Content determination of seventeen amino acids in Gualoupi Injection and its intermediates and research on their change laws / 中成药

AIM To determine the contents of aspartic acid,glutamic acid,serine,glycine,threonine,citrulline,arginine,alanine,γ-amino-butyric acid,tyrosine,valine,phenlalanine,isoleucine,ornithine,leucine,lysine and proline in Gualoupi Injection and its intermediates,and to analyze their change laws.METHODS The OPA-FMOC online derivatization analysis was performed on a 45℃ thermostatic Waters XBridge C18 column(4.6 mm×100 mm,3.5 μm),with the mobile phase comprising of phosphate buffer solution-[methanol-acetonitrile-water(45 : 45 : 10)]flowing at 1 mL/min in a gradient elution manner,and the detection wavelengths were set at 262,338 nm.Principal component analysis and heatmap analysis were adopted in chemical pattern recognition for the corresponding intermediates in ten processes of six batches of samples.RESULTS Seventeen amino acids showed good linear relationships within their own ranges(R2>0.998 0),whose average recoveries were 83.4％-119.5％with the RSDs of 0.91％-7.94％.Different batches of samples in the same process were clustered,and the corresponding intermediates in different processed were clustered into three groups.Alcohol precipitation and cation exchange column demonstrated the biggest influences on amino acid composition.CONCLUSION This experiment can provide important references for the critical factors on quality control of Gualoupi Injection,thus ensure the stability and uniformity of final product.

Simultaneously quantitative analysis of 35 components in gualoupi injection using hydrophilic interaction liquid chromatography tandem mass spectrometry / 药学学报

A hydrophilic interaction chromatography tandem mass spectrometry method was developed for simultaneous quantification of 35 components in gualoupi injection. The analytes were separated with an ACQUITY XBridge Amide column using 20 mmol·L-1 ammonium formate aqueous solution (pH 3.0) as mobile phase A and 20 mmol·L-1 ammonium formate (pH 3.0)∶acetonitrile (1∶9) as mobile phase B for gradient elution. Mass spectrometry with dynamic multiple reaction monitoring and external standard method were used for quantitative analysis. A total of 35 components were determined in 10 batches of gualoupi injection. The results showed that the 35 compounds had a good linear relationship within their respective concentration ranges with the correlation coefficients (R2 > 0.998 0), the recoveries ranged from 76.6% to 118.5%. The results showed that γ-aminobutyric acid, trigonelline, alanine, threonine, homoserine, citrulline, and leucine were abundant in gualoupi injection, while nicotinamide, methylsuccinic acid, cytosine and choline account for a low percentege. The present study provides an important reference for elucidation of the effective material basis and the improvement of quality standard of gualoupi injection.

Characterization of natural peptides in Pheretima by integrating proteogenomics and label-free peptidomics / 药物分析学报

Pheretima,also called"earthworms",is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines(CPMs)in Chinese Pharmacopoeia(2020 edi-tion).However,its zoological origin is unclear,both in the herbal market and CPMs.In this study,a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing al-gorithms(restricted search,open search,and de novo)was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima,including Pheretima aspergillum(PA),Pheretima vulgaris(PV),and Metaphire magna(MM).We identified 10,477 natural peptides in the PA,7,451 in PV,and 5,896 in MM samples.Five specific signature peptides were screened and then validated using synthetic peptides;these demonstrated robust specificity for the authentication of PA,PV,and MM.Finally,all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills,revealing the inconsistent Pheretima species used in these CPMs.In conclusion,our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines,especially non-model species with poorly annotated protein databases.

Qualitative screening of 52 illegally adulterated weight-losing compounds in foods by ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry / 上海预防医学

ObjectiveAn analytical method was developed for the qualitative screening of 52 illegally adulterated weight-losing compounds in foods by ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. MethodsThe samples were extracted by methanol. Waters CORTECS T3 column （100 mm×2.1 mm， 2.7 μm） was used with acetonitrile -0.1% formic acid aqueous solution as mobile phases by gradient elution. The compounds were detected by electrospray ionization mass spectrometry and Q-TOF-MS detector in positive and negative ion mode. A standard spectrum library was established by reference standards， and the qualitative analysis was finished by the comparison of the retention time， parent ion and fragment ion accurate molecular mass of each compound in the sample and the library. ResultsThe method was specific without interference of blank matrix， and repeatable in sextuplicate. The detection limits of 52 compounds in 5 matrix were 1‒100 mg‧kg-1. The method was successfully applied to the analysis of actual samples，and 16 compounds were checked out in 246 samples. ConclusionThe method is accurate， specific and sensitive， which can be used to combat the illegal adulteration behavior effectively.

Determination of 7 aminoglycoside antibiotics residues in bear bile powder by mix-mode SPE-hydrophilic interaction liquid chromatography tandem mass spectrometry / 药学学报

An analytical method was developed for determination of 7 aminoglycosides antibiotics in bear bile powder by hydrophilic interaction liquid chromatography tandem mass spectrometry. The samples were purified by mix-mode weak cation exchange and reversed-phase SPE. Waters ACQUITY UPLC BEH Amide column (100 mm × 3.0 mm, 1.7 μm) was used with 0.2% formic acid aqueous solution-0.2% formic acid acetonitrile solution as mobile phases by gradient elution. The aminoglycosides were detected by electrospray ionization mass spectrometry in positive mode with multiple reaction monitoring (MRM) mode. Spectinomycin, streptomycin, amikacin, kanamycin, tobramycin, apramycin and neomycin possessed good linear correlation in the respective concentration ranges, with the correlation coefficients more than 0.99. The mean recoveries at 3 spiked levels were in the range of 61.3%～127.3%, and the RSDs were 0.1%～1.9%. The limits of quantification were 0.2～1.0 mg·kg-1. The method had been applied to the analysis of actual samples.

Differential Regulation of Integrin α5 and ß4 in Normal and Psoriatic Epidermal Keratinocytes

Abstract Psoriasis is a chronic skin inflammation, characterized by impaired differentiation, hyperproliferation of keratinocytes involving pro-inflammatory factors interleukin (IL)-13/17A, tumor necrosis factor (TNF)-α, interferon (IFN)-γ. Among the integrin family, α5 is important for blood vessel formation, and ß4 for proliferation, differentiation of keratinocytes. To investigate the expression and regulation of integrin α5 and ß4 in psoriatic keratinocytes. Skin biopsies were obtained from 14 psoriatic patients and 12 normal volunteers. We compared the immunolocalization and regulation of α5 and ß4 between the psoriatic and normal ones, before and after incubation with MEK/ERK pathway inhibitor U0126 by immunohistochemistry and western blot separately. Immunohistochemistry showed psoriatic keratinocytes had higher α5 than normal ones. According to western blot, IL-17A and IL-13 increased normal keratinocytes' α5 and ß4 respectively, but psoriatic keratinocytes were the exact opposite. Incubated with U0126, normal keratinocytes' α5 was enhanced by the 5 cytokines ; while IL-13/17A, IFN-γ suppressed ß4. Psoriatic keratinocytes' α5 was increased by IL-13/17A, decreased by IFN-γ; but ß4 increased by IL-17A, IFN-γ. IL-13/17A, TNF-α, IFN-γ regulate α5 and ß4 through ERK pathway whether normal or psoriasis. The normal and psoriatic keratinocytes respond to the same cytokines differently

Plant metabolomics for studying the effect of two insecticides on comprehensive constituents of Lonicerae Japonicae Flos / 中国天然药物

Pesticides' overuse and misuse have been reported to induce ingredient variations in herbal medicine, which is now gaining attention in the medicinal field as a form of alternative medicine. To date, available studies on pesticide-induced ingredient variations of herbal medicine are limited only on a few compounds and remain most others unexamined. In this study, a plant metabolomics-based strategy was performed to systematically explore the effects of two frequently used insecticides on the comprehensive constituents of Lonicerae Japonicae Flos (LJF), the flower buds of Lonicera japonica Thunb. Field trials were designed on a cultivating plot of L. japonica with controls and treatments of imidacloprid (IMI) and compound flonicamid and acetamiprid (CFA). Unbiased metabolite profiling was conducted by ultra-high performance liquid chromatography/quadrupole-Orbitrap mass spectrometer. After data pretreatment by automatic extraction and screening, a data matrix of metabolite features was submitted for statistical analyses. Consequently, 29 metabolic markers, including chlorogenic acids, iridoids and organic acid-glucosides were obtained and characterized. The relative quantitative assay was subsequently performed to monitor their variations across flowering developments. This is the first study that systematically explored the insecticide-induced metabolite variations of LJF while taking into account the inherent variability of flowering development. The results were beneficial for holistic quality assessment of LJF and significant for guiding scientific use of pesticides in the large-scale cultivation.

Discovery and identification of a tadalafil analogue / 药学学报

A tadalafil analogue was detected during routine screenings from two "fatigue reliever, immunity enhancer" dietary supplements by using UHPLC/Q-TOF HRMS. The MS2 spectrum of this compound was almost identical to that of 2-hydroxypropylnortadalafil. However, the retention time of this analogue was different from that of the 2-hydroxypropylnortadalafil isomers. The analogue was purified by using preparative HPLC and the structure was elucidated by mass spectrometric and NMR spectroscopic experiments. The spectral data suggested that the analogue bore a 3-hydroxypropyl group instead of the N-methyl group in tadalafil. The structure was further confirmed by comparison of the 1H NMR spectra data with those of the reference standard, and thus named as 3-hydroxypropylnortadalafil. The structure is first reported in China.

Qualitative screening of 169 veterinary drug residues in bearbile powder by QuEChERS-ultra high performance liquid chromatography-tandem mass spectrometry / 药学学报

A QuEChERS-ultra high performance liquid chromatography-tandem mass spectrometry method was developed for qualitative screening of 169 veterinary drug residues in bear bile powder, including β-agonists and inhibitors, antibiotics (penicillins, β-lactams, sulfomamides, quinolones, chloramphenicals, tetracyclines, nitroimidazoles, macrolides, polyethers, etc.), antiviral drugs, anthelminitics, steroid hormones, nonsteroidal antiinflammatory drugs (NSAIDs) and sedatives. The samples were extracted by Na2EDTA-McIlvaine buffer solution and 5% fomic acid-acetonitrile solution, then purified by dispersive solid phase extraction. Detection of veterinary drug residues by ultra high performance liquid chromatography-triple quadrupole mass spectrometry was conducted and qualitative confirmed by ion ratios. The limits of detection of 169 veterinary drugs were 1-1 000 μg·kg-1. The method is simple and fast, which had been used for the analysis of actual samples, and can be extended to the detection of similar matrix.

Study on proteins in Guangdilong by nano LC/orbitrap fusion lumos HR-MS / 中国中药杂志

In the present study,fresh Guangdilong( GD),originating from Pheretima aspergillum,was taken as the object. The total proteins from GD were firstly separated by SDS-PAGE according to their molecular weights and in-gel digestion was then performed.After that,the peptides were analyzed by nano LC/orbitrap fusion lumos high resolution mass spectrometry( nano LC/orbitrap fusion lumos HR-MS). Protein identification was implemented by comparison with Annelida. fasta database using Proteome Discoverer software.As a result,386 proteins were tentatively identified,including chain F,globin B chain,glyceraldehyde-3-phosphate dehydrogenase,fibrinolytic protein,and so on. Most of the proteins took part in cell structure and energy metabolism,and fibrinolytic protein and lombricine kinase might be related to fibrinolytic activity. Protein classification based on gene ontology was carried out using PANTHER and KEGG for metabolic pathway enrichment. The results indicated that these proteins were related to diverse signal transduction pathways,including metabolic pathways,central carbon metabolism,biosynthesis of amino acids,ribosome,glycolysis,citrate cycle( TCA cycle),and so on. This study would lay the foundation for the further research on the proteins in GD and also their functions.

Determination of ginkgolic acids in Yinxing Tongzhi Dropping Pills by mix-mode SPE-UHPLC/MS/MS / 药学学报

An analytical method was developed for determination of ginkgolic acids in Yinxing Tongzhi Dropping Pills by ultra high performance liquid chromatography-triple quadrupole mass spectrometry. The samples were purified by mix-mode anion exchange and reversed-phase SPE. A chromatographic column, Waters Cortecs T3 (50 mm×2.1 mm, 2.7 μm), was used with acetonitrile-methanol-1% acetic acid (44:44:12) as the mobile phase. The ginkgolic acids were detected by electrospray ionization mass spectrometry in negative mode with multiple reaction monitoring (MRM) mode. Ginkgolic acid C13:0, C15:1 and C17:1 possessed good linear correlation in the mass concentration range from 0.2 to 200 μg·L-1, 2 to 200 μg·L-1, 4 to 200 μg·L-1, respectively, with the correlation coefficients more than 0.999. The mean recoveries at spiked levels of 50, 250 and 600 μg·kg-1 were in the range of 70.8%-95.1%, and the RSDs were 0.7%-8.6%. The limits of quantification were 1, 10, 20 μg·kg-1, respectively. The method could be applied to the analysis of ginkgolic acids in complex matrix samples.

Rapid and intelligentized identification of the constituents in Gualoupi injection by HILIC/ESI-Orbitrap HRMS combined with Compound Discoverer software / 药学学报

A rapid identification of the constituents in Gualoupi injection was developed by HILIC/Orbitrap Fusion Lumos HRMS. ACQUITY XBridge Amide column was used to isolate the constituents with large polarity. ESI with positive ion mode was employed and "Top Speed" DDA was applied in the MS2 scan. Compound identification was carried out by using Compound Discoverer software through comparing the precursor and product ions information with those in ChemSpider and mzCloud database. As a result, 48 compounds was identified, including alkaloids, amino acid, nucleosides, nucleobases, etc., among which 25 was unambiguously identified by comparing the retention time and mass spectra information with those of reference standards. In general, the main constituents from Gualoupi injection were explained in this study. Additionally, full-scan mass spectra of 21 batches of the injection were collected by the established method. Subsequently, principal component analysis (PCA) with the peak area as the observation ID was employed to analyze the discrimination of different bathes of samples. The results indicated that the batch-to-batch difference was generally from the crude drug, and the preparation process of this injection was relatively stable. In conclusion, a rapid and effective method for the identification of compounds in Gualoupi injection was established by using UHPLC-Orbitrap Fusion Lumos HRMS and the inter-bath stability was also investigated, which may make a great contribution to the study of the bioactive ingredients in Gualoupi injection and also provide vital evidence for the standard promotion.

Control and Study on the Visible Particles in Shengmai Injection / 中国药师

Objective:To understand the basic situation of visible particles in Shengmai injection,and study the visible particles control and inspection for Shengmai injection. Methods: The samples of Shengmai injection were collected and the visible particles were checked by light inspection and light scattering,and the results were summarized and analyzed. The exogenous factors and endog-enous factors affecting the visible particles in Shengmai injection were investigated. Results:The positive inspection rate of visible par-ticles in Shengmai injection in market was low. Light,temperature and pH value had mild influence on the visible particles in Sheng-mai injection. Conclusion:The control measurement for the visible particles in Shengmai injection in every enterprise is effective,and the quality control is promising.

Ustekinumab treats psoriasis by suppressing RORC and T-box but its suppression of GATA restrains its efficacy

Psoriasis is a T-cell mediated disease that involves IL-23/Th17 and IL-12/Th1 axes. Ustekinumab, a fully human monoclonal antibody targeting the p40 subunit of both IL-12 and IL-23, has proven to be efficient and safe for treating patients with psoriasis. Yet, there have been no reports with human skin/blood samples that would elucidate the molecular mechanisms by which ustekinumab calms psoriasis skin lesions. To investigate the efficacy and molecular pathway (RORC, t-BOX and GATA) of ustekinumab in treating patients with psoriasis skin lesions. A total of 30 patients with psoriasis were randomized into placebo group and treatment group. PASI of each patient was calculated at 0, 12 and 24 weeks post-treatment. The mRNA levels of RORC, t-BOX and GATA in peripheral blood mononuclear cells separated from patients' whole blood were analyzed using qPCR. Decreased mRNA of RORC, t-BOX and GATA were observed after continuous injections, indicating that ustekinumab exerts its effect by interacting with these molecules; while no significant difference in foxp3 mRNA levels were found between placebo group and treatment group.

Effect of dexmedetomidine on stress response during tracheal intubation in patients with ICU / 药物评价研究

Objective To investigate the effect of dexmedetomidine on tracheal intubation in patients with ICU.Methods A total of 76 severe cases of patients treated in ICU of Kunming General Hospital of Chinese PLA from January 2015 to January 2016 were selected as the subjects,randomly divided into control and observation groups,38 cases in each group.The two groups underwent radial artery puncture before the trachea intubation,and the venous passage was established.The observation group received iv infusion of dexmedetomidine hydrochloride (0.5 g/kg) for 10 min,and the control group received iv injection of normal saline.Then the two groups were given iv infusion of appropriate atracurium and propofol.The anesthesia dosages of two groups were observed;The levels of angiosthenia (MAP),heart rate (HR) and plasma corticosterone were detected before intubation (T1),after intubation (T2),3 min after intubation (T3) and 5 min after intubation (T4) in two groups.Results Propofol and CIS atracurium anesthesia dose of the two groups had no significant difference.The levels of MAP,HR and plasma corticosterone in observation group were significantly lower than those in the control group at T4 and T3 time (P ＜ 0.05).Conclusion Application of dexmedetomidine anesthesia could reduce the stress response in patients with endotracheal intubation in ICU and maintain stable hemodynamics and plasma corticosterone.

Quality Standard for Anweiyang Capsules / 中国药师

Objective:To improve the quality standard for Anweiyang capsules. Methods: TLC was used to identify Glycyrrhiza inflata Bat. Licochalcone A was determined by RP-HPLC. Using a C18 Column, the mobile phase consisted of acetonitrile-0. 1% phos-phoric acid(40:60), and the detection wavelength was at 376 nm. Results:The herb could be identified by TLC. For licochalcone A, the linear range was from 25. 563 to 1 533. 798 ng, and the average recovery was 99. 8%(n=9). Conclusion:The method is simple and accurate, which can be used to improve and control the quality of Anweiyang capsules.

Determination of Deoxynivalenol in Four Traditional Chinese Herbal Medicines by LC-MS-MS / 中国药师

Objective:To develop a method for determining deoxynivalenol in four kinds of traditional Chinese herbal medicines. Methods:After being extracted by water, purified by an immunoaffinity column, deoxynivalenol was analyzed by LC-MS-MS. Results:The calibration curve was linear within the range of 2-50 ng·ml-1 for deoxynivalenol. The recovery was 68. 7%-88. 3%. Conclusion:The method is simple, sensitive and accurate in the determination of deoxynivalenol in Semen Ziziphi Spinosae, Semen Sojae Praepara-tum, Semen Coicis and Fructus Psoraleae.

Identification of a new sildenafil analogue based on Q-TOF-MS / 药学学报

The drugs such as sildenafil adulterated in herbal products and dietary supplements may endanger human health. The number of the new modified derivatives is increasing recently. Based on Q-TOF-MS, a new sildenafil analogue was found. It was isolated and purified by preparative liquid chromatography. Its structure was determined by NMR, as 1-[4-propoxy-3-(6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4, 3-d] pyrimidin-5-yl)phenylsulfonyl]-4-methylpiperazine. Compared with sildenafil, the ethoxy group of the benzene ring moiety was moved to the propoxy group, which had not been reported in China. The mass spectrometric behavior pattern of the structure type was summarized, which can greatly accelerate the structural analysis of novel analogues.

Determination of sulfur dioxide residues in sulfur fumigated Chinese herbs with headspace gas chromatography / 药学学报

This paper aims to establish a method for the determination of sulfur dioxide in sulfur fumigation Chinese herbs. Sample powder and hydrochloric acid solution were isolated by paraffin layer in order to avoid early reactions, with the generation of sulfur dioxide, headspace with airtight needle was used to transfer sulfur dioxide into gas chromatograph, and detected with thermal conductivity detector. The analytical performance was demonstrated by the analysis of 12 herbs, spiked at four concentration levels. In general, the recoveries ranging from 70% to 110%, with relative standard deviations (RSDs) within 15%, were obtained. The limit of detection (LOD) was below 10 mg x kg(-1). Standard addition can be used for low recovery samples. The method is simple, less time-consuming, specific and sensitive. Methods comparison revealed that gas chromatography is better than traditional titration in terms of method operability, accuracy and specificity, showing good application value.

Determination of sulfur dioxide residues in sulfur fumigated Chinese herbs with headspace gas chromatography / 药学学报

This paper aims to establish a method for the determination of sulfur dioxide in sulfur fumigation Chinese herbs. Sample powder and hydrochloric acid solution were isolated by paraffin layer in order to avoid early reactions, with the generation of sulfur dioxide, headspace with airtight needle was used to transfer sulfur dioxide into gas chromatograph, and detected with thermal conductivity detector. The analytical performance was demonstrated by the analysis of 12 herbs, spiked at four concentration levels. In general, the recoveries ranging from 70% to 110%, with relative standard deviations (RSDs) within 15%, were obtained. The limit of detection (LOD) was below 10 mg x kg(-1). Standard addition can be used for low recovery samples. The method is simple, less time-consuming, specific and sensitive. Methods comparison revealed that gas chromatography is better than traditional titration in terms of method operability, accuracy and specificity, showing good application value.