Non-targeted chemical analysis of consumer botanical products labeled as blue cohosh (Caulophyllum thalictroides), goldenseal (Hydrastis canadensis), or yohimbe bark (Pausinystalia yohimbe) by NMR and MS.

Consumers have unprecedented access to botanical dietary supplements through online retailers, making it difficult to ensure product quality and authenticity. Therefore, methods to survey and compare chemical compositions across botanical products are needed. Nuclear magnetic resonance (NMR) spectroscopy and non-targeted mass spectrometry (MS) were used to chemically analyze commercial products labeled as containing one of three botanicals: blue cohosh, goldenseal, and yohimbe bark. Aqueous and organic phase extracts were prepared and analyzed in tandem with NMR followed by MS. We processed the non-targeted data using multivariate statistics to analyze the compositional similarity across extracts. In each case, there were several product outliers that were identified using principal component analysis (PCA). Evaluation of select known constituents proved useful to contextualize PCA subgroups, which in some cases supported or refuted product authenticity. The NMR and MS data reached similar conclusions independently but were also complementary.

Caulophyine A, a Rare Azapyrene Alkaloid from the Roots of Caulophyllum robustum.

A novel alkaloid caulophyine A (1) was isolated from the roots of Caulophyllum robustum Maxim., along with six known alkaloids 2-7. The structure of 1 was elucidated by extensive NMR and high resolution-time-of-flight (HR-TOF)-MS analyses, it is a rare nitrogen containing polycyclic aromatic hydrocarbon. The in vitro bioassays revealed that 2 presented remarkable cytotoxicity against A549 with an IC50 value of 3.83 µM in comparison with the positive control etoposide (IC50 = 11.63 µM). Compounds 1 and 2 also displayed weak Acetylcholinesterase (AChE) inhibitory activity with IC50 values of 123.03 and 80.74 µM respectively.

HPLC-MS/MS method for the determination and pharmacokinetic study of six compounds against rheumatoid arthritis in rat plasma after oral administration of the extract of Caulophyllum robustum Maxim.

Caulophyllum robustum Maxim (CRM) is a well-known traditional Chinese medicine (TCM) mainly present in the northeast, northwest and southwest regions of China, which is belong to the family Berberidaceae. The roots and rhizomes of CRM have been used as a famous TCM for the treatment of rheumatoid arthritis (RA). The selective, sensitive and accurate high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the determination and pharmacokinetic study cauloside H, leonticin D, cauloside G, cauloside D, cauloside C and magnoflorine in rat plasma was developed and validated in this paper. Chromatographic separation was achieved by using a Waters ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.7 µm) with gradient elution using a mobile phase consisting of acetonitrile and 0.1 % formic acid in water at a flow rate of 0.4 mL/min. The detection was performed in multiple reaction monitoring (MRM) mode and electrospray ionization (ESI) in positive and negative modes. The linearity, precision, accuracy, extraction recovery, matrix effects and stability were assessed to validate the current high-performance liquid chromatography/mass spectrometry (HPLC-MS) assay. Good linearity was achieved for each analyte with a correlation coefficient (r2) > 0.99). All the precision (RSD) data were less than 12.20 %, the accuracies ranged from -12.39 % to 10.55 %, the recovery rates from the rat plasma ranged from 85.48%-98.69 %, and the matrix effects ranged from 80.96 % to 91.35 %. The validated approach was successfully applied to study the pharmacokinetic characteristics of saponins and alkaloids in plasma after administering CRME to rats, and this assay provides a platform for studying the active components of multicomponent traditional Chinese medicines and provides useful information for further clinical studies.

Systematic screening and characterization of prototype constituents and metabolites of triterpenoid saponins of Caulopphyllum robustum Maxim using UPLC-LTQ Orbitrap MS after oral administration in rats.

Triterpenoid saponins are the main bioactive components in Caulopphyllum Robustum Maxim (CRM), and they have been reported to have extensive pharmacological properties, such as anti-inflammatory, immunomodulatory, and anti-tumor effects. Cauloside C, Cauloside D, Leonticin D and Cauloside H are the main active chemical constituents of CRM in the treatment of rheumatoid arthritis (RA). However, their metabolic processes and products remain unclear. Therefore, the purpose of this study was to analyze the metabolic components and metabolic pathways of total saponins after oral administration of CRM effective part (CRME) in rats. In this work, we collected plasma, bile, urine and feces of rats at different sampling time points after intragastric administration. The saponins and reference substances were separated from CRME and analyzed via Thermo Scientific™ Ultra Performance Liquid Chromatography-Orbitrap Elite Combined High resolution Mass Spectrometry. According to the structural characteristics of the compounds in CRM, the pyrolysis behavior of various components was inferred in the negative ion mode. Twenty-two components were found in rat plasma, bile, urine and stool; among these components, there were 8 prototypes and 14 metabolites. Seven prototypes and 8 metabolites were found in rat plasma; no prototype and 6 metabolites were found in bile; 5 prototypes and 8 metabolites were found in urine; and 4 prototypes and 9 metabolites were found in stool. The metabolites include deglycosylation products, sapogenin products, sulfides, and glucuronide conjugates. The same metabolites were also found in biological samples, and these products may be important metabolic pathways of triterpene saponins in rats. The current findings clarified the metabolic pathways of the main active ingredients in CRME and further elucidated the anti-RA drug-responsive substance basis of CRM.

Piperidine alkaloids and xanthone from the roots of Caulophyllum robustum Maxim.

Two undescribed piperidine racemates, (±)-caulophines A and B (1 and 2), a new N-containing xanthone derivative (3), together with six known piperidines, were isolated from the roots of Caulophyllum robustum Maxim. Their structures were determined by extensive spectroscopic techniques. Compounds 3 and 7 exhibited weak cytotoxicities against human palace cancer hela cell line with inhibitory rates of 32.2% and 39.7%, respectively, at the concentration of 40 µM.

Anti-Inflammatory Triterpenoids from the Caulophyllum robustum Maximin LPS-Stimulated RAW264.7 Cells.

Caulophyllum robustum Maxim is widely distributed in China and used as a traditional herbal medicine to induce childbirth, ease the pain of labor, rectify delayed or irregular menstruation, alleviate heavy bleeding and pain during menstruation, and treat external injuries and irregular menses. According to our detailed chemical investigation, three new triterpene derivatives (1⁻3), together with seven known compounds, were isolated from the root and rhizome of C. robustum Maxim. Their structures were elucidated by 1D- and 2D-NMR spectroscopic analysis and physio-chemical methods. They were identified as (1) 23-hydroxy-3,19-dioxo-olean-12-en-28-oic-acid; (2) 23-hydroxy-3,11-dioxo-olean-12-en-28-oic acid; and (3) 16α,23-dihydroxy-3-oxo-olean-12-en-28-oic acid. Compounds (1⁻10) inhibited the LPS-activated NO production in RAW264.7 cells. Furthermore, the anti-inflammatory characteristics of these compounds were confirmed on the basis of decreases in iNOS and NF-κB protein expression in RAW264.7 cells.

Spectrum-Effect Relationships between Fingerprints of Caulophyllum robustum Maxim and Inhabited Pro-Inflammation Cytokine Effects.

Caulophyllum robustum Maxim (CRM) is a Chinese folk medicine with significant effect on treatment of rheumatoid arthritis (RA). This study was designed to explore the spectrum-effect relationships between high-performance liquid chromatography (HPLC) fingerprints and the anti-inflammatory effects of CRM. Seventeen common peaks were detected by fingerprint similarity evaluation software. Among them, 15 peaks were identified by Liquid Chromatography-Mass Spectrometry (LC-MS). Pharmacodynamics experiments were conducted in collagen-induced arthritis (CIA) mice to obtain the anti-inflammatory effects of different batches of CRM with four pro-inflammation cytokines (TNF-α, IL-ß, IL-6, and IL-17) as indicators. These cytokines were suppressed at different levels according to the different batches of CRM treatment. The spectrum-effect relationships between chemical fingerprints and the pro-inflammation effects of CRM were established by multiple linear regression (MLR) and gray relational analysis (GRA). The spectrum-effect relationships revealed that the alkaloids (N-methylcytisine, magnoflorine), saponins (leiyemudanoside C, leiyemudanoside D, leiyemudanoside G, leiyemudanoside B, cauloside H, leonticin D, cauloside G, cauloside D, cauloside B, cauloside C, and cauloside A), sapogenins (oleanolic acid), ß-sitosterols, and unknown compounds (X3, X17) together showed anti-inflammatory efficacy. The results also showed that the correlation between saponins and inflammatory factors was significantly closer than that of alkaloids, and saponins linked with less sugar may have higher inhibition effect on pro-inflammatory cytokines in CIA mice. This work provided a general model of the combination of HPLC and anti-inflammatory effects to study the spectrum-effect relationships of CRM, which can be used to discover the active substance and to control the quality of this treatment.

Energy-resolved technique for discovery and identification of malonyl-triterpene saponins in Caulophyllum robustum by UHPLC-electrospray Fourier transform mass spectrometry.

Malonyl-triterpene saponins (MTSs) attract scientific attentions because of their structural diversities and valuable bioactivities. However, its thermal instability brings a huge amount of challenges for isolation and purification of this class of compounds. To our best knowledge, there has been no report on isolation and analysis of MTSs from genus Caulophyllum. In this study, a strategy combining data acquisition using an energy-resolved technique and the narrow widow extracted ion chromatograms as data mining method was developed for discovery and identification of MTSs in Caulophyllum robustum hair roots by ultra high liquid chromatography coupled to electrospray ionization Fourier transform mass spectrometry. The method was performed at an independent MS full scan using our bottom-up energies by in-source collision induced dissociations with 0, 25, 50 and 100 eV in both positive and negative modes. Precursor ion as well as fragment ion information was simultaneously collected from four energy-resolved MS spectra in a single run of 18 min. The fragmentation pathways of intact deprotonated, protonated and sodium ions of MTSs were proposed for the structural elucidation of Caulophyllum MTSs. A flowchart involving a stepwise procedure based on key fragments from ESI- /ESI+ -FT-MS(1, 1) to MS(1, 4) spectra was constructed for the identification of structural elements in the MTSs. As a result, a total of 23 MTSs were discovered and tentatively identified, which had not been reported from Caulophyllum species before. All of these were potentially new compounds. This study provides an excellent example for discovery and identification of MTSs in herb medicines. Copyright © 2016 John Wiley & Sons, Ltd.

Analysis of oligosaccharide sequences of trace Caulophyllum robustum saponins by direct infusion multiple-stage tandem mass spectrometry.

The saponins in Caulophyllum robustum have not yet been fully characterized. Furthermore these saponins are often present in trace amounts and are structurally complex. Here, a simple direct infusion electrospray ion trap multiple-stage tandem mass spectrometry (DI-ESI-IT-MS(n)) method was described for the characterization of trace C. robustum saponins. Eight reference saponins from the C. robustum hairy root were investigated by DI-ESI-IT-MS(n) in positive ion mode. Some fragmentation approaches were proposed through analysis of the [M+Na](+) ions: (1) preferential cleavage of the C-28 ester glycosidic bond to provide complementary [Y0α+Na](+) and [Bα+Na](+) ions for bidesmosidic saponins; (2) diagnostically neutral loss of CO2 from free carboxyl groups at C-28 for monodesmosidic saponins; and (3) the ion intensity ratio between [C2ß+Na](+) and [B2ß+Na](+), which is sensitive to the structural differences between the two isomeric ß-sugar chains (Glc → (2)Ara and Glc → (3)Ara). The DI-ESI-IT-MS(n) method was successfully used for the analysis of trace C. robustum saponins with [M+Na](+) ions at m/z 1745.6, 1729.5, 1583.7, 1567.7, 1421.7 and 1405.7. This article highlights the discovery and identification of complex α- and ß-oligosaccharide moieties in Caulophyllum saponins by glycosidic product ions along with cross ring cleavage product ions. Five oligosaccharide moieties were unambiguously or tentatively identified as Rha → (4)Glc → (6)Glc → (4)Rha → (4)Glc → (6)Glc, Glc → (4)Glc → (6)Glc → (4)Rha → (4)Glc → (6)Glc, Rha → Glc → Glc (Glc) → (2,3)Ara, Glc → Glc (Glc) → (2,3)Ara and Glc (Glc) → (2,3)Ara. Accuracy of the analytical procedure was demonstrated by structural identification of two saponins isolated using 1D and 2D-NMR spectroscopy. The DI-ESI-IT-MS(n) method facilitates rapid discovery and analysis of trace Caulophyllum saponins and is a powerful and practical tool for structural characterization of complex oligosaccharide moieties in triterpene saponins.

A strategy for characterization of triterpene saponins in Caulophyllum robustum hairy roots by liquid chromatography with electrospray ionization quadrupole time-of-flight mass spectrometry.

Triterpene saponins are important bioactive constituents widely distributed in many plants. Saponins present in Caulophyllum (Berberidaceae) have not been fully characterized. In this study, we studied triterpene saponins from Caulophyllum robustum using liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-qTOF-MS). Rapid identification of Caulophyllum saponins was facilitated using low and high MS cone voltages to induce controlled fragmentation in positive mode. The full scan spectra at low cone voltage of 40V provided considerable structural information relating to aglycone skeletons, sugar types, and linked sequences for Caulophyllum saponins. Seven Caulophyllum aglycones were differentiated and identified by their diagnostic fragment ions combined with accurate mass measurements and characteristic fragmentation pathways. Peak intensity ratio of [aglycone+H-2H2O](+) to [aglycone+H-H2O](+) in full scan spectra acquired with low cone voltage is correlated with structural features of hederagenin and echinocystic acid and is useful for the discrimination of these positional isomers. However, at a high voltage of 200V, the saponin [M+H](+) ion and its fragmentation ions were not present; and the single saponin [M+Na](+) generated [Bα+Na](+) and [Y0α+Na](+) by in-source fragmentation, which provided structural information on the α- and ß-sugar chains in the saponins. This approach enabled simultaneous acquisition of structural information on both aglycones and sugar chains from full scan spectra in one injection. Based on the developed strategy, 51 triterpene saponins of seven different classes were fully characterized or tentatively identified, of which 32 constituents were the first to be reported in genus Caulophyllum and 18 compounds were characterized as potentially new compounds.

Toxins in botanical dietary supplements: blue cohosh components disrupt cellular respiration and mitochondrial membrane potential.

Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA "black box" warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3), exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage.

Primary constituents of blue cohosh: quantification in dietary supplements and potential for toxicity.

Dietary supplements containing dried roots or extracts of the roots and/or rhizomes of blue cohosh (Caulophyllum thalictroides) are widely available. This botanical has a long history of use by Native Americans and its use continues to the present day. The primary constituents of blue cohosh are its alkaloids and saponins. The structures of the alkaloids magnoflorine, baptifoline, anagyrine, and N-methylcytisine have been known for many years. The last 10 years have seen a great increase in isolation and identification of the large number of saponins present in blue cohosh. Important developments in nuclear magnetic resonance techniques have contributed substantially to the increase in elucidation of the structures of the complex saponins. Several authors have described quantitative methods for both the alkaloids and saponins in blue cohosh. Such methods have made it possible to quantify these constituents in dietary supplements containing this botanical ingredient. Concentrations of both alkaloids and saponins vary substantially in dietary supplements of blue cohosh. The nicotinic alkaloid, N-methylcytisine, a potent toxicant, has been found in all dietary supplements of blue cohosh analyzed. The teratogenic alkaloid anagyrine has been found in some but not all dietary supplements.

[Effect of taspine derivatives on human liver cancer SMMC7721].

OBJECTIVE: To analyse the inhibition effect of taspine derivatives on human Liver cancer SMMC7721 cell and its mechanism. METHODS: The effects of five taspine derivatives on SMMC7721 cell growth were determined by MTT. The flow cytometry was used to determine the cell cycle. The effects of Tas-D1 on the EGF and VEGF in SMMC7721 cell were determined by ELISA. The mRNA level of EGF and VEGF in SMMC7721 cell was determined by RT-PCR. RESULTS: The MTT assay demonstrated that the taspine derivative Tas-D1 significantly inhibited the growth of SMMC7721 cell in a dose-dependent manner. Cell was stopped at S phase by Tas-D1. Tas-D1 inhibited the expression of EGF and VEGF and their mRNA in a dose-dependent manner (P<0.05). CONCLUSIONS: The taspine derivative Tas-D1 can inhibit the growth of human Liver cancer SMMC7721 cell and change cell cycle, which may be related to the inhibition of EGF and VEGF expression.

Analytical methods for determination of magnoflorine and saponins from roots of Caulophyllum thalictroides (L.) Michx. using UPLC, HPLC and HPTLC.

Analytical methods including HPLC, UPLC and HPTLC are presented for the determination of major alkaloid and triterpene saponins from the roots of Caulophyllum thalictroides (L.) Michx. (blue cohosh) and dietary supplements claiming to contain blue cohosh. A separation by LC was achieved using a reversed phase column, PDA with ELS detection, and ammonium acetate/acetonitrile gradient as the mobile phase. Owing to their low UV absorption, the triterpene saponins were detected by evaporative light scattering. The eight triterpene saponins (cauloside H, leonticin D, cauloside G, cauloside D, cauloside B, cauloside C, cauloside A and saponin PE) and the alkaloid magnoflorine could be separated within 35 min using HPLC method and within 8.0 min using UPLC method with detection limits of 10 µg/mL for saponins and 1 µg/mL for magnoflorine. The detection wavelength was 320 nm for magnoflorine and ELS detection was used for the eight saponins. The methods were also successfully applied to analyze different dietary supplements. For the products claiming to contain blue cohosh, there was a significant variability in the amounts of triterpene saponins detected. Calculations based on the analysis results for dietary supplements showed that maximum daily intake of alkaloid and saponins vary with the form (solids/liquids) and recommended doses according to the products label. Intakes varied from 0.57 to 15.8 mg/day for magnoflorine and from 5.97 to 302.4 mg/day for total saponins. LC-mass spectrometry coupled with electrospray ionization (ESI) method is described for the identification and confirmation of nine compounds in plant samples and dietary products. A HPTLC method was also developed for the fast chemical fingerprint analysis of C. thalictroides samples.

Four new fluorenone alkaloids and one new dihydroazafluoranthene alkaloid from Caulophyllum robustum Maxim.

Four new fluorenone alkaloids, caulophylline A-D (1-4), and one new dihydroazafluoranthene alkaloid, caulophylline E (5) were isolated from the roots of Caulophyllum robustum Maxim. Their structures were elucidated by spectroscopic analysis. Among the isolated alkaloids, Caulophylline E showed good scavenging effects against DPPH radical with IC(50) of 39 µM.

Establishment of A431 cell membrane chromatography-RPLC method for screening target components from Radix Caulophylli.

We describe here an analytical method of A431 cell membrane chromatography (A431/CMC) (CMC, cell membrane chromatography) combined with RPLC for recognition, separation, and identification of target components from traditional Chinese medicines (TCMs) Radix Caulophylli. The A431 cells with high expressed epidermal growth factor receptor (EGFR) were used to prepare the stationary phase in the CMC model. Retention fractions on the A431-CMC model were collected using an automated fraction collection and injection module (FC/I). Each fraction was analyzed by RPLC under the optimized conditions. Gefitinib and erlotinib were used as standard compounds to investigate the suitability and reliability of the A431 cell membrane chromatography-RPLC method prior to screening target component from Radix Caulophylli total alkaloids. The results indicated that caulophine and taspine were the target component acting on the epidermal growth factor receptor. This method could be an efficient way in drug discovery using natural medicinal herbs as a source of novel compounds.

A combined A431 cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening compounds from total alkaloid of Radix Caulophylli acting on the human EGFR.

We have developed an online analytical method that combines A431 cell membrane chromatography (A431/CMC) with high performance liquid chromatography and mass spectrometry (LC/MS) for identifying active components from Radix Caulophylli acting on human EGFR. Retention fractions on A431/CMC model were captured onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using Sorafenib tosylate as a positive control, taspine and caulophine from Radix Caulophylli were identified as the active molecules which could act on the EGFR. This A431/CMC-online-LC/MS method can be applied for screening active components acting on EGFR from traditional Chinese medicines exemplified by Radix Caulophylli and will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds.

Teratogenic effects of blue cohosh (Caulophyllum thalictroides) in Japanese medaka (Oryzias latipes) are probably mediated through GATA2/EDN1 signaling pathway.

Blue cohosh (Caulophyllum thalictroides) (BC) has been used widely to induce labor and to treat other uterine conditions. However, the safety and effectiveness of this herbal product has not yet been evaluated by the US Food and Drug Administration (FDA). Several conflicting reports indicated that the root extract of BC is a teratogen and, by some unknown mechanisms, is able to induce cardiovascular malfunctions in new-born babies. To understand the mechanism, we have used Japanese medaka (Oryzias latipes) embryo-larval development as the experimental model and the methanolic extract of BC root as the teratogen. The embryo mortality, hatching efficiency, and morphological abnormalities in craniofacial and cardiovascular systems are considered for the evaluation of BC toxicity. Our results indicate that BC is able to disrupt cardiovascular and craniofacial cartilage development in medaka embryo in a dose and developmental stage-specific manner. Moreover, embryos in precirculation are to some extent more resistant to BC than ones with circulation. By using subtractive hybridization, we have observed that gata2 mRNA was differentially expressed in the circulating embryos after BC treatment. As GATA-binding sequences are required for the expression of the endothelin1 (edn1) gene and edn1 expressed in blood vessels and craniofacial cartilages, we have extended our investigations to edn1 gene expression regulation by BC. We found that edn1, furin1, and endothelin receptor A (ednrA) genes are developmentally regulated; endothelin converting enzyme mRNA (ece1) maintained a steady-state level throughout development. Circulating medaka embryos (3 days post fertilization, dpf) exposed to BC (10 microg/mL) for 48 h have increased levels of gata2, ece1, and preproenodthelin (preproedn1) mRNA contents; however, other mRNAs (furin and ednrA) remained unaltered. Therefore, the enhanced expression of gata2 mRNA followed by ece1 and preproedn1 mRNA by BC might be able to induce vasoconstriction and cardiovascular defects and disrupt craniofacial cartilages in medaka embryos. We conclude that cardiovascular and craniofacial defects in medaka embryogenesis by BC are probably mediated through a GATA2-EDN1 signaling pathway.

Leiyemudanosides A-C, three new bidesmosidic triterpenoid saponins from the roots of Caulophyllum robustum.

Three new oleanane bidesmosidic triterpenoid saponins, named leiyemudanosides A-C (1-3) were isolated from the roots of Caulophyllum robustum Maxim. Their structures were established by chemical and detailed spectroscopic analysis as 3-O-alpha-L-arabinopyranosyl-caulophyllogenin-28-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (1), 3-O-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranosyl-caulophyllogenin-28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (2), and 3-O-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranosyl-echinocystic acid-28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (3), respectively.

A combined cell membrane chromatography and online HPLC/MS method for screening compounds from Radix Caulophylli acting on the human alpha(1A)-adrenoceptor.

We have developed an online analytical method that combines alpha(1A)-adrenoceptor (alpha(1A)AR) cell membrane chromatography (alpha(1A)AR-CMC) with high performance liquid chromatography and mass spectrometry (HPLC/MS) for the identification of active components from Radix Caulophylli acting on the human alpha(1A)AR. Fractions retained by the alpha(1A)AR-CMC column were captured into a loop and the components were directly analyzed by combining an 8 port column switcher with an HPLC/MS system for separation and preliminary identification. Using methoxamine as a positive control drug, magnoflorine and caulophine from Radix Caulophylli were identified as the active molecules acting on the alpha(1A)AR. This new alpha(1A)AR-CMC-online-HPLC/MS method can be applied for screening active components acting on alpha(1A)AR from traditional Chinese medicines exemplified by Radix Caulophylli. This method will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds.