Global characterization of modifications to the charge isomers of IgG antibody / 药物分析学报

Posttranslational modifications of antibody products affect their stability,charge distribution,and drug activity and are thus a critical quality attribute.The comprehensive mapping of antibody modifications and different charge isomers(CIs)is of utmost importance,but is challenging.We intended to quanti-tatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity.The CIs of antibodies were fraction-ated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection,followed by stepwise structural characterization at three levels.First,the differences between CIs were explored at the intact protein level using a top-down mass spectrometry approach;this showed differences in glycoforms and deamidation status.Second,at the peptide level,common modifications of oxidation,deamidation,and glycosylation were identified.Peptide mapping showed nonuniform deamidation and glycoform distribution among CIs.In total,10 N-glycoforms were detected by peptide mapping.Finally,an in-depth analysis of glycan variants of CIs was performed through the detection of enriched glycopeptides.Qualitative and quantitative analyses demonstrated the dynamics of 24 N-glycoforms.The results revealed that sialic acid modification is a critical factor ac-counting for charge heterogeneity,which is otherwise missed in peptide mapping and intact molecular weight analyses.This study demonstrated the importance of the comprehensive analyses of antibody CIs and provides a reference method for the quality control of biopharmaceutical analysis.

Evaluation and optimization of content determination method of Chrysanthemi Flos in Chinese Pharmacopoeia / 中国中药杂志

This study discovered that the resolution of 3,5-O-dicaffeoylquinic acid(isochlorogenic acid A) in the content determination method of Chrysanthemi Flos in Chinese Pharmacopoeia(ChP)(2020 edition) was poor, which affected accurate quantification. We tested the method in ChP with chromatographic columns of seven brands to clarify the problems in the existing method, optimized the chromatographic conditions by adjusting the mobile phase composition and elution ratio and replacing the chromatographic column packing, and carried out the reproducibility assay for the new method. The two methods were compared for the content determination results of Chrysanthemi Flos prepared from six different varieties. As evaluated by the resolution based on different chromatographic columns of seven brands, the existing method failed to separate isochlorogenic acid A and isochlorogenic acid D well. The peaks of the two components were not completely separated on three chromatographic columns, and isochlorogenic acid A and isochlorogenic acid D generated a co-effluent peak in the other four columns. Isochlorogenic acid A and isochlorogenic acid D could be completely separated under the optimized chromatographic conditions. The difference in the peak areas of isochlorogenic acid A+isochlorogenic acid D obtained by the optimized method and the method in ChP was not significant, with deviation less than 3.0%, which further proved that the result measured by the method in ChP was the co-effluent of isochlorogenic acid A and isochlorogenic acid D. The optimized method can ensure the accurate quantification of isochlorogenic acid A. The existing content determination method of Chrysanthemi Flos has the problem of poor resolution. It is recommended to revise the chromatographic conditions for the content determination method of Chrysanthemi Flos to improve the resolution of isochlorogenic acid A and ensure its accurate quantification.

Effects of GYP mRNA alternative splicing on cell surface localization of MNS blood group glycoprotein GPA and GPB / 中国输血杂志

【Objective】 To analyze the polymorphisms of GYPA and GYPB mRNA spliceosomes associated with MNS blood group, and to explore the mechanism of subcellular localization of GPA and GPB protein isomerism encoded by various spliceosomes as well as the expression of MNS blood group antigen. 【Methods】 Ten blood samples of voluntary blood donors were randomly selected. The total mRNA of peripheral blood was extracted and reversed into cDNA. Nested PCR was used to amplify reading open frame of GYPA and GYPB gene, and sequencing was performed by Sanger. The base sequence obtained was compared with GYPA(NCBI: NM_002099) and GYPB(NCBI: Nm_002100.5). After the wild type and various splicing isomer of the open reading frame of GYPA and GYPB had been obtained, they were fused with the encoding gene of green fluorescent protein (GFP) by fusion PCR technology, then cloned and transfected into HEK293 cells for over expression. The subcellular localization of GPA-GFP and GPB-GFP fused fluorescent proteins was monitored by focusing laser scanning microscope. 【Results】 Exon-1 and Exon-2 were missing in GYPA mRNA of the 2 samples, and 2~26 amino acids were missing in the predicted GPA isomer, and the full length sequence of GYPB mRNA was complete. GYPA mRNA was intact in 6 samples, exon-2 was missing in GYPB mRNA, 13~45 amino acids were missing in the predicted GPB protein isomer, and other exon sequences were intact. One sample had intact GYPA mRNA, and 364~385 bases in exon-5 of GYPB mRNA were replaced by AG, indicating truncation of amino acid signal peptide. The GYP mRNA sequences of other samples were complete. The fluorescence signal of GP-GFP fusion protein showed that all GPA and GPB glycoprotein isomers, cloned according to various RNA splicing, could demonstrate the orientation distribution on the cell membrane surface, while some alternative splicing leaded to different degrees of protein dispersion in the cell, and affected the distribution speed and proportion of protein on the cell surface, which might be one of the reasons for the strength variation of MNS antigen. 【Conclusion】 The GYP mRNA spliceosome is obviously polymorphic, but the partial deletion of GYP mRNA fragment does not affect the localization and distribution of the protein isomers encoded by GYP mRNA on the cell surface, which can ensure the expression of MNS antigen characteristics.

Cytotoxic diaporindene and tenellone derivatives from the fungus Phomopsis lithocarpus / 中国天然药物

Nine new compounds, including five natural rarely-occurring 2, 3-dihydro-1H-indene derivatives named diaporindenes E-I (1-5), and four new benzophenone analogues named tenellones J-M (6-9) were isolated from the deep-sea sediment-derived fungus Phomopsis lithocarpus FS508. All the structures for these new compounds were fully characterized on the basis of spectroscopic data, NMR spectra, and ECD calculation and single-crystal X-ray diffraction analysis. The potential anti-tumor activities of compounds 1-9 against four tumor cell lines SF-268, MCF-7, HepG-2, and A549 were evaluated using the SRB method. Compound 7 exhibited cytotoxic activity against the SF-268 cell line with an IC

Rapid and simultaneous determination of 10 active components of Psoraleae Fructus in beagle dog plasma using UPLC-MS/MS and its application in pharmacokinetic study / 中国中药杂志

An UPLC-MS/MS method for rapid and simultaneous determination of psoralen, isopsoralen, apigenin, genistein, bavaisoflavone, neobavaisoflavone, bavachin, bavachinin, psoralenoside, and isopsoralenoside of Psoraleae Fructus in beagle dog plasma was established, and then the method was applied in the pharmacokinetic study after oral administration of Psoraleae Fructus extract to beagle dogs. The pharmacokinetic parameters were calculated by the software of WinNonlin. A Waters HSS-T3 column(2.1 mm×100 mm,1.8 μm)was used for liquid chromatography separation with acetonitrile-water(containing 0.004% formic acid) as the mobile phase for gradient elution.The mass spectrometry was detected using electrospray ion source(ESI) under multi-reaction monitoring mode(MRM), as well as positive ion mode. Analysis time only takes 8.5 min. The methodological study in terms of specificity, accuracy, precision, linear range, recovery, matrix effect, and stability, was validated. The LC-MS analysis method established in this experiment was simple, specific, accurate, reliable, and meet the requirement of pharmacokinetic study in plasma after administration of Psoraleae Fructus extract to beagle dogs. Six beagle dogs received intragastric administration of Psoraleae Fructus extract, T_(max) of 10 chemical components is 1.92-5.67 h; among them, C_(max) of psoralen, isopsoralen, psoralenoside and isopsoralenoside is 383-3 613 ng·mL~(-1), and AUC_(0-∞) is 3 556-18 949 ng·h·mL~(-1), t_(1/2) is 2.45-4.83 h. C_(max) of the remaining six compounds is 0.81-19.9 ng·mL~(-1), AUC_(0-∞ )is 6.54-178 ng·h·mL~(-1), t_(1/2) is 2.95-7.29 h. The UPLC-MS/MS analysis method established in this study was proved to be accurate and sensitive that it can be applied to the pharmacokinetic study of beagle dogs after oral administration of Psoraleae Fructus extract.

Antibacterial spectrum of silibinin and isomers and its antibacterial effect with antibiotics / 中草药

Objective To investigate the in vitro antibacterial spectrum of silibinin and its isomers and the combined antibacterial effect of silibinin and commonly-used clinical antibiotics. Methods Micro-broth dilution method was used to determine the minimum inhibitory concentration (MIC) of silibinin and its isomers against six standard strains and six clinical isolates (74 strains) of common bacteria infections. Plate count method was used to measure the growth inhibition curves of different concentrations of silibinin against six standard strains. Checkerboard micro-broth dilution method was used to carry out the combined susceptibility test of silibinin and clinical antibiotics. The fractional inhibitory concentration (FIC) was calculated to determine the combined antibacterial effect of silibinin and antibiotics. Results The MICs of silibinin against standard strains of Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium were between 50 μg/mL and 400 μg/mL, and those of silibinin for standard strains of Escherichia coli and Pseudomonas aeruginosa were both greater than 400 μg/mL. The MICs of silibinin for clinical strains, Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium were all within the range of 100 μg/mL to 400 μg/mL, and MICs of silibinin against clinical strains of Escherichia coli and Pseudomonas aeruginosa were greater than 400 μg/mL. The MICs of other isomers of silibinin against six standard strains were greater than or equal to 400 μg/mL. Silibinin had a significant inhibitory effect on the growth curve of standard strains of Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium, which increased with increasing drug concentration, and it had no effect on that of Escherichia coli and Pseudomonas aeruginosa. The FIC of silibinin combined with penicillin/erythromycin for gram-positive test bacteria was in the range of 0.5 2 or 1 < FIC ≤ 2. Conclusion Silibinin has a notable antibacterial activity to gram-positive bacteria with the best inhibitory effect on Staphylococcus epidermidis. The antibacterial activities of silibinin is higher than those of other isomers. The combined antibacterial effect of silibinin and penicillin/erythromycin is mainly additive and irrelevant, while that of silibinin and ciprofloxacin or gentamicin is mainly antagonistic or irrelevant.

Spectral-effect relationship between HPLC fingerprint and cell proliferation and differentiation of crude and sweated Dipsaci Radix / 中草药

Objective: To study the spectrum-effect relationships of fingerprints of sweated and crude Dipsaci Radix on cell proliferation and differentiation, and find out the material basis of efficacy before and after sweating in order to provide the basis for the impact of the efficacy. Methods: The DAD detector was used to establish HPLC fingerprints of sweated and crude Dipsaci Radix, and the relationship between the spectrum and efficiency was established by gray relational analysis. Results: The chemical composition of peaks 14 and 4 were highly correlated with the proliferation and differentiation of osteoblasts and the proliferation of MG-63 cells. The correlations were all above 0.7. The three pharmacophore indicators associated much with the characteristic peaks 14, 4, 6, 16, 13, 11, and 5. Combined with the previous analysis, these peaks represented respectively asperosaponin VI, chlorogenic acid, loganin, asperosaponin IV isomers, dipsacoside X, chlorogenic acid C, and caffeic acid. Conclusion: Asperosaponin VI, chlorogenic acid, loganin, asperosaponin IV isomers, dipsacoside X, chlorogenic acid C, and caffeic acid may be the main material basis for the effect of cell proliferation and differentiation, thus affecting its efficacy.

Reverse phase high-performance liquid chromatography (RP-HPLC) system for analysis of vitamin D3 soft capsules with soybean oil matrix / 复旦学报（医学版）

Objective To develop a method for the determination and impurity analysis of vitamin D3 soft capsules with soybean oil matrix in reverse phase high performance liquid chromatography (RP-HPLC) system.Methods RP-HPLC system had Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm,5 ttm) with detection wavelength of 265 nm,column temperature of 25 ℃ and flow rate of 1 mL/min.Retention behaviors of vitamin D3 and its 3 isomers were studied by altering the mobile phase.Firstly,acetonitrile was mixed with different proportions of methanol,water and ethanol as the mobile phase to investigate the effects of these 4 mobile phase components on the retention behavior of vitamin D3 and its 3 main related substances (isomers) on a C18 column.Then,a suitable mobile phase was selected for content determination and impurity analysis according to the retention behavior study.Results The recovery was only 80.55％-84.37％ with 100％ acetonitrile as the mobile phase.The addition of ethanol in acetonitrile was found to make remarkably significant improvement.Recovery rate was achieved between 98.07 ％ and 103.23 ％ with V (acetonitrile) ∶V (ethanol) =90∶10 as the mobile phase,while improving pealk shape.The method showed good linearity [(0.52-5.2) x 10-4 t mol/L,R2＞0.999] and fine density (RSD＜2.32％) which can be used for determination.For impurities profile,it could be achieved using V (acetonitrile):V (water) =95:5 as the mobile phase which can obviate interference from soybean oil matrix.Conclusions The method established in this experiment can easily and accurately determine the content and impurity analysis of vitamin D3 soft capsules with soybean oil matrix in a RP-HPLC system.

Chemical profiling for bile acid derivatives in yak bile / 中国中药杂志

Bile acids( BAs),the major constituents of bile,are also known to be potential biomarkers of various diseases,especially liver disease. The systematic analysis of BAs is believed to be of great importance towards the clarification of the effective material basis for bile-type medicines,and the diagnosis and therapy of related diseases as well. As a part of systematic study on bile-type medicine ongoing in our group,this study lays emphasis on the isomer discrimination,and the improvement of analytical method of BAs. Further,this method was subsequently applied to elucidate in depth the chemical profile of BAs in yak bile. Regarding isomer discrimination for BAs,we constructed relative response-collision energy curves( RRCECs) by high performance liquid chromatographyion trap-time of flight-mass spectrometry( HPLC-IT-TOF-MS) in combination with high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry( HPLC-Qtrap-MS). As a result,both the optimum collision energy( OCE) and CE_(50) exhibited great correlations with structural characteristics,thus enabling the isomer distinguishing,such as unconjugated BAs,glycine-conjugated BAs,and taurine-conjugated BAs. According to information provided by mass spectrometry,the comparison of OCE and CE_(50),retention time matching,combined with reference substances and database retrieval,a total of 30 bile acid derivatives were observed and identified in yak bile. The newly developed method could serve as a feasible tool for the in-depth characterization of BAs in bile and biological samples.

Three resin glycosides isolated from Argyreia acuta, including two isomers / 中国天然药物

In the present study, three compounds were isolated from Argyreia acuta, among them, compounds 1 and 2 were new and Compounds 1 and 3 were isomers. They were separated by several types of columns, such as normal phase, RP, size exclusion and preparative HPLC, and their structures were elucidated by several spectroscopic methods, such as 1D- and 2D-NMR and HR-TOF-MS.

Determination of three pair of triterpenoid isomers in leaf of Ilex hainanensis by HPLC-ELSD / 中国中药杂志

The present study is to develop an HPLC-ELSD method for simultaneous determination of three pairs of triterpenoid isomers, Ilexsaponin A₁, Ilexhainanoside D, Ilexgenin A, 3β, 19α-dihydroxyolean-12-ene-24, 28-dioic acid (ilexhainanin E) ursolic acid and oleanic acid in the leaf of Ilex hainanensis, which could provide evidence to the quality control of this herb. The six constituents were measured on a Waters XBridge C₁₈ column (4.6 mm×250 mm, 5 μm), with a mobile phase consisting of methanol (A)- 0.5% formic acid in water (B) at a flow rate of 1.0 mL·min⁻¹ (0-18 min，70%-85% A，18-20 min，85%-95% A；20-35 min，95% A). The carrier gas was N₂, and the pressure was 2.8 L·min⁻¹. The drift tube in this experiment were set at 70 °C. The injection volume was 10 μL. The contents of the six triterpenoids in 6 samples were 3.7-8.5, 10.3 -22.1, 2.8-5.9, 7.8-14.1, 2.6-3.8 and 8.8-11.9 mg·g⁻¹, respectively. The established method is proved to be accurate and sensitive for the determination of triterpenoids in Ilicis Hainanensis Folium, and may be used for the quality improvement of this herb.

Anesthesia of tambaqui Colossoma macropomum (Characiformes: Serrasalmidae) with the essential oils of Aniba rosaeodora and Aniba parviflora and their major compound, linalool

The aim of this study was to determine the anesthetic efficacy of the essential oils (EOs) of Aniba rosaeodora (EOAR) and Aniba parviflora (EOAP) and one of their main compounds, linalool, in two forms: synthetic and extracted from EOAR (linalool-AR) in tambaqui (Colossoma macropomum). In the first experiment, the anesthetic induction and recovery of juveniles exposed to 25- 200 µL L-1 of EOAR or 50- 300 µL L-1 of EOAP or synthetic linalool or linalool-AR was evaluated. The second experiment observed the behavioral effects of long-term exposure (12h) of these EOs and linalools (5 and 10 µL L-1). Fish exposed to 50-200 µL L-1 of EOAR and 100-300 µL L-1 of EOAP and both linalools reached deep anesthesia between 1-10 min. Induction time for all anesthesia stages decreased with the increasing concentration of the anesthetics. Linalool-AR showed lengthier time for anesthesia induction in some stages and for recovery at 100 and 200 µL L-1 in comparison to synthetic linalool. Normal equilibrium and swimming behavior was observed in fish exposed to the EOs and linalools throughout the 12 h of exposure. In conclusion, both EOs and linalools can be used as anesthetics and sedatives in tambaqui.(AU)

O objetivo deste trabalho foi determinar a eficácia anestésica dos óleos essenciais (OEs) de Aniba rosaeodora (OEAR) Aniba parviflora (OEAP) e um de seus compostos majoritários, linalol, em duas formas: sintética e extraída a partir de OEAR (linalol-AR) em tambaqui (Colossoma macropomum). No primeiro experimento avaliou-se a indução anestésica e a recuperação de juvenis expostos a 25- 200 µL L-1 de EOAR ou 50- 300 µL L-1 de EOAP ou linalol sintético ou linalol-AR. No segundo experimento observaram-se os efeitos comportamentais de uma longa exposição (12h) a estes OEs e linalois (5 e 10 µL L-1). Os peixes expostos a 50-200 µL L-1 de EOAR e 100-300 µL L-1 de OEAP e ambos os linalois alcançaram anestesia profunda entre 1-10 min. Tempo de indução a todos os estágios de anestesia diminuiu com o aumento na concentração dos anestésicos. Linalol-AR levou um maior tempo para induzir anestesia e para recuperação com 100 e 200 µL L-1 em comparação ao composto sintético. Peixes expostos aos OES e linalois por 12 h apresentaram equilíbrio e comportamento natatório normais. Em conclusão, tanto os OEs como o linalol sintético ou natural de AR podem ser usados como anestésicos e sedativos em tambaqui.(AU)

Three resin glycosides isolated from Argyreia acuta, including two isomers / 中国天然药物

In the present study, three compounds were isolated from Argyreia acuta, among them, compounds 1 and 2 were new and Compounds 1 and 3 were isomers. They were separated by several types of columns, such as normal phase, RP, size exclusion and preparative HPLC, and their structures were elucidated by several spectroscopic methods, such as 1D- and 2D-NMR and HR-TOF-MS.

8,4'-Oxyneolignane glucosides from an aqueous extract of "ban lan gen" (root) and their absolute configurations

Three pairs of glycosidic 8,4'-oxyneolignane diastereoisomers, named isatioxyneolignosides A-F (-), were isolated from an aqueous extract ofroots. Their structures and absolute configurations were elucidated by comprehensive spectroscopic data analysis and enzyme hydrolysis. The validity of Δvalues to distinguishandaryl glycerol units and Cotton effects at 235±5 nm to determine absolute configurations at C-8 in-and their aglycones (-) are discussed.

Determination of Optical Isomers in Bortezomib by HPLC / 中国药师

Objective:To establish a method for the determination of three optical isomers in bortezomib. Methods: An HPLC method was used with a ChiralPAKAY-H normal phase chiral column (250 mm × 4. 6 mm, 5 μm). The mobile phase was n-hexane-ethanol-methanol-trifluoroaceticacid (90 ∶7. 5∶2. 5∶0. 1) and the flow rate was 0. 8 ml·min-1 . The detection wavelength was set at 270 nm. The column temperature was 40℃ and the injection volume was 5 μl. Results: The separation of bortezomib from the three optical isomers was more than 2. 0. The linear range of the three optical isomers was 0. 6-20μg·ml-1(r≥0. 999 7). The average re-covery was 104. 1%, 105. 5% and 92. 0% with RSD of 2. 3%, 2. 4% and 2. 7%, respectively (n=9). The limit of quantification and detection limit was 3 ng and 1 ng, respectively. Conclusion:The method is rapid and accurate, and can be used for the determi-nation of optical isomers in bortezomib.

Research Progresses of Determination of Perfluorinated Compounds in Environmental Water and Solid Samples / 分析化学

Perfluorinated compounds (PFCs), a group of persistent organic pollutants, have been widely detected in environmental media and posed great threat to human health. The researches on environmental pollution and health concern of PFCs are the hotspot areas. Because PFCs contain lots of homologs and isomers which are detected at trace levels (ng/g or μg/L) in environment, advanced and reliable analytical methods for determination of PFCs in environment are urgently needed. At present, studies on analytical methods of trace PFCs in environmental samples have been widely carried out in China and abroad. However, systematic review on the sample pretreatment, analytical method, and matrix effect of PFCs determination in complex environmental matrixes is relatively scarce. Therefore, this paper reviews the pretreatment methods, martix effects, and detection techniques (especailly isomers) of PFCs in environment samples (water, sediment/sluge, soil and plant). We hope that this review may provide valuable reference for the enviromental researches on PFCs.

Study on monogalactosylmonoacylglycerol in marine algae (Sargassum fusiforme) / 中草药

Objective To indentify monogalactosylmonoacylglycerol (MGMG) and analyze the chemical characteristics of sn-1 and sn-2 type isomers in Marine TCM Sargassum fusiforme. Methods The MGMG compounds were isolated by multiple chromatography methods and their structures were determined by spectroscopic methods such as NMR and HRESI-MS. The interconversion and chromatography-mass spectrometry feature of MGMG positional isomers were analyzed through HPLC and HPLC-ESI-MS/MS. Results Five MGMGs were isolated from Sargassum fusiforme, and the fatty acyl compositions were stearidonoyl, linolenoyl, arachidoyl, linoleyl, and oleyl. The sn-1 and sn-2 type of MGMG were unstable. The interconversion tended to be sn-1 type rapidly, and kept balanced content at 85:15. The relative retention time of sn-2 type was less than sn-1 type on C18 reverse phase chromatography. The abundant fragment ions of MS/MS were significantly differences. The base peak was [M+Na-Gal]+ (100%), and the peak intensity of [M+Na-RCOOH]+ was always more than 50% from sn-2 type while less than 20% from sn-1 type. Conclusion Five MGMGs were isolated from brown algae for the first time. This was the first report about the interconversion and chromatography-mass spectrometry feature of sn-1 and sn-2 type. It can use to determine the fatty acyl attachment in each MGMG.

Determination of Optical Isomers in Raltitrexed Raw Material by Modified HPLC / 中国药房

OBJECTIVE:To improve HPLC method for determining optical isomers in raltitrexed raw material.METHODS:After improved,the column was CHIRALPAK AD-H with mobile phase consisted of n-hexane-anhydrous ethanol-trifluoroacetate (70∶30∶0.1,V/V/V).The sample size was 100 μL.The flow rate of 1.0 mL/min.The detection wavelength was set at 226 rm,and column temperature was 35 ℃.RESULTS:The linear range of optical isomers was 99.68-398.7 ng(r=0.999 9).The quantitation limit was 4.945 ng,and detection limit was 1.648 ng.RSDs of precision,stability and reproducibility tests were all lower than 1.0％;the recoveries ranged 99.43％-100.14％ (RSD=0.25％,n=9).CONCLUSIONS:The optimized method is simple,accurate,sensitive and reproducible,which can be used for the determination of optical isomers in raltitrexed raw material.

Antagonistic Activity of α-Conotoxin TxIB Isomers on Rat and Human α6 /α3β2β3 Nicotinic Acetylcholine Receptors / 中国药学杂志

OBJECTIVE: To investigate antagonistic activities of three isomers of α-conotoxin TxIB on rat and human α6 /α3β2β3 nicotinic acetylcholine receptors (nAChRs). METHODS: Three disulfide bond isomers were synthesized using Fmoc chemistry, which were identified by ultra performance liquid chromatography (UPLC)and confirmed by MALDI-TOF mass spectrometry. Rat and human α6/α3β2β3 nAChRs were expressed in oocytes of Xenopus laevis, which were used to test the antagonistic abilities of the 3 isomers. RESULTS: The three isomers of α-conotoxin TxIB were synthesized successfully. The retention time of each isomer of α-conotoxin TxIB was different each other significantly. The observed molecular masses of three isomers were the same, which were consistent with their theoretical molecular mass. Their hydrophilicity orders were globular > ribbon > bead. Both rat and human α6/α3β2β3 nAChRs were expressed in oocytes well. Inhibition of three isomers of α-conotoxin TxIB on rat and human α6 /α3β2β3 nAChRs were evaluated respectively. Among the three isomers of TxIB, the activity of the globular isomer was the most potent one, which had almost same activity at rat and human α6/α3β2β3 nAChRs with corresponding IC50 of 28.2 and 32.0 nmol·L-1 respectively. However, the other two isomers, ribbon and bead isomers displayed little antagonistic effect on both rat and human α6/α3β2β3 nAChRs only with an IC50 of > 10 μmol·L-1. CONCLUSION: The synthesized globular isomer of α-conotoxin TxIB in this work has a high selectivity and potent antagonistic activity on rat and human α6/α3β2β3 nAChRs, which would be helpful for its new drug development.

GC fingerprint of volatile components in Qishen Yiqi Dropping Pills / 中草药

Objective :To establish the GC fingerprint of Qishen Yiqi Dropping Pills(QYDP)and Dalbergiaodorifera oiland identify the common chromatographic peaks by GC-MS. Methods: The GC analysis was performed on an Agilent HP-INNOWaxcapillary gas chromatography column (30 m × 0.25 mm, 0.25 μm). The temperature program was as follows: 100 ℃ for 2 min, 160 ℃ at 3 ℃/min, 190 ℃ at 1 ℃/min, 220 ℃ at 5 ℃/min, then 250 ℃ at 10 ℃/min; Nitrogen was used as the carrier gas with its flow rate 0.5 mL/min; The sample injection temperature was 250 ℃ and the split ratio was 5:1; The detector was FID with its temperature of 300 ℃. MS parameters: MS electron energy of 70 eV, ion source temperature of 230 ℃, full-ion scan mass rangem/z 35-600, solvent delay for 8 min. Results: Thirty-three common peaks were found from QYDP and 25 common peaks were found from D.odorifera oil in their GC fingerprints. Twenty-three common peaks in the fingerprint ofD.odorifera oil,such as trans-nerolidol,3,7,11-trimethyl-3,6-epoxy-1,10-dodecadien-7-ol isomers, farnesol isomers, α-santalene, norbornane, andβ-bisabolene were identified. The similarity of all batches of QYDP and D.odorifera oil were both over 0.979. Conclusion: This method is simple and reliable and can be used to evaluate the quality of QYDP and D.odorifera oil.