Novel Approaches for the Analysis and Isolation of Benzylisoquinoline Alkaloids in Chelidonium majus.

Benzylisoquinoline alkaloids are the major bioactive components in Chelidonium majus, a plant that has a long usage history for the treatment of gastrointestinal ailments in European and Asian phytomedicine. This study reports on the development and application of a supercritical fluid chromatography technique for the simultaneous qualitative and quantitative determination of seven benzylisoquinoline alkaloids in under six minutes using a Viridis BEH 2-EP column and a modifier comprising methanol with 30% acetonitrile and 20 mM ammonium formate. The method was fully validated according to ICH guidelines showing, e.g., excellent linearity (≥ 0.9997) and maximum deviations for intraday and inter-day precision of 2.99 and 2.76%, respectively. The new supercritical fluid chromatography assay was not only employed for the analysis of several C. majus samples but was also used for the subsequent development of a fast centrifugal partition chromatography technique, whereby five benzylisoquinoline alkaloids could be isolated within approximately 2.5 h, with only two of them, protopine and chelidonine, requiring an additional purification step. To achieve this, a solvent system composed of chloroform/methanol/0.3 M hydrochloric acid was used in descending mode. By injecting 500 mg of crude extract, stylopine (1.93 mg), sanguinarine (0.57 mg), chelidonine (1.29 mg), protopine (1.95 mg), and coptisine (7.13 mg) could be obtained. The purity of compounds was confirmed by supercritical fluid chromatography and MS.

Development of an artificial biosynthetic pathway for biosynthesis of (S)-reticuline based on HpaBC in engineered Escherichia coli.

Benzylisoquinoline alkaloids (BIAs) are an important class of plant secondary metabolites with a variety of pharmacological activities. Although they are widely used, traditionally these compounds are extracted from natural sources because their structure is too complicated to achieve economically feasible chemical synthesis. Thus, microbial biosynthesis of BIAs is expected to reduce dependence on natural extracts. (S)-Reticuline is an important precursor for BIAs biosynthesis. Therefore, it is an attractive engineering target. In this study, we reported the development of a novel (S)-reticuline biosynthetic pathway based on 4-hydroxyphenylacetate 3-hydroxylase (HpaBC) in Escherichia coli. Then, we further improved the (S)-reticuline production to 307 ± 26.8 mg/L by increasing the availability of the precursor 3, 4-dihydroxyphenylacetaldehyde. The E. coli cell factory developed in this study can be used as a potential platform for further efficient biosynthesis of BIAs derivatives.

Pharmacokinetics, bioavailability and metabolism of neferine in rat by LC-MS/MS and LC-HRMS.

In this study, a simple and sensitive analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the determination of neferine in rat plasma. After acetonitrile-mediated protein precipitation, the samples were separated on an Acquity BEH C18 column (2.1 × 50 mm, 1.7 µm) maintained at 40°C. The mobile phase comprising 0.1% formic acid in water and acetonitrile was delivered at a flow rate of 0.4 ml/min. The mass detection was conducted using multiple reaction monitoring mode with ion transitions at 625.4 > 206.3 and m/z 622.9 > 380.9 for neferine and internal standard, respectively. The assay was demonstrated to be linear over the concentration range of 0.5-1,000 ng/ml, with correlation coefficient >0.999 (r > 0.999). The validated method was further applied to the pharmacokinetic study of neferine in rat plasma. In addition, the metabolism of neferine was investigated using high-resolution mass spectrometry. A total of six metabolites from rat liver microsomes and plasma were detected and their structures were identified according to their fragment ions. The proposed metabolic pathways of neferine were demethylation, dealkylation, dehydrogenation and glucuronidation.

Chemical Evaluation of the Effects of Storage Conditions on the Botanical Goldenseal using Marker-based and Metabolomics Approaches.

Hydrastis canadensis, commonly known as goldenseal, is a botanical native to the southeastern United States that has been used for the treatment of infection. The activity of goldenseal is often attributed to the presence of alkaloids (cyclic, nitrogen-containing compounds) present within its roots. Chemical components of botanical supplements like goldenseal may face degradation if not stored properly. The purpose of the research was to analyze the stability of known and unknown metabolites of H. canadensis during exposure to different storage conditions using mass spectrometry. Three abundant metabolites of H. canadensis, berberine, canadine, and hydrastine, were chosen for targeted analysis, and the stability of unknown metabolites was evaluated using untargeted metabolomics. The analysis and evaluation of H. canadensis samples were performed utilizing LC-MS and Principal Component Analysis (PCA). The research project focused on identifying the chemical changes in the metabolite content of H. canadensis under different temperature conditions (40°C ± 5°C, 20°C ± 5°C , and 4°C ± 5°C), different light:dark (hr:hr) cycles (16:8, 12:12, and 0:24), and different sample conditions (powdered roots versus whole roots) over a six month period. The results of this 6-month study revealed that the storage conditions evaluated had no significant effects on the chemical composition of H. canadensis roots. Hence, as long as H. canadensis roots are stored within the storage conditions tested in the study, no significant changes in chemical compositions of metabolites are expected.

A rapid method for simultaneous quantification of berberine, berbamine, magnoflorine and berberrubine in mouse serum using UPLC-MS/MS.

Berberidis cortex, the dry bark of Berberis L., is used to treat diabetes and contains at least three bioactive components: berberine (BBR), berbamine (BBM) and magnoflorine (MGF). BBR in turn is metabolized into berberrubine (BRB). Although it is possible to quantify each of these components individually in serum, there are currently no methods for simultaneously quantifying all four. Here, we developed a specific and rapid method for simultaneously quantifying BBR, BBM, MGF and BRB in mouse serum using ultra high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Samples were pretreated by protein precipitation, separated using an ACQUITY UPLC® BEH C18 column and detected by a triple quadrupole mass spectrometer with electrospray ionization. The compound [9,10-(OC2H3)2]-BBR (d6-BBR) was used as internal standard for BBR and BRB, boldine (BOL) for MGF and tetrandrine (TET) for BBM. The m/z transitions for precursor/product ion pairs were 336.1/320.2 for BBR, 305.2/566.3 for BBM, 342.0/297.1 for MGF, 322.1/307.2 for BRB, 342.2/294.3 for d6-BBR, 312.2/580.3 for TET and 328.1/265.2 for BOL. We validated our method in terms of selectivity, linearity and lower limit of quantification, accuracy, precision, matrix effect and recovery, dilution integrity and stability. This method showed good linearity from 0.1 to 40 ng/mL for BBR, 8 to 3200 ng/mL for BBM, 5 to 2000 ng/mL for MGF and 0.2 to 80 ng/mL for BRB. The chromatographic run time was 3.9 min, and sample preparation took approximately 15 min per batch. Finally, we used our method to measure BBR, BBM, MGF and BRB in serum from diabetic mice after gavage administration of BBR hydrochloride, BBM hydrochloride, and MGF. This method is precise, accurate and suitable for high-throughput sample analysis.

A single residue determines substrate preference in benzylisoquinoline alkaloid N-methyltransferases.

N-methylation is a recurring feature in the biosynthesis of many plant specialized metabolites, including alkaloids. A crucial step in the conserved central pathway that provides intermediates for the biosynthesis of benzylisoquinoline alkaloids (BIAs) involves conversion of the secondary amine (S)-coclaurine into the tertiary amine (S)-N-methylcoclaurine by coclaurine N-methyltransferase (CNMT). Subsequent enzymatic steps yield the core intermediate (S)-reticuline, from which various branch pathways for the biosynthesis of major BIAs such as morphine, noscapine and sanguinarine diverge. An additional N-methylation yielding quaternary BIAs is catalyzed by reticuline N-methyltransferase (RNMT), such as in the branch pathway leading to the taxonomically widespread and ecologically significant alkaloid magnoflorine. Despite their functional differences, analysis of primary sequence information has been unable to accurately distinguish between CNMT-like and RNMT-like enzymes, necessitating laborious in vitro screening. Furthermore, despite a recent emphasis on structural characterization of BIA NMTs, the features and mechanisms underlying the CNMT-RNMT functional dichotomy were unknown. We report the identification of structural variants tightly correlated with function in known BIA NMTs and show through reciprocal mutagenesis that a single residue acts as a switch between CNMT- and RNMT-like functions. We use yeast in vivo screening to show that this discovery allows for accurate prediction of activity strictly from primary sequence information and, on this basis, improve the annotation of previously reported putative BIA NMTs. Our results highlight the unusually short mutational distance separating ancestral CNMT-like enzymes from more evolutionarily advanced RNMT-like enzymes, and thus help explain the widespread yet sporadic occurrence of quaternary BIAs in plants. While this is the first report of structural variants controlling mono-versus di-methylation activity among plant NMT enzymes, comparison with bacterial MT enzymes also suggests possible convergent evolution.

Determination of berberine-upregulated endogenous short-chain fatty acids through derivatization by 2-bromoacetophenone.

Short-chain fatty acids (SCFAs) are a major group of endogenous metabolites generated by the gut microbiota and have been reported to play an important role in physical health, such as improving energy metabolism. Here, using 2-bromoacetophenone as the derivatization reagent (BP, 10 mg/mL, 40 °C for 20 min), a sensitive liquid chromatography-tandem mass spectrometric method was established for the quantitative determination of seven short-chain fatty acids in plasma and feces. The analyses were performed on a C18 column in positive multiple reaction monitoring mode. Specificity, linearity, accuracy, precision, recovery, and stability were observed within the quantitative limits of biological sample analysis. The established method has largely improved the sensitivity by 200- to 2000-fold than that in gas chromatography (GC). Especially for butyrate, the lower quantitative limit of 1 ng/mL, 1600-fold higher in sensitivity than that of GC (1.6 µg/mL), ensured the accurate determination of its low level in blood or feces (88 ± 29 ng/mL in blood, 176 ± 18 µg/g in feces). Then, the validated method was applied for therapeutic studies of berberine in hyperlipidemia hamsters in vivo and screening of 13 compounds (including five metabolites of berberine and eight typical isoquinoline alkaloids) in vitro. After berberine treatment (oral, 200 mg/kg, 2 weeks) to hyperlipidemia hamsters, the levels of butyrate were significantly upregulated in blood (77 ± 10 ng/mL vs. 117 ± 13 ng/mL, *P < 0.05) and feces (132 ± 11 µg/g vs. 547 ± 57 µg/g, ***P < 0.001), which further verified butyrate as an active endogenous metabolite in coordination with berberine to lower the blood lipids. Additionally, the berberine metabolites (M1, M2, M3), as well as two isoquinoline alkaloids (tetrandrine and dauricine), could also obviously induce the production of SCFAs (butyrate, etc.) in gut microbiota. In total, we have successfully established a new derivative LC-MS/MS method for the targeted quantitative determination of seven SCFAs in biological samples. Graphical abstract.

A method using angiotensin converting enzyme immobilized on magnetic beads for inhibitor screening.

Angiotensin converting enzyme (ACE), fusing with FLAG tag, was overexpressed in human embryonic kidney 293T cells. This recombinant FLAG-tagged ACE was immobilized on anti-FLAG antibody coated magnetic beads by affinity method in crude cell lysate for the first time. The enzyme-immobilized magnetic beads (ACE-MB), without further cleavage procedure, were used directly to establish a cost-effective and reliable method for screening ACE inhibitors by coupling with fluorescence detection. The enzymatic activity of the ACE-MB was validated based on its Michaelian kinetic behavior towards hippuryl-histidyl-leucine by UHPLC-MS/MS method firstly. Then, several conditions were optimized including amount of magnetic beads, incubation temperature and time in the procedure of ACE immobilization and amount of ACE-MB in the microplate operation. Moreover, this screening assay was validated with Z' factors between 0.71 and 0.81 using four known ACE inhibitors (captopril, lisinopril, fosinopril and fosinoprilat). The developed method was applied for the screening of ACE inhibitors from a small compound library of 45 natural products. As a result, epiberberine and fangchinoline with certain ACE inhibitory activities were screened out in the assay and validated. The results demonstrate the usefulness of this screening method using ACE immobilized on magnetic beads and the advantage of great efficiency with respect to both time and reagents for screening ACE inhibitors.

Quantification of natural products in herbal supplements: A combined NMR approach applied on goldenseal.

Authentication of natural products is of major relevance in the context of manufactured drugs or herbal supplements since such active products generate a lucrative market. The analytical method to identify and quantify valuable natural products is critical for quality control and product assignment of herbal supplements. In this framework, we propose to apply a recently developed quantitative 2D NMR approach called Q QUIPU (Quick QUantItative Perfected and pUre shifted) in combination with 1D 1H NMR capable to access the concentration of three major alkaloids, berberine, ß-hydrastine and canadine, in the root extract of goldenseal (Hydrastis canadensis), one of the 20 most popular herbal supplements used worldwide. We highlight the complementarity of 1D and 2D quantitative NMR to accurately assess the amount of alkaloids with different range of concentrations and stability within extracts. In particular, unstable natural products having non-overlapped signals like berberine could only be quantified by sensitive and fast 1D 1H, while overlapped signals of ß-hydrastine and low intense ones of canadine could only be quantified with the recent 2D Q QUIPU HSQC. Results obtained from this combined approach have led to a good accuracy (<10%) as compared with coupled UHPLC-MS/UV techniques. This quantitative NMR approach paves the way to numerous applications where the accurate quantification of targeted compounds in complex mixtures is required, for instance in agricultural, food and pharmaceuticals products.

Otoprotective Effects of Stephania tetrandra S. Moore Herb Isolate against Acoustic Trauma.

Noise is the most common occupational and environmental hazard, and noise-induced hearing loss (NIHL) is the second most common form of sensorineural hearing deficit. Although therapeutics that target the free-radical pathway have shown promise, none of these compounds is currently approved against NIHL by the United States Food and Drug Administration. The present study has demonstrated that tetrandrine (TET), a traditional Chinese medicinal alkaloid and the main chemical isolate of the Stephania tetrandra S. Moore herb, significantly attenuated NIHL in CBA/CaJ mice. TET is known to exert antihypertensive and antiarrhythmic effects through the blocking of calcium channels. Whole-cell patch-clamp recording from adult spiral ganglion neurons showed that TET blocked the transient Ca2+ current in a dose-dependent manner and the half-blocking concentration was 0.6 + 0.1 µM. Consistent with previous findings that modulations of calcium-based signaling pathways have both prophylactic and therapeutic effects against neural trauma, NIHL was significantly diminished by TET administration. Importantly, TET has a long-lasting protective effect after noise exposure (48 weeks) in comparison to 2 weeks after noise exposure. The otoprotective effects of TET were achieved mainly by preventing outer hair cell damage and synapse loss between inner hair cells and spiral ganglion neurons. Thus, our data indicate that TET has great potential in the prevention and treatment of NIHL.

Tissue-specific metabolite profiling of benzylisoquinoline alkaloids in the root of Macleaya cordata by combining laser microdissection with ultra-high-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Tissue-specific metabolite profiling helps to find trace alkaloids masked during organ analysis, which contributes to understanding the alkaloid biosynthetic pathways in vivo and evaluating the quality of medical plants by morphology. As Macleaya cordata contains diverse types of benzylisoquinoline alkaloids (BIAs), the alkaloid metabolite profiling was carried out on various tissues of the root. METHODS: Laser microdissection with fluorescence detection was used to recognize and dissect different tissues from the root of M. cordata. Ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry was applied to analyze the trace alkaloids in tissues. These detected alkaloids were elucidated using their accurate molecular weights, MS/MS data, MS fragmentation patterns and the known biosynthetic pathways of BIAs. Finally, the distribution of alkaloids in dissected tissues and whole sections was mapped. RESULTS: Forty-nine alkaloids were identified from five microdissected tissues, and 24 of them were detected for the first time in M. cordata. Some types of alkaloids occurred specifically in dissected tissues. More alkaloids were detected in the cork and xylem vascular bundles which emit strong fluorescence under fluorescence microscopy. Some of the screened alkaloids were intermediates in sanguinarine and chelerythrine biosynthetic pathways, and others were speculated to be involved in the new branches of biosynthetic pathways. CONCLUSIONS: The integrated method is sensitive, specific and reliable for determining trace alkaloids, which is also a powerful tool for metabolite profiling of tissue-specific BIAs in situ. The present findings should contribute to a better understanding of the biosynthesis of BIAs in M. cordata root and provide scientific evidence for its quality evaluation based on morphological characteristics. Copyright © 2016 John Wiley & Sons, Ltd.

Measurement of some Benzylisoquinoline Alkaloids in Different Organs of Persian Poppy during Ontogenetical Stages.

Papaver bracteatum, a perennial species, has been known as a rich source of thebaine and a potential alternative to Papaver somniferum for the production of codeine and some semisynthetic antagonist drugs. In this study, ion mobility spectrum (IMS) of the root, leaf, bottom part of stem, upper part of stem, capsule wall, petal, and capsule content during developmental stages of P. bracteatum including annual rosette, perennial rosette, bud initiation, pendulous bud, preflowering, and lancing were investigated. The IMS revealed thebaine, papaverine, and noscapine as the major components of the extracted alkaloids. Based on the results of the study it appears that, at least in part, there is a competition among the biosynthesis pathways of papaverine, noscapine, and morphinan alkaloids from a common source. Root and capsule wall were the most potent organs for extraction of thebaine, while lancing stage was the best developmental stage for thebaine exploitation. However, it seems that total biomass of root and capsule wall plays a key role in the final selection of favorite organ. Although papaverine and noscapine in the stem at preflowering stage had the most quantity, significant amounts were found in the capsule wall. In general, total alkaloid content of leaf was lower than the other plant parts.

Simultaneous Determination of Fangchinoline and Tetrandrine in Qi-Fang-Xi-Bi-Granules by RP-HPLC.

Knee Osteoarthritis is one of the most common diseases of elder worldwide. Qi-Fang-Xi-Bi-Granules (QFXBG) is a new Chinese medicine granules employed for the treatment of Knee Osteoarthritis. Fangchinoline (FAN) and tetrandrine (TET) are used as targets of quality control of QFXBG. A simple, practical high-performance liquid chromatographic method was developed for the simultaneous quantitation of FAN and TET in Stephaniae tetrandrae radix and QFXBG. The analysis was performed on a Athena C18 column (250 × 4.6 mm, 5 µm) with mobile phase of methanol-water (65 : 35, v/v, pH 3.0, adjusted by glacial acetic acid) containing 1.0 g/L sodium 1-octanesulfonate. This method was validated in terms of linearity, precision, accuracy and recovery. Results showed that this method had good linearity with R(2) at >0.999. The limit of detection and limit of quantification for FAN were 0.13 and 0.35 mg/L, while for TET were 0.28 and 0.76 mg/L, respectively. The relative standard deviations of precision were 0.1-1.3% for intraday and 0.5-2.4% for interday. The recovery was 94.56-98.81% of FAN and 94.07-99.12% of TET, respectively. The chromatographic analytical time was 14 min. This method was successfully applied for the quantitative analysis of FAN and TET in Stephaniae tetrandrae radix and QFXBG, so that it could be extended to quality control of QFXGB in commercial.

Anti-nociceptive, anti-inflammatory and toxicological evaluation of Fang-Ji-Huang-Qi-Tang in rodents.

BACKGROUND: Fang-Ji-Huang-Qi-Tang (abbreviated as FJHQT), composed by six medicinal herbs including Radix Stephania Tetrandra, Radix Astragali, Rhizoma Atractylodis Macrocephalae, Radix Glycyrrhizae, Rhizoma Zingiberis and Fructus Ziziphi Jujubae, is a frequently Chinese prescription for treating painful and inflammatory disorders such as rheumatoid arthritis. When Radix Stephania Tetrandra was misused with Aristolochia species, acute or chronic nephropathy caused by aristolochic acid was happened. Thus, the present study was aimed to identify Radix Stephania Tetrandra and performed the pharmacological and toxicological evaluation of FJHQT extract in rodents. METHODS: Radix Stephania Tetrandra was identified by macroscopic and microscopic observation, and the content of tetrandrine in FJHQT extract was measured by high performance liquid chromatography. Then, the pharmacological activities of FJHQT extract with respect to clinical use was investigated with acetic acid-induced writhing response, formalin-induced licking response and carrageenan-induced paw edema. Finally, we evaluated the subacute toxicology of FJHQT extract after 28-day repeated oral administration in rats. RESULTS: Radix Stephania Tetrandra was correctly used in FJHQT extract, and the content of tetrandrine in FJHQT extract was 2.5 mg/g. FJHQT extract produced a pronounced and dose-dependent antinociceptive and anti-inflammatory effects in three above models. FJHQT extract after 28-day repeated administration did not caused any hematological, biochemical and histological change in rats. CONCLUSIONS: We suggest that FJHQT extract is a high safety index Chinese medicine for antinociceptive and anti-inflammatory application when Radix Stephania Tetrandra was correctly used in FJHQT. Its antinociceptive and anti-inflammatory mechanism might be related to peripheral nociceptive pathway such as prostaglandins.

Chiral differentiation of the noscapine and hydrastine stereoisomers by electrospray ionization tandem mass spectrometry.

Energy-dependent collision-induced dissociation (CID) of the dimers [2 M + Cat](+) of the noscapine and hydrastine stereoisomers was studied where Cat stands for Li(+), Na(+), K(+) and Cs(+) ions. These dimers were generated 'in situ' from the electrosprayed solution. The survival yield (SY) method was used for distinguishing the noscapine and hydrastine dimers. Significant differences were found between the characteristic collision energies (CE50, i.e. the collision energy necessary to obtain 50% fragmentation) of the homo- (R,R; S,S) and heterochiral (R,S; S,R) stereoisomers. To distinguish the enantiomer pairs L-, D-tyrosine ([M + Tyr + Cat](+)) and L-, D-lysine ([M + Lys + Cat](+)) were used as chiral selectors. Furthermore, these heterodimers [M + amino acid + Cat](+) were also applied to determine the stereoisomeric composition. It was found that the characteristic collision energy (CE50) of the noscapine and hydrastine homodimers ([2 M + Cat](+)) was inversely proportional to the ionic radius of the cations. Furthermore, the structures of the dimers [2 M + Cat](+) were studied by high level quantum chemical calculations.

P­glycoprotein inhibition increases the transport of dauricine across the blood­brain barrier.

Dauricine is the major bioactive component isolated from the roots of Menispermum dauricum D.C. The aim of the present study was to investigate the role of P­glycoprotein in the transport of dauricine across the blood­brain barrier by pre­treatment with the P­glycoprotein inhibitor verapamil. Sprague Dawley rats were divided into a verapamil group (pretreated with verapamil at a dose of 20 mg/kg) and a control group (pretreated with the same volume of normal saline). After 90 min, the animals were injected intravenously with dauricine (10 mg/kg). At 15, 30 and 60 min after dauricine administration, the levels of dauricine in the blood and brain were detected by high­performance liquid chromatography. Compared with the control group, the dauricine concentration in the brains of the rats in the verapamil group was significantly increased. Furthermore, the brain­plasma ratio of dauricine in the rats pretreated with verapamil was significantly higher than that of the animals in the control group. However, there was no difference identified between dauricine levels in the plasma of the verapamil and the control groups. The results indicated that dauricine is able to pass the blood­brain barrier, and that P­glycoprotein has an important role in the transportation of dauricine across the blood­brain barrier.

[Effect of berberine, liensinine and neferine on HERG channel expression].

OBJECTIVE: Immunofluorescence and Western blot methods were adopted for qualitative and quantitative detections of the effect of different concentrations of berberine, liensinine and neferine on the expression of stable transfection in HERG potassium channel in HEK-293 cells, as well as the effect of different concentrations of berberine on protein expression of Ikr channel in cardiac muscular tissues, in order to investigate the anti-arrhythmic mechanism of berberine, liensinine and neferine. METHOD: Western blot method was used to detect protein expression of HERG channel in HERG-HEK cells. Immunofluorescence method as well as confocal laser microscope were used to detect the effect of different concentrations of berberine, liensinine and neferine on protein expression of HERG channel. Western blot method was used to detect the effect of different concentrations of berberine on protein expression of Ikr channel in cardiac muscular tissues as well as the effect of berberine, liensinine and neferine on protein expression of stable transfection in HERG potassium channel in HEK-293 cells. RESULT: Western blot experiment manifested that stable transfection of HEK293 cells containing HERG genes could increase protein expression of HERG channel. Berberine (10, 30 micromol x L(-1)) remarkably inhibited protein expression of HERG channel in HERG-HEK cells (P < 0.01). Berberine (10, 20 mg x kg(-1)) also inhibited protein expression of Ikr channel in rat ventricular tissues (P < 0.05). Liensinine (3, 10, 30 micromol x L(-1)) increased protein expression of HERG channel in HERG-HEK cells (P < 0.05). Neferine showed no effect on protein expression of HERG channel in HERG-HEK cells. CONCLUSION: The stably transfection of HERG-HEK cells can increase protein expression of HERG channel. Berberine shows inhibitory effect on protein expressions of in vitro HERG-HEK cells and Ikr channel in rat ventricular tissues. Liensinine improves protein expression of HERG channe in HERG-HEK cells. Neferine shows no effect on protein expression of HERG channel.

Simultaneous determination of fangchinoline and tetrandrine in Stephania tetrandra S. Moore by using 1-alkyl-3-methylimidazolium-based ionic liquids as the RP-HPLC mobile phase additives.

A reversed phase high performance liquid chromatography (RP-HPLC) method for simultaneous determination of fangchinoline (FAN) and tetrandrine (TET) in Stephania tetrandra S. Moore was established by using 1-hexyl-3-methylimidazolium tetrafluoroborate as the mobile phase additives in this paper. Four types of 1-alkyl-3-methylimidazolium-based ionic liquids (ILs) were used as additives of the mobile phase to separate FAN and TET by RP-HPLC. The effects of the length of the alkyl group on the imidazolium ring and its counterion, the concentrations of IL and the pH of the mobile phase, which influenced the chromatographic behaviors of FAN and TET, were investigated in detail. The linearity, sensitivity, accuracy and repeatability of the proposed method were also investigated. The probable mechanism of the separation with ILs as the mobile phase additives was explored and discussed.

Interaction of cepharanthine with immobilized heat shock protein 90α (Hsp90α) and screening of Hsp90α inhibitors.

Heat shock protein 90α (Hsp90α) immobilized on aminopropyl silica gels was prepared via the N- or C-terminal, which was termed Hsp90α-NT or Hsp90α-CT, respectively. Binding interactions of biscoclaurine alkaloids (cepharanthine (CEP), berbamine (BBM), isotetrandrine (ITD), and cycleanine (CCN)) with Hsp90α were examined using the Hsp90α-NT or -CT columns by frontal and zonal chromatography studies. The dissociation constants of CEP, BBM, ITD, and CCN to Hsp90α-NT were estimated to be 5.3, 18.6, 46.3, and 159 µM, respectively, by frontal chromatography techniques. Similar results were obtained with the Hsp90α-CT column. These data suggest that these biscoclaurine alkaloids interact with the middle domain of Hsp90α. This was confirmed by demonstrating that CEP competed with endothelial nitric oxide synthase at the middle domain of Hsp90α, where it was shown to have a dissociation constant of 15 nM. Furthermore, the Hsp90α-NT column was applied for preliminary screening of natural Hsp90α inhibitors by zonal chromatography studies.

Identification of pavinane alkaloids in the genera Argemone and Eschscholzia by GC-MS.

The genera Eschscholtzia and Argemone (Papaveraceae) represent a rich source of pavinane alkaloids, the identification of which in alkaloid extracts is generally problematic without standards. The alkaloid extracts of three Argemone and four Eschscholtzia species were analyzed using GC-MS. The alkaloids were identified based on comparison of their mass spectra with commercial libraries, with reported data in the literature and with spectra of reference compounds. A total of 23 alkaloids of six structural types (pavinane, protopine, benzylisoquinoline, benzophenanthridine, aporphine and protoberberine) were identified. The fragmentation pathway of pavinane alkaloids was used for their identification. O-Methylneocaryachine has been reported for the first time from a natural sources and the alkaloid pattern of Eschscholzia pulchella has been analyzed and described for the first time.