quality control.•A multi heart-cutting 2D-LC (MHC-2D-LC) assay method was developed and validated.•The quality of eight Chinese patent medicines was assessed by the MHC-2D-LC method.•The MHC-2D-LC method was proven as superior over the China Pharmacopoeia methods.Current China Pharmacopoeia (ChP) standards employ diversified and case-dependent assay methods to evaluate the quality of different Chinese patent medicines (CPMs) that contain Panax notoginseng as the monarch drug. These conventional, HPLC-based approaches, utilizing a complex sample preparation procedure, can easily result in low analytical efficiency and possible component loss. Here, a “monomethod-heterotrait matrix” (MHM) strategy is proposed, that is, developing a universal multi heart-cutting two-dimensional liquid chromatography (MHC-2D-LC) approach that facilitates the simultaneous quantitation of five P. notoginseng saponins (noto-R1, Re, Rg1, Rb1, and Rd) in eight different CPMs. The MHC-2D-LC system was constructed on a dual-gradient liquid chromatography instrument equipped with a Poroshell SB C18 column and a Zorbax SB-Aq column for respective 1D and 2D separation. Method validation was performed in terms of specificity, linearity (r2 and F-test), intra-/inter-day precision (0.4–7.9%), stability (1.2–3.9%), and recovery (90.2–108.7%), and the LODs and LOQs (loaded masses) of the five analytes varied between 4.0–11.0 ng and 6.0–33.0 ng, respectively. The validated MHC-2D-LC approach was subsequently applied to quantify the five saponins in thirty batches of different CPMs. The method demonstrated superiority over the current ChP assay methods in respect of specificity (avoiding co-elution), resolution (Rs > 1.5), sample preparation (easy-to-implement ultrasonic extraction without repeated re-extraction), and transfer rate (minimum component loss). This is the first application of an MHC-2D-LC method for the quantitative assessment of the constituents of CPMs. The MHM approach represents a new, strategically significant methodology for the quality control of CPMs that involve complex chemical matrix.

Undoubtedly, metabonomics can reveal the comprehensive efficacies of traditional Chinese medicine (TCM) formulae and its complex mechanism at the molecular biological level. In this study, an attempt was made to address the pretreatment effect of a TCM formula. In this case, as a critical point, we should first know how to really reflect the various endogenous metabolites in a disease status before a TCM formula is employed in a therapeutic procedure. Here, we explored an approach that combined high resolution LTQ-Orbitrap mass spectrometry with a spike-in method to characterize endogenous metabolites in acute myocardial ischemia (AMI) rats. As a result, 19 potential biomarkers in rat plasma were identified and 10 related disturbed pathways were perturbed in the early stages of AMI development. Subsequently, the metabonomics method was applied to investigate the pretreatment effect of the TCM formula named the Danqi Tongmai tablet (DQTM). The results revealed that the DQTM pretreatment could reduce the AMI injury and partially regulate the perturbed TCA cycle and amino and nucleotide metabolism, which were presumable related to energy metabolism and myocardial cells apoptosis/necrosis. In conclusion, UHPLC-LTQ-Orbitrap MS combined with a spike-in method were successfully applied to the metabonomics analysis of DQTM, which demonstrated that not only a comprehensive metabolic profile in the early stages of AMI development was achieved, but also that the underlying holistic efficacies were assessed and it was helpful to understand the possible mechanism of pretreatment with DQTM.

•A single standard to determine multi-components (SSDMC) method was innovated on HPLC–UV detector with C8 core–shell column.•Three jatrophane diterpenoids, eight ingenane diterpenoids and two triterpenes in kansui radix were simultaneously assayed first.•The contents of its three processing products were first compared and discriminated.Kansui radix is a famous poisonous traditional Chinese medicine. However, due to its different types of constituents with broad polarity, a variety of UV absorptions and lack of the reference standards, it was difficult to simultaneously determine the main component in kanui radix. A single, multi-faceted, enhanced strategy, exogenous reference standard – single standard to determine multi-components method (ERS-SSDMC), was proposed. Thirteen major components of kansui radix, including three jatrophane diterpenoids, eight ingenane diterpenoids and two triterpenes, among which there were three pairs of isomers, were simultaneously assayed. A C8 column, packed with 2.7 μm core–shell particles, was optimized to separate these constituents in 25 min on HPLC instrument detected at a program wavelength. Ethyl benzoate employed as single exogenous reference standard. The method was fully validated with respect to linearity (r2 > 0.9995), LOQs (0.1–0.4 μg/mL), precision, accuracy (92–114%, RSD < 4.4%) and stability. The robustness of the method was performed by Plackett–Burmantest tests which eight primary chromatographic parameters were investigated. It was found that the two factors, wavelength and flow rate, should be strictly controlled. A total of 75 batches of kansui radix and its three different processing products were successfully analyzed and discriminated by applying the proposed method. This work demonstrates an effective strategy for the SSDMC method making the simultaneous assay of complex multi-component TCM system achievable.

Four new alkaloids, comprising three 3-oxo-3,7-seco-oxindole alkaloids (hirsutanine D–F, 1–3) and one oxindole alkaloid N-oxide (uncarine B N-oxide, 4), together with four known heteroyohimbine-type oxindole alkaloids, were isolated from the stems of Uncaria hirsuta Havil. Structures of 1–4 were elucidated by extensive NMR and HR-ESIMS data analyses. Compound 3 is the first 3-oxo-3,7-seco-oxindole alkaloid with ring B opened and degraded isolated from the Uncaria genus. Compounds 1–3 exhibited slight inhibition effect on the proliferation of the breast cancer cell MDA-MB-231. The positive mode collision-induced dissociation of the 3-oxo-3,7-seco-oxindole alkaloids (1–3) was featured by the β-cleavage and α-cleavage of the amido bond, while the N-oxide (4) showed characteristic neutral eliminations of ·OH and H2O.Download high-res image (220KB)Download full-size image

Ethnopharmacological relevanceVenenum Bufonis (VB), also called toad venom, has been widely used in clinic as a cardiotonic, anohyne and antineoplastic agents both in China and other Asian countries. However, its neurotoxicity and cardiotoxicity limit its wide clinical application. Compared with extensive attention attracted with cardiotoxicity, the toxic effect of VB on Central Nervous System (CNS) is much less studied.Aim of the researchThis study was performed to examine the neurotoxicity caused by VB on Sprague Dawley (SD) rats, then to clarify the mechanism in vivo by investigating its action on the neuroinflammation which possibly attributed to the activation of nuclear factor κB (NF-κB) pathway and the attenuation of brain-derived neurotrophic factor (BDNF).Materials and methodsRats administrated with 0.5% carboxymethyl cellulose sodium salt (CMC-Na) aqueous solution and VB (100 mg/kg, 200 mg/kg and 400 mg/kg) were sacrificed at 2 h, 4 h, 6 h, 8 h, 24 h and 48 h. The brain level of neurotransmitters and their corresponding receptors, pro-inflammatory cytokines, BDNF/TrkB and NF-κB pathway-related proteins were examined, respectively.ResultsVB administration induced severe neurologic damage and neuroinflammation, as indicated by the disordered 5-hydroxytryptamine (5-HT), dopamine (DA) and their corresponding receptors, together with the over production of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). VB also notably promoted the expression of p-NF-κBp65, p-IκBα, p-IKKα and p-IKKβ and down-regulated the expression of BDNF and TrkB.ConclusionThis study demonstrates that VB triggers neurotoxicity which probably is induced by neuroinflammation via activating of NF-κB pathway and attenuating the expression of BDNF.Download high-res image (296KB)Download full-size image

Ethnopharmacological relevancePearl and nacre are valuable traditional medicines to treat palpitations, convulsions or epilepsy in China for thousands of years. However, the active ingredients are not clear till now.Aim of the studyThe main purpose of the current investigation was to assess the anticonvulsant and sedative–hypnotic activity of pearl powder and nacre powder, including their corresponding 6 protein extracts.Material and methodsDetermination of the amino acid composition of the obtained protein was carried out by ultra-performance liquid chromatography (UPLC) combined with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) pre-column derivatisation. The influence of the tested drugs on locomotor activity and convulsions latency was recorded. The contents of 5-Hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) in brain were detected by enzyme-linked immunesorbent assay (ELISA) kits. In addition, immunohistochemistry was carried out to evaluate the changes of 5-HT3 and GABAB. In parallel, the expressions of them were demonstrated by western blot.ResultsThe obtained data suggested that pearl original powder (1.1 g/kg), pearl water-soluble protein (0.2 g/kg), pearl acid-soluble protein (0.275 g/kg), pearl conchiolin protein (1.1 g/kg), nacre original powder (1.1 g/kg), nacre water-soluble protein (0.2 g/kg), nacre acid-soluble protein (0.7 g/kg) and nacre conchiolin protein (1.1 g/kg) could down-regulate the expression of 5-HT3 and up-regulate the level of GABAB to varying degrees compared with the control group. Besides, drug administration also reduced the locomotor activity and increased convulsions latency with a certain mortality.ConclusionsThese findings correlated with the traditional use of pearl and nacre as sedation and tranquilization agents, thus making them interesting sources for further drug development and also providing critical important evidence for the selection of quality control markers.Download high-res image (297KB)Download full-size image

•An IMC-NLF-based nontargeted metabolomics approach was developed.•It enables the characterization and statistical analysis of target components.•Dual neutral loss filtering facilitates the exact screening of malonyl-ginsenosides.•101 malonyl-ginsenosides were primarily characterized from three Ginseng species.•Ten potential markers were discovered for discriminating three Ginseng species.The simultaneous identification and quantification of target metabolites from herbal medicines are difficult to implement by the full-scan MS based nontargeted metabolomics approaches. Here an in-source multiple collision-neutral loss filtering (IMC-NLF) based nontargeted metabolomics approach is developed and applied to identify and quantify the variations of malonyl-ginsenosides, a common group of acyl saponins with potential anti-diabetic activity, among Panax ginseng, P. quinquefolius, and P. notoginseng. The key steps of the IMC-NLF strategy are the acquisition of specific high-resolution neutral loss data and the efficient filtering of target precursor ions from the full-scan spectra. Using a hybrid LTQ-Orbitrap mass spectrometer after UHPLC separation, abundant in-source product ions, [M-H-CO2]- (due to the vulnerability of the carboxyl group) and [M-H-Mal.]-, were generated at the energies of 70 V and 90 V, respectively. After spectral deconvolution, the generated peak list was screened by dual NLF using a Neutral Loss MS Finder software (NL of 43.9898 Da for CO2 and 86.0004 Da for the malonyl substituent). By combining the precursor ions list-triggered HCD-MS/MS and basic hydrolysis, a total of 101 malonyl-ginsenosides (including 69 from P. ginseng, 52 from P. quinquefolius, and 44 from P. notoginseng) were identified or tentatively characterized. The variations of 81 characterized malonyl-ginsenosides among 45 batches of Ginseng samples were statistically analyzed disclosing ten potential markers. It is the first systematic analysis of malonyl-ginsenosides. The IMC-NLF approach by a single analytical platform is promising in targeted analyses of modification-specific metabolites in metabolomics and drug metabolism.