Tissue distribution of Qingfei Paidu Decoction based on HPLC-MS/MS / 中国中药杂志

The tissue distribution of Qingfei Paidu Decoction was studied by HPLC-MS/MS in vivo. Hypersil GOLD C_(18) column(2.1 mm×50 mm, 1.9 μm) was used for gradient elution with acetonitrile as the mobile phase A and 0.1% formic acid solution as the mobile phase B. High-resolution liquid chromatography-mass spectrometry in both positive and negative ion scanning mode and multiple response monitoring(MRM) mode was employed to analyze the behaviors of the active components of Qingfei Paidu Decoction in diffe-rent tissues. The results showed that 19, 9, 17, 14, 22, 19, 24, and 2 compounds were detected in plasma, heart, liver, spleen, lung, kidney, large intestine, and brain, respectively. The compounds belonged to 8 groups, covering 14 herbs in the prescription. After administration with Qingfei Paidu Decoction, the compounds were rapidly distributed in various tissues, especially in the lung, liver, large intestine, and kidney. The majority of the compounds displayed secondary distribution. This study comprehensively analyzed the distribution rules of the main active components in Qingfei Paidu Decoction and provided a basis for the clinical application.

Clinical application value of Huanglian Jiedu Pills in improving syndrome of excess heat and fire toxin based on phase Ⅱ clinical trial study on plasma ATP, 4-HNE, and ACTH levels / 中国中药杂志

A randomized, double-blind, placebo-controlled, multi-center phase Ⅱ clinical trial design was used in this study to recruit subjects who were in line with the syndrome of excess heat and fire toxin, and were diagnosed as recurrent oral ulcers, gingivitis, and acute pharyngitis. A total of 240 cases were included and randomly divided into a placebo group and a Huanglian Jiedu Pills group. The clinical efficacy of Huanglian Jiedu Pills in treating the syndrome of excess heat and fire toxin was evaluated by using the traditional Chinese medicine(TCM) syndrome scale. Enzyme-linked immunosorbent assay(ELISA) was used to determine and evaluate the levels of adenosine triphosphate(ATP), 4-hydroxynonenal(4-HNE), and adrenocorticotropic hormone(ACTH) in plasma of the two groups before and after administration and to predict their application value as clinical biomarkers. The results showed that the disappearance rate of main symptoms in the Huanglian Jiedu Pills group was 69.17%, and that in the placebo group was 50.83%. The comparison between the Huanglian Jiedu Pills group and the placebo group showed that 4-HNE before and after administration was statistically significant(P<0.05). The content of 4-HNE in the Huanglian Jiedu Pills group decreased significantly after administration(P<0.05), but that in the placebo group had no statistical significance and showed an upward trend. After administration, the content of ATP in both Huanglian Jiedu Pills group and placebo group decreased significantly(P<0.05), indicating that the energy metabolism disorder was significantly improved after administration of Huanglian Jiedu Pills and the body's self-healing ability also alleviated the increase in ATP level caused by the syndrome of excess heat and fire toxin to a certain extent. ACTH in both Huanglian Jiedu Pills group and placebo group decreased significantly after administration(P<0.05). It is concluded that Huanglian Jiedu Pills has a significant clinical effect, and can significantly improve the abnormal levels of ATP and 4-HNE in plasma caused by the syndrome of excess heat and fire toxin, which are speculated to be the effective clinical biomarkers for Huanglian Jiedu Pills to treat the syndrome of excess heat and fire toxin.

Mechanism of Xiaoer Chiqiao Qingre Granules in clearing heat and removing food stagnation in suckling rats with fever and food accumulation based on metabolomics / 中国中药杂志

Children's fever is often accompanied by food accumulation. Traditional Chinese medicine believes that removing food stagnation while clearing heat of children can effectively avoid heat damage. To systematically evaluate the efficacy of Xiaoer Chiqiao Qingre Granules(XRCQ) in clearing heat and removing food accumulation and explore its potential mechanism, this study combined suckling SD rats fed with high-sugar and high-fat diet with injection of carrageenan to induce rat model of fever and food accumulation. This study provided references for the study on the pharmacodynamics and mechanism of XRCQ. The results showed that XRCQ effectively reduced the rectal temperature of suckling rats, improved the inflammatory environment such as the content of interleukin-1β(IL-1β), interleukin-2(IL-2), interferon-γ(IFN-γ), white blood cells, and monocytes. XRCQ also effectively repaired intestinal injury and enhanced intestinal propulsion function. According to the confirmation of its efficacy of clearing heat, the thermolytic mechanism of XRCQ was further explored by non-targeted and targeted metabolomics methods based on LTQ-Orbitrap MS/MS and UPLC-QQQ-MS/MS. Non-target metabolomics analysis of brain tissue samples was performed by QI software combined with SIMCA-P software, and 22 endogenous metabolites that could be significantly regulated were screened out. MetaboAnalyst pathway enrichment results showed that the intervention mechanism was mainly focused on tyrosine metabolism, tricarboxylic acid cycle, inositol phosphate metabolism, and other pathways. At the same time, the results of targeted metabolomics of brain tissue samples showed that XRCQ changed the vitality of digestive system, and inhibited abnormal energy metabolism and inflammatory response, playing a role in clearing heat and removing food stagnation from multiple levels.

Component profile analysis of Sanhan Huashi Formula based on UHPLC-LTQ-Orbitrap-MS, GC-MS, and UHPLC-QQQ-MS/MS / 中国中药杂志

This study comprehensively analyzed the active components of Sanhan Huashi Formula using qualitative and quantitative mass spectrometry techniques, laying the foundation for understanding its pharmacological substance basis. UHPLC-LTQ-Orbitrap-MS and GC-MS technologies were used to analyze and identify the volatile and non-volatile components in Sanhan Huashi Formula. UHPLC-QQQ-MS/MS technology was used to simultaneously determine the content of 27 major active components in the formula. The results showed that 308 major chemical components were identified in Sanhan Huashi Formula, among which 60 compounds were identified by comparing with reference standards, mainly including alkaloids, flavonoids, coumarins, triterpenoid saponins, amino acids, and nucleosides. GC-MS technology preliminarily identified 52 volatile compounds, with γ-eudesmol and β-eudesmol as the main components. The quantitative results demonstrated good linearity(r>0.99) for the 27 active components, indicating the stability, simplicity, and reliability of the established method. Among them, amygdalin, nodakenin, arecoline, ephedrine, and pseudoephedrine had relatively high content and were presumably the main pharmacologically active substances. In conclusion, this study systematically and comprehensively characterized the major chemical components and patterns in Sanhan Huashi Formula, providing a basis for understanding its pharmacological mechanisms and clinical applications.

Study on biomarkers of acteoside in treating puromycin aminonucleoside nephropathy in young rats based on non-targeted urine metabolomics technology / 中国中药杂志

This study aims to reveal the endogenous metabolic characteristics of acteoside in the young rat model of purinomycin aminonucleoside nephropathy(PAN) by non-targeted urine metabolomics and decipher the potential mechanism of action. Biochemical indicators in the urine of rats from each group were determined by an automatic biochemical analyzer. The potential biomarkers and related core metabolic pathways were identified by ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) combined with principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). MetaboAnalyst 5.0 was used to establish the receiver operating characteristic(ROC) curve for evaluating the clinical diagnostic performance of core metabolites. The results showed that acteoside significantly decreased urinary protein-to-creatinine ratio in PAN young rats. A total of 17 differential metabolites were screened out by non-targeted urine metabolomics in PAN young rats and they were involved in phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. Thirtten differential metabolites were screened by acteoside intervention in PAN young rats, and they were involved in phenylalanine metabolism and arginine and proline metabolism. Among them, leucylproline and acetophenone were the differential metabolites that were significantly recovered after acteoside treatment. These pathways suggest that acteoside treats PAN in young rats by regulating amino acid metabolism. The area under the curve of two core biomarkers, leucylproline and acetophenone, were both greater than 0.9. In summary, acteoside may restore amino acid metabolism by regulating endogenous differential metabolites in PAN young rats, which will help to clarify the mechanism of acteoside in treating chronic glomerulonephritis in children. The characteristic biomarkers screened out have a high diagnostic value for evaluating the treatment of chronic glomerulonephritis in children with acteoside.

Application of Three Blood Stasis Models in Zebrafish in Evaluation of Anti-thrombosis and Anti-myocardial Hypoxia Activities of Notoginseng Radix et Rhizoma / 中国实验方剂学杂志

ObjectiveTo establish blood stasis models in zebrafish using three inducers and select the optimal model for evaluating the activity of Notoginseng Radix et Rhizoma in promoting blood circulation. MethodArachidonic acid （AA）， ponatinib， and isoprenaline （ISO） were used to induce blood stasis models in zebrafish. A normal group， a model group， a positive drug group， and Notoginseng Radix et Rhizoma water extract freeze-dried powder groups at different concentrations were set up. The staining intensity of cardiac erythrocytes and the fluorescence intensity of cardiac apoptotic cells were calculated， the anti-thrombotic effect and anti-myocardial hypoxia activity of Notoginseng Radix et Rhizoma were evaluated. The activities of water extract and 70% methanol extract of Notoginseng Radix et Rhizoma were compared based on the preferred AA- and ISO-induced blood stasis models in zebrafish and the difference in the chemical composition was analyzed by UHPLC LTQ-Orbitrap MS/MS. ResultAfter induction by AA and ponatinib， the staining intensity of cardiac erythrocytes was reduced （P<0.01）， and the fluorescence intensity of cardiac apoptotic cells increased after the induction by ISO （P<0.01）. The freeze-dried powder of the water extract of Notoginseng Radix et Rhizoma could antagonize the thrombosis in the AA-induced model （P<0.01） and the myocardial apoptosis in the ISO-induced model （P<0.05）， while no significant improvement in the thrombosis was observed in the ponatinib-induced model. The freeze-dried powder of 70% methanol extract of Notoginseng Radix et Rhizoma could inhibit myocardial apoptosis in the ISO-induced blood stasis model （P<0.01）， and the effect was stronger than that of the freeze-dried powder of Notoginseng Radix et Rhizoma water extract. The difference in chemical composition lay in some saponins （such as ginsenoside Re）， amino acids， and acetylenic alcohols. ConclusionAA， ponatinib， and ISO all can serve as inducers for the blood stasis model in zebrafish. AA- and ISO-induced models can be used to evaluate the activity of freeze-dried powder of Notoginseng Radix et Rhizoma water extract in promoting blood circulation. The chemical compositions of the freeze-dried powders of Notoginseng Radix et Rhizoma extracted with water and 70% methanol are quite different. For the ISO-induced blood stasis model， the freeze-dried powder of Notoginseng Radix et Rhizoma extracted with 70% methanol has a stronger ability against myocardial hypoxia. Saponins and acetylenic alcohols may be closely related to the effects of promoting blood circulation and resolving blood stasis.

Comprehensive evaluation system for quality of Chinese medicinal decoction pieces based on "experience-ingredients-activity-electronic sensing" / 中国中药杂志

Quality evaluation of Chinese medicinal decoction pieces is vital for the development of the downstream industries, and is an important channel for implementing the strategy of "higher quality, higher price, and priority for the high quality" for traditional Chinese medicine. At the moment, the quality of Chinese medicinal decoction pieces is mainly evaluated based on chemical component examination. Considering the weak preliminary research foundation and poor research conditions, traditional experience-based evaluation is undervalued in the quality rating of Chinese medicinal decoction pieces. However, traditional experience is a summary of the quality of Chinese medicinal materials based on clinical experience, which thus can be a potential basis for the quality evaluation of the decoction pieces. It is a challenge in the evaluation of Chinese medicinal decoction pieces to objectify the traditional experience-based evaluation from multiple aspects such as chemistry, effect, and characterization via modern techniques. Therefore, this study developed the "experience-ingredients-activity-electronic sensing" evaluation system for Chinese medicinal decoction pieces on the basis of experience-based assessment, chemical ingredients that can truly reflect the traditional experience, biological effect assessment, and electronic sensory evaluation, which is expected to quantify the traditional experience of quality evaluation of Chinese medicinal decoction pieces via chemistry, biology, and sensory simulation. The evaluation system can serve as a reference for clinical experience-based quality evaluation of Chinese medicinal decoction pieces.

Historical Evolution and Modern Clinical Application of Qingxin Lianzi Yin / 中国实验方剂学杂志

Qingxin Lianzi Yin （QXLZY）， as an ancient classical formula for clearing the heart and nourishing the Yin， was composed of nine herbs （Scutellariae Radix， Ophiopogonis Radix， Lycii Cortex， Plantaginis Semen， Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle， Nelumbinis Semen， Poria， Astragali Radix and Ginseng Radix et Rhizoma）， coming from Prescriptions of the Bureau of Taiping People's Welfare Pharmacy. It could clear away the heart-fire， promote the interaction of the heart and kidney， replenish Qi and Yin， and stop strangury-turbidity. It was used to treat gonorrhea of urination， seminal emission， restlessness， wasting-thirst and so on. At present， the usage and dosage of QXLZY and its addition and subtraction are different in clinical practice. Most of the studies just focus on its clinical efficacy， and there is few review literature reflecting its historical evolution. Based on this， this paper systematically clarified the historical evolution， composition， preparation， interpretation， function， and modern clinical application of QXLZY. This work has been explained the historical evolution of QXLZY， and found that it was wildly used in modern clinical， especially suitable for the treatment of chronic urinary system diseases. At the same time， QXLZY also had significant therapeutic effects on neurasthenia， stomatitis， diabetic nephropathy and other aspects. Through the comprehensive analysis of ancient and modern literature， this work explores the true connotation of QXLZY from the perspective of traditional Chinese medicine theory， which can point out the direction of the clinical application and positioning of this famous classical formula after it comes into the market， and also can provide reference basis for its subsequent in-depth research and development.

Effect of Different Extracts and Monomers of Lepidium meyenii on Proliferation of Mouse Splenic Lymphocyte and Induction of IL-2 and TNF-α / 中国实验方剂学杂志

Objective:To explore effects of different extracts and monomers of <italic>Lepidium meyenii </italic>（Maca） on the proliferation of mouse splenic lymphocytes and induction of interleukin-2 （IL-2） and tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） by observing their immunomodulatory effects. Method:An octadecylsilyl （ODS） column was used to enrich the methanol extract of <italic>L. meyenii</italic> in stages to obtain six fractions and three monomers. Different groups of extracts and monomers of <italic>L. meyenii </italic>at different doses were set up. Cell counting Kit-8 （CCK-8） was used to detect the effect on the proliferation of mitogen-free， concanavalin A （Con A）-induced， and lipopolysaccharides （LPS）-induced mouse splenic lymphocytes. Enzyme-linked immunosorbent assay （ELISA） was used to determine the levels of IL-2 and TNF-<italic>α</italic>. Result:<italic>L. meyenii </italic>extracts Fr₃ and Fr₆， and monomers <italic>N</italic>-benzyl hexadecanamide and 1，2-dihydro-4-carboxaldehyde-3-benzyl-<italic>N</italic>-hydroxypyridine slightly promoted the proliferation of Con A-induced T lymphocytes and LPS-induced B lymphocytes （<italic>P</italic><0.01） as compared with the conditions in the model group. <italic>L. meyenii</italic> extracts and monomers significantly induced the secretion of IL-2 and TNF-<italic>α</italic> by splenic lymphocytes （<italic>P</italic><0.01）. Conclusion:<italic>L. meyenii</italic> extracts and monomers can achieve immunological enhancement by promoting the secretion of IL-2 and TNF-<italic>α</italic>， and facilitate the proliferation of splenic lymphocytes. The active components are presumedly macamides and pyridine alkaloids， and the specific mechanism still needs to be further explored.

Study on anti-inflammatory activity and mechanism of indolealkylamines in toad skin on LPS-activated neutrophils / 中国中药杂志

Indolealkylamines(IAAs) are the main hydrophilic substances in toad skin, mainly including free N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine, dehydrobufotenine, and binding bufothionine. In this study, the LPS-activated neutrophils were used to investigate the structure-activity relationship and anti-inflammatory mechanism of the above-mentioned five monomers from the toad skin in vitro. The neutrophils were divided into the control group, model group(1 μg·mL~(-1) LPS), positive drug group(100 μg·mL~(-1) indometacin), as well as the low-(50 μg·mL~(-1)), medium-(100 μg·mL~(-1)) and high-dose(200 μg·mL~(-1)) free N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine, dehydrobufotenine, and binding bufothionine groups. The levels of IL-6, TNF-α and IL-1β in the neutrophil supernatant of each group was measured by enzyme-linked immunosorbent assay(ELISA) after LPS stimulation, followed by the detection of apoptosis in each group after Annexin V/PI staining. The protein expression levels of caspase-3, Bax, Bcl-2, beclin1, LC3-I, and LC3-Ⅱ were assayed by Western blot. The results showed that IAAs reduced the excessive secretion of inflammatory cytokines caused by LPS compared with the model group. Besides, the activity of each free IAAs(N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine and dehydrobufotenine), especially bufotenine, was stronger than that of the binding bufothionine. As revealed by Annexin V/PI staining, LPS delayed the early apoptosis of neutrophils compared with the control group, while bufotenine promoted the apoptosis of neutrophils in a dose-dependent manner, which might be related to the elevated expression of apoptosis-related protein Bax/Bcl-2. In addition, LPS activated the autophagy pathways in neutrophils. This study confirmed the efficacy of IAAs in reducing the secretion of inflammatory cytokines in neutrophils induced by LPS for the first time. For instance, bufotenine exerts the anti-inflammatory effect possibly by inducing the apoptosis of neutrophils.

Identification of chemical constituents of Xiaoer Chiqiao Qingre Granules based on UHPLC-LTQ-Orbitrap-MS/MS and network pharmacology analysis / 中国中药杂志

This study aimed to qualitatively analyze the chemical components in Xiaoer Chiqiao Qingre Granules(XRCQ) by UHPLC-LTQ-Orbitrap-MS/MS and identify its material basis. The absorbed components in plasma were combined for exploring the potential action mechanism by integrated network pharmacology. ACQUITY UPLC HSS T3(2.1 mm×100 mm, 1.8 μm) column and mobile phase system of 0.1% formic acid solution(A)-acetonitrile(B) were used for gradient elution, followed by high resolution liquid chromatography-mass spectrometry in both positive and negative ion scanning modes. According to the precise relative molecular mass and MS/MS fragment ions, a total of 124 chemical components were identified in XRCQ by the comparison with references and literature reports, among which 29 compounds were completely confirmed by comparison with reference substances. Then, the main absorbed components of XRCQ in plasma were also analyzed and clarified by UHPLC-LTQ-Orbitrap-MS/MS. BATMAN-TCM and SwissTargetPrediction were used for target prediction of absorbed components in plasma. Following the plotting of association network with Cytoscape 3.8.2, the core targets were subjected to GO and KEGG pathway enrichment analysis and a component-target-pathway network was constructed. A total of eight main targets of XRCQ against fever in children were obtained together with eight absorbed components in plasma, including glycyrhydinic acid, hesperidin, emodin, reticuline, daidzein, magnolignan C, magnolignan A, and magnolaldehyde D. It was inferred that XRCQ might improve alimentary system abnormality, inflammatory response, oxidative stress, and endocrine disorder through tumor necrosis factor, PI3 K-AKT, and other signaling pathways. The present study comprehensively expounded the chemical profiles of XRCQ and the main absorbed components in plasma and predicted the potential mechanism of XRCQ based on integrated network pharmacology, which has provided certain theoretical reference for the clinical application of XRCQ.

Identification and attribution of chemical constituents of Qingfei Paidu Decoction based on UHPLC-LTQ-Orbitrap-MS technology / 中国中药杂志

UHPLC-LTQ-Orbitrap-MS was developed for the identification of chemical constituents in Qingfei Paidu Decoction, which will clarify its material basis. ACQUITY UHPLC HSS T3 chromatography column(2.1 mm×100 mm, 1.8 μm) was used with 0.1% formic acid(B)-acetonitrile(A) as the mobile phase in gradient elution. The decoction was detected by high-resolution liquid chromatography-mass spectrometry equipped with an ESI ion source in positive and negative mode. Based on the accurate mass measurements, retention time, mass fragmentation patterns combined with comparison of reference and literature reports, a total of 87 major compounds including 43 flavonoids, 9 alkaloids, 4 triterpenoid saponins, 1 sesquiterpene, 2 coumarins, 10 phenolic acids and 18 other compounds were tentatively screened and characterized. UHPLC-LTQ-Orbitrap-MS was employed to comprehensively elucidate the chemical components in Qingfei Paidu Decoction, which basically covered 20 Chinese medicines except gypsum in Qingfei Paidu Decoction. These collective results provide a scientific basis for further research on the quality control standard of Qingfei Paidu Decoction.

Pharmacokinetics of active ingredients of Pudilan Xiaoyan Oral Liquid in rats and effect on liver drug-metabolizing enzymes / 中国中药杂志

This study is aimed to establish a method for the determination of baicalin,baicalin and purpurin in the plasma of rats after oral administration of Pudilan Xiaoyan Oral Liquid( PDL) by using liquid chromatography-mass spectrometry( LC-MS),analyze the pharmacokinetics of three components in rats,and investigate the effects of PDL on drug-metabolizing enzymes in rat liver. C18 column was used for liquid chromatography separation,with acetonitrile-water( containing 0. 2% formic acid) as the mobile phase for gradient elution. The mass spectrometry was detected by electrospray ion source( ESI) under multi-reaction monitoring mode( MRM),as well as positive and negative ion alternating mode. Plasma sample collection was performed by using an automatic blood collection meter for small animals. The pharmacokinetic parameters were calculated by Win Nonlin software. The total protein concentration of rat liver microsomes and the total enzyme content of CYP450 were determined by BCA method and spectrophotometry respectively. The methodological study in terms of linear range,recovery rate,precision and sample stability,was used to confirm that the LC-MS analysis method established in this experiment was simple,exclusive,accurate and reliable,and can meet the requirement of determining the content of baicalin,oroxindin and corynoline in plasma after PDL administration in rats. The drug-time curve showed that baicalin and oroxindin had a bimodal phenomenon,and the pharmacokinetic parameters indicated that baicalin,oroxindin and corynoline in PDL had certain drug-like properties. After 7 consecutive days of PDL administration,the rat liver coefficient,total liver microparticle protein and CYP450 enzyme content were increased,but there was no significant difference,indicating that PDL was less likely to develop drug-drug interaction based on CYP enzyme. The results of this experiment can provide reference for the research on in vivo efficacy and drug interaction of PDL as well as on its clinical application.

Expression and Significance of Serum Mir-132 in Patients with Obstructive Sleep Apnea with Cognitive Impairment / 中山大学学报(医学科学版)

@#【Objective】To explore the relationship between the miR- 132 expression in serum and cognitive deficits of OSA. 【Methods】 66 Chinese adults age 30 to 60 years old were enrolled and categorized into two groups based on Montreal Cognitive Assessment（MoCA）scores： OSA patients with cognitive impairment（OSAI，n=36），OSA patients without cognitive impairment（OSAN，n=30），and thirty adults without OSA as healthy control group（HC，n=30）. Out- of- center cardiopulmonary sleep testing （OCST） and MoCA assessment were performed and the relative expression of miR-132 in serum was detected by PCR.【Results】No significant difference was observed in age，education，gender and hypertension（P>0.05）. The relative expression level of miR-132 was significantly up-regulated in OSAI patient′s serum compared to the OSAN and HC patients （P<0.001），and had a positive correlation with MoCA score （r=- 0.726，P<0.001）. ROC analyses showed that the areas under the curve（AUC）were statistically significant from the line of identity in OSA with cognitive impairment（AUC=0.935，95% CI：0.890- 0.981，P<0.001）and in OSA（AUC=0.787，95% CI：0.695-0.879，P<0.001）.【Conclusions】Elevated serum miR-132 expression levels are closely related to the diagnosis of OSA and its cognitive dysfunction. Detection of serum miR- 132 may be a potential indicator of cognitive dysfunction and diagnosis in OSA patients.

Identification and attribution of chemical compounds of Pudilan Antiphlogistic Oral Liquid / 中国中药杂志

UPLC-ESI-Orbitrap-MS/MS was used to analyze,identify and attribute the chemical constituents in Pudilan Antiphlogistic Oral Liquid. The analysis was performed on an Agilent Eclipse XDB-C18(4.6 mm × 150 mm,3.5 μm) with a gradient mobile phase of methanol-0.1% formic solution system at the flow rate of 0.5 m L·min-1. The sample volume was 2 μL. The column temperature was30 ℃. The high-resolution orbitrap mass spectrometry was used as detector,with electrospray ion source in both positive and negative models,and the MS scanning ranged between m/z 50 and 2 000. Based on the analysis of mass spectrometry and literature reports,79 compounds were confirmed,including 30 alkaloids,28 organic acids,18 flavonoids and 3 coumarins. Finally,39 compounds,such as rutin,esculetin,gallic acid,caffeic acid,cichoric acid,were identified from Taraxacum mongolicum; 11 compounds,such as baicalin,baicalein,apigenin,chrysin,oroxylin A,were identified from Scutellaria baicalensis; 13 compounds,such as arginine,proline,hypoxanthine,epigoitrin,indirubin,were identified from Isatis indigotica; and 18 compounds,such as dehydrocheilanthifoline,oxysanguinarine,corynoline,protopine,spallidamine,were identified from Corydalis bungeana. After the analysis of chemical model and attribution,the contents of some compounds were high in Pudilan Antiphlogistic Oral Liquid,such as baicalin,wogonoside,baicalein,wogonin,apigenin,chrysin,skullcapflavonⅡ,oroxylin A,cichoric acid,chlorogenic acid,caffeic acid,esculetin,dehydrocheilanthifoline,dihydrosanguinarine,protopine,corynoline and indirubin. The established method is simple,accurate,rapid,sensitive and reproducible,and thus suitable for the qualitative identification and quantitative determination of Pudilan Antiphlogistic Oral Liquid,which lays a foundation for the systematic quality control and the establishment of whole-course traceability system of active ingredients.

Regulation of miR-133b on Methamphetamine-induced Neuronal Apoptosis in PC12 Cells / 中山大学学报(医学科学版)

[Objective]To investigate the expression changes of miR-133b in methamphetamine(MA)-induced neuro-nal injury in PC12 cells and its regulative effects on cellular apoptosis.[Methods]PC12 cells were cultured and divided into control group and MA treated group.In MA treated group,PC12 cells were insulted with 800μmol/L MA in culture medium. The cellular injury of PC12 cells was observed under microscope. The cellular apoptosis was detected by Hoechst33342/PI double staining,and the expression level of miR-133b was examined by real-time quantitative PCR(RT-PCR). Further-more,miR-133b mimic and inhibitors were transfected into PC12 cells to analyze miR-133b's function in MA-induced cell apoptosis.[Results]The data showed that 800 μmol/L MA could induce obvious cellular injury,cause neurite shortened and increase the cell apoptosis. The RT-PCR data showed that the expression of miR-133b of PC12 cells treated with MA de-creased significantly.The apoptosis rate of PC12 cells decreased after transfection of miR-133b mimic,while increased after transfection of miR-133b inhibitors.[Conclusions]High concentration of MA causes neuron damage and induces neuronal apoptosis,and also decreases the levels of miR-133b expression. Whereas,overexpression of miR-133b can reduce the apoptosis of cultured PC12 cells.Thus,miR-133b plays a crucial role in MA mediated neurotoxicity.This study provides a theoretical basis for elucidating the mechanism of MA-induced neurotoxicity and may provide a new strategy for treating MA addiction.

Pharmacodynamics of Huanglian Jiedu decoction in Alzheimer's disease (AD) model rats and effect on improvement of inflammation microenvironment in brain / 中国中药杂志

To study the pharmacokinetics of active ingredients (alkaloids, iridoids and flavonoids) in Huanglian Jiedu decoction (HLJDD) in Alzheimer's disease (AD) model rats, and investigate its mechanism in treatment of AD. All of rats were divided into normal control group (=6), shame operation group (=6) and model group (=12). Rats in shame operation group received daily subcutaneous injection of D-galactose (D-gal 50 mg·kg⁻¹) for a total of 45 d to induce subacute aging model. Based on the operation of shame operation group, the rats in model group were given with an injection of Aβ₂₅₋₃₅ (4.0 g·L⁻¹)-ibotenic acid (2.0 g·L⁻¹) into the nucleus basalis magnocellularis. Then rats in model group were divided into HLJDD one day administration group (=6) and HLJDD one week group (=6). The plasma concentration of alkaloids, iridoid and flavonoids was determined by liquid chromatography tandem mass spectrometry (LC-QQQ-MS) at different time points. The levels of seven inflammatory factors (MIP-2, IL-1β, IL-2, IL-8, IL-10, IL-13, TNF-α) in cerebrospinalfluid (CSF) were measured by Bio-Plex multi-factor detection technology. Iridoids in AD model rats, with high bioavailability, were easily absorbed and eliminated. The plasma concentration of alkaloid was lower, and the AUC (area under the curve) was higher in HLJDD one week group than that in HLJDD one day group. The plasma concentration-time curves of flavonoids showed obvious bimodal phenomena. After the gastric administration of HLJDD, the inflammatory factors in CSF of AD rats demonstrated a callback trend, including IL-1β/IL-10 (<0.05) with significant difference. The pharmacokinetic behaviors of iridoids, alkaloids and flavonoids (41 compounds) in AD model rats were fundamentally elucidated, and HLJDD can improve the central inflammatory status of AD rats by regulating the levels of inflammatory factors.

Chemical studieson hydrophilic constituents of toad skin / 中国中药杂志

Fifteen compounds were isolated from the toad skin by a combination of various chromatographic methods including macroporous resin, silica gel, ODS and semi-preparative HPLC. Their structures were identified as 4,5-dimethyl-1,3,4,5-tetrahydropyrrolo[4,3,2-de]quinolin-6-ol(1), serotonin(2), N-methyl serotonin(3), O-methyl bufotenine(4), 1,2,3,4-tetrahydro-6-hydroxy-β-carboline(5), O-methylserotonin(6), glycinebetaine(7), caffeine(8), bufotenine(9), shepherdine(10), tryptophan(11), (5-hydroxy-1H-indol-3-yl)acetic acid(12), 5-hydroxy tryptophol(13), 2-methyl-6-hydroxy-1,2,3,4-tetrahydro-β-carboline(14), bufothionine(15). Among them, compound 1 was a new compound,compound 5 was a new natural product. Compounds 4-8 and 10-14 were separated from toad skin for the first time.

Study on characteristic spectrum of ingredients from different species Cordyceps and its anti-fibrotic activity on human embryonic fibroblasts / 中国中药杂志

In this study, 10 samples of parasites, cursive, and the whole from six different species of Cordyceps were determined and compared by HPLC and LC-MS methods. Uridine, adenosine, and cordycepin were selected as the main evaluation index. The anti-fibrotic activity of different species Cordyceps extracts was observed using in vitro TGF-β1-induced ECM accumulation in human embryonic fibroblasts CCC-ESF-1. The results demonstrated that the number of atoms and hyphae ingredients of different species showed little difference, however, the content distribution of each component has obvious significance. The in vitro anti-fibrotic activities of different species were as follow： Cordyceps flower > Cicada Cordyceps (Cicada flower)> Silkworm Cordyceps> Tussah Cordyceps>natural Cordyceps. Our preliminary data could serve as reference for the discovery of artificial alternatives of natural Cordyceps.

Study of screening pseudoallergenic substances of Shuanghuanglian injection / 中国中药杂志

In this study, chemistry, biology and pharmacology were combinated to screen pseudoallergenic substances of Shuang-huanglian injection (SHLI) so that to establish a scientific and systematic approach to screen pseudoallergenic substances of traditional Chinese medicine injections. The mouse pseudoallergic reaction models were used to screen the pseudoallergic reaction of SHLI's intermediate extract and the intermediate extract's component or ingredient. Among the three intermediates of Shuanghuanglian injection (extract of Scutellaria baicalensis, extract of Lonicera japonica, extract of Forsythia suspensa) , pseudoallergic action of Forsythia suspensa was the strongest, Forsythia suspesnsa's pseudoallergic reaction mainly associated with the composition with largerchemical polarity. Further it was found that forsythiaside A and arctiin which existed in the the composition with largerchemical polarity caused obvious pseudoallergic reactions. SHLI with removal forsythoside A with the technology of HPLC-MS displayed reduced pseudoallergic reaction and a significant improved safety. This study provided a scientific basis for SHLI process improvements and also offered idea and research foundation for screening pseudoallergenic substances injections in other TCM injections.