New insights into the chemistry and anticancer activity of Ophiocordyceps xuefengensis.

Ophiocordyceps xuefengensis (O. xuefengensis), the sister taxon of Ophiocordyceps sinensis (O. sinensis), is consumed as a "tonic food" due to its health benefits. However, little is known regarding the chemistry and bioactivity of O. xuefengensis. In this study, we characterized 80 indole-based alkaloids in the ethyl acetate fraction of O. xuefengensis by high performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS/MS), of which 54 indole-based alkaloids were identified as possibly new compounds. Furthermore, 29 of these compounds were established as potential anti-cancer compounds by ligand fishing combined with HPLC-Q-TOF-MS/MS. Moreover, molecular docking identified the NH- and OH- groups of these compounds as the key active groups. The present study has expanded the knowledge on the characteristic indole-based alkaloids and anti-cancer activity of O. xuefengensis.

Exploration of potential active ingredients and mechanism of action of Xihuang pill-medicated serum against glioma based on HPLC-Q-TOF-MS/MS, network pharmacology and experimental verification / 药学学报

Qualitative analysis of the ingredients absorbed into blood and their metabolites of Xihuang pill (XHP) were conducted using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS) technology. Network pharmacology was used to explore the potential anticancer mechanisms of the ingredients against glioma, and their specific mechanisms were validated through molecular docking and experimental verification. SD rats were intragastrically administered with XHP, and rat serum samples were collected. Ingredients absorbed into blood and their metabolites were identified based on the retention time of chromatographic peaks, accurate molecular mass, characteristic fragment ions, and comparisons with reference substances and literature data. PharmMapper and SwissTarget Prediction databases were used to obtain the targets of the XHP-medicated serum, while GeneCards, OMIM, PharmGKB, TTD, and DrugBank databases were used to obtain glioma disease targets. The "component-target" network relationship diagram was constructed using Cytoscape 3.9.1 software. The protein-protein interaction (PPI) network diagram was constructed using the STRING database, and the targets were analyzed using GO and KEGG analyses. Molecular docking was used to verify the binding ability of core targets with their corresponding compounds in XHP-medicated serum. The potential mechanism of the anti-glioma effect of 11-keto-β-boswellic acid (KBA), a representative component of XHP-medicated serum, was verified using CCK-8 and Western blot assays. A total of 40 compounds were identified in the XHP-medicated serum, including 28 prototype components and 12 metabolites. The network pharmacology results showed that elemonic acid, 3-acetyl-β-boswellic acid, KBA, α-boswellic acid, and other 5 compounds might be the active ingredients of XHP-medicated serum in the treatment of glioma. Glutathione reductase (GSR), glucose-6-phosphate dehydrogenase (G6PD), ATP-citrate lyase (ACLY), aldo-keto reductase family 1 member B1 (AKR1B1) and glutaredoxin (GLRX) were identified as key targets, involving pathways such as glutathione metabolism and the pentose phosphate pathway. Further cell experiments showed that KBA significantly inhibited the proliferation of T98G cells with an IC50 of 30.96 μmol·L-1, and KBA (30 μmol·L-1) significantly downregulated the protein expression levels of GSR in T98G cells. In summary, XHP-medicated serum may exert its anti-glioma effect by regulating GSR and G6PD-targeted pathways involved in glutathione metabolism. These results provide valuable evidence for further investigating the mechanism of XHP in treating glioma. The animal welfare and experimental procedures were approved by the Ethical Committee of Laboratory Animals at Nanjing University of Chinese Medicine (approval No. ACU221001).

The metabolite profiling of YR-1702 injection in human plasma, urine and feces by HPLC-Q-TOF-MS/MS.

YR-1702, a hybrid µ/κ/δ receptor agonist, is modified from the traditional opioid analgesic dezocine. It had shown both excellent analgesic effect and lower addiction in phase I clinical trial in China, however, the metabolic pathway of YR-1702 in humans remains unelucidated.The goals of this study are to characterise the metabolism of YR-1702 in human liver microsomes (HLMs) and patients with chronic non-cancer pain by high performance liquid chromatography-coupled with quadrupole-time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS).The results showed that a total of twelve metabolites were identified in HLMs, in which 7, 6 and 5 metabolites were also found in human plasma, urine and feces, respectively. And the major metabolic pathways include mono-hydroxylation, di-hydroxylation, dehydrogenation and glucuronidation. The locations of hydroxylation and dehydrogenation were identified by the signature fragments of the metabolites.The relative contents of the metabolites in human plasma were also evaluated, in which the main metabolite M1 notably accounting for more than 14% of the total drug exposure. This study would contribute to the understanding of the in vivo metabolite profile of YR-1702 injection for future use.

The potential mechanism of Choulingdan mixture in improving acute lung injury based on HPLC-Q-TOF-MS, network pharmacology and in vivo experiments.

Choulingdan mixture (CLDM) is an empirical clinical prescription for the adjuvant treatment of acute lung injury (ALI). CLDM has been used for almost 30 years in the clinic. However, its mechanism for improving ALI still needs to be investigated. In this study, high-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS) was applied to characterize the overall chemical composition of CLDM. A total of 93 ingredients were characterized, including 25 flavonoids, 20 organic acids, 11 saponins, nine terpenoids, seven tannins and 21 other compounds. Then network pharmacology was applied to predict the potential bioactive components, target genes and signaling pathways of CLDM in improving ALI. Additionally, molecular docking was performed to demonstrate the interaction between the active ingredients and the disease targets. Finally, animal experiments further confirmed that CLDM significantly inhibits pulmonary inflammation, pulmonary edema and oxidative stress in lipopolysaccharide-induced ALI mice by inhibiting the PI3K-AKT signaling pathway. This study enhanced the amount and accuracy of compounds of CLDM and provided new insights into CLDM preventing and treating ALI.

Dynamic changes of serum metabolite profiling in septic mice based on high performance liquid chromatography of quadrupole time of flight mass spectrometry analysis.

The objective of this study is to gain insights into the underlying metabolic transformations that occurred during the whole progression of cecal ligation and puncture (CLP)-induced sepsis, thus providing new targets for its treatment. High-performance liquid chromatography of quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS/MS) combined with multivariate statistical techniques was used to detect the s in serum from septic mice. Fifty male mice were divided into two groups, including the sham group (n = 7) and the CLP-induced sepsis group (n = 43). Animals were sacrificed at 1, 3, 5, and 7 days post-CLP and then serum were collected for metabolomic analysis. Multivariate regression analysis was carried out through MetaboAnalyst 5.0, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), to identify the s and screen out the related differential metabolites. Besides, the KEGG pathway analysis was used to analyze the related metabolic pathways in which the identified metabolites were involved. Based on the fold change (FC > 2.0 or <0.5), variable important in projection (VIP > 1.2), and P value (P < 0.05), we found 26, 17, 21, and 17 metabolites in septic mice at 1, 3, 5, and 7 days post-CLP, respectively, compared with that of the sham group. The PCA and PLS-DA pattern recognition showed a cluster-type distribution between the sham group and the CLP group. Dysregulated amino acid metabolism, as well as disturbed nucleotide metabolism, is observed. Several important metabolic pathways were identified between the sham group and the CLP group. Among them, phenylalanine metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis showed striking at day 1 post-CLP. At day 3, phenylalanine, tyrosine, and tryptophan biosynthesis changed significantly. However, as the disease process, only pyrimidine metabolism showed the most significant alternation, compared to the sham group. Several differential metabolites were identified in the CLP group compared with that of the sham group and they were presented with dynamic alternation at different time points post-CLP, indicating metabolic disturbance occurred throughout the whole sepsis progression.

Study of Active Phytochemicals and Mechanisms of Cnidii Fructus in Treating Osteoporosis Based on HPLC-Q-TOF-MS/MS and Network Pharmacology.

INTRODUCTION: This study aimed to clarify the anti-osteoporosis mechanism of Cnidii Fructus (CF) via network pharmacology and experimental verification.\ Methods: HPLC fingerprints combined with HPLC-Q-TOF-MS/MS analysis confirmed common components (CCS) of CF. Then, network pharmacology was used to investigate the anti-OP mechanism of CF, including potential anti-OP phytochemicals, potential targets, and related signalling pathway. Molecular docking analysis was carried on investigating the protein-ligand interactions. Finally, in vitro experiments were performed to verify anti-OP mechanism of CF. RESULTS: In this study, 17 compounds from CF were identified by HPLC-Q-TOF-MS/MS and HPLC fingerprints and then were further screened key compounds and potential targets by PPI analysis, ingredient-target network and hub network. The key compounds were SCZ10 (Diosmin), SCZ16 (Pabulenol), SCZ6 (Osthenol), SCZ8 (Bergaptol) and SCZ4 (Xanthotoxol). The potential targets were SRC, MAPK1, PIK3CA, AKT1 and HSP90AA1. Molecular docking further analysis indicated that the five key compounds have a good binding affinity with related proteins. CCK8 assays, TRAP staining experiments, and ALP activity assays concluded that osthenol and bergaptol inhibited osteoclast formation and promoted osteoblast bone formation to improve osteoporosis. CONCLUSION: Based on network pharmacology and in vitro experiments analysis, this study revealed that CF possessed an anti-OP effect, and its potential therapeutic effect may be involved with osthenol and bergaptol from CF.

Integrating HPLC-Q-TOF-MS/MS, network pharmacology and experimental validation to decipher the chemical substances and mechanism of modified Gui-shao-liu-jun-zi decoction against gastric cancer.

Background and aim: Gastric cancer (GC) is a common malignant tumor worldwide. Modified Gui-shao-liu-jun-zi decoction (mGSLJZ) is a clinically effective traditional Chinese medicine (TCM) compound in GC treatment. This study aimed to analyze main chemical substances of mGSLJZ and investigate active ingredients and molecular mechanism of mGSLJZ against GC. Experimental procedure: HPLC-Q-TOF-MS/MS was used to analyze chemical substances of mGSLJZ, and potential active ingredients were screened from TCMSP. The target set of mGSLJZ for GC was obtained based on SwissTargetPrediction. The PPI network was constructed to screen out core targets. GO and KEGG enrichment analyses were conducted to identify BPs, CCs, MFs and pathways. The "active ingredient-core target-pathway" regulatory network was constructed to obtain core substances. Subsequently, Oncomine, Proteinatlas and molecular docking were performed to validate these findings. The cell experiments were conducted to confirm the anti-GC effects of mGLSJZ. Results and conclusion: Forty-one potential active ingredients were filtered out from 120 chemical substances in mGSLJZ, including various organic acids and flavonoids. The top 10 key targets, 20 related pathways and 6 core medicinal substances were obtained based on network pharmacology analysis. Molecular docking results indicated that the core substances and key targets had good binding activities. The cell experiments validated that mGSLJZ and the core substances inhibited the proliferation in multiple GC cells and that mGLSJZ restrained the migration of GC. Meanwhile, the top 5 targets and top 2 pathways were verified. The rescue experiments demonstrated that mGSLJZ suppressed the proliferation and migration of GC through the PI3K/AKT/HIF-1 pathway.

[Mechanism of Didang Decoction in prevention of anti-atherosclerosis and hyperlipidemia by HPLC-Q-TOF-MS/MS and network pharmacology based on theory of "nutrients return to heart and fat accumulates in channels"].

Atherosclerosis(AS) is caused by impaired lipid metabolism, which deposits lipids in the intima, causes vascular fibrosis and calcification, and then leads to stiffening of the vascular wall. Hyperlipidemia(HLP) is one of the key risk factors for AS. Based on the theory of "nutrients return to the heart and fat accumulates in the channels", it is believed that the excess fat returning to the heart in the vessels is the key pathogenic factor of AS. The accumulation of fat in the vessels over time and the blood stasis are the pathological mechanisms leading to the development of HLP and AS, and "turbid phlegm and fat" and "blood stasis" are the pathological products of the progression of HLP into AS. Didang Decoction(DDD) is a potent prescription effective in activating blood circulation, removing blood stasis, resolving turbidity, lowering lipids, and dredging blood vessels, with the functions of dispelling stasis to promote regeneration, which has certain effects in the treatment of atherosclerotic diseases. This study employed high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS) to screen the main blood components of DDD, explored the targets and mechanisms of DDD against AS and HLP with network pharmacology, and verified the network pharmacological results by in vitro experiments. A total of 231 blood components of DDD were obtained, including 157 compounds with a composite score >60. There were 903 predicted targets obtained from SwissTargetPrediction and 279 disease targets from GeneCards, OMIM, and DisGeNET, and 79 potential target genes of DDD against AS and HLP were obtained by intersection. Gene Ontology(GO) analysis suggested that DDD presumably exerted regulation through biological processes such as cholesterol metabolism and inflammatory response, and Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis suggested that signaling pathways included lipid and atherosclerosis, insulin resistance, chemo-carcinogenesis-receptor activation, and AGE-RAGE signaling pathways in diabetic complications. In vitro experiments showed that DDD could reduce free fatty acid-induced lipid accumulation and cholesterol ester content in L02 cells and improve cellular activity, which might be related to the up-regulation of the expression of PPARα, LPL, PPARG, VEGFA, CETP, CYP1A1, and CYP3A4, and the down-regulation of the expression of TNF-α and IL-6. DDD may play a role in preventing and treating AS and HLP by improving lipid metabolism and inflammatory response, and inhibiting apoptosis with multi-component, multi-target, and multi-pathway characteristics.

Discovery of the material basis of Jiuwei Xifeng granules using pharmaco-chemistry and pharmacokinetics.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiuwei Xifeng granules (JWXF) is primarily used for the treatment of Tourette syndrome (TS) with kidney-Yin deficiency and internal stirring of liver wind. However, few studies have focused on this issue. AIM OF THE STUDY: This study aimed to clarify chemical composition of JWXF using in vitro and in vivo pharmaco-chemistry and to provide a basis for the clinical use of JWXF using a strategy of pharmacokinetics. MATERIALS AND METHODS: In this study, the chemical constituents and in vivo metabolism of JWXF were evaluated using high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS), and the time-dependent processes of the three main components in rats were detected using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS). RESULTS: A total of 75 constituents were identified, including 22 alkaloids, 21 terpenes, 15 organic acids and their derivatives, and 17 other compounds. After administration, 12 compounds were identified in rat plasma, including 11 prototypes and one metabolite. Pharmacokinetic analysis showed that the effects of gentiopicroside, gastrodin, and sweroside in rats were dose-dependent when the dose of JWXF was 1-4 g/kg. They were rapidly absorbed and did not accumulate in the plasma after 7-day continuous intragastric administration. CONCLUSIONS: JWXF consists of 75 components, including alkaloids, terpenes, and organic acids. The three main compounds, gastrodin, gentiopicroside, and sweroside, undergo rapid absorption, elimination, and dose-dependent pharmacokinetics.

Mechanism of Didang Decoction in prevention of anti-atherosclerosis and hyperlipidemia by HPLC-Q-TOF-MS/MS and network pharmacology based on theory of "nutrients return to heart and fat accumulates in channels" / 中国中药杂志

Atherosclerosis(AS) is caused by impaired lipid metabolism, which deposits lipids in the intima, causes vascular fibrosis and calcification, and then leads to stiffening of the vascular wall. Hyperlipidemia(HLP) is one of the key risk factors for AS. Based on the theory of "nutrients return to the heart and fat accumulates in the channels", it is believed that the excess fat returning to the heart in the vessels is the key pathogenic factor of AS. The accumulation of fat in the vessels over time and the blood stasis are the pathological mechanisms leading to the development of HLP and AS, and "turbid phlegm and fat" and "blood stasis" are the pathological products of the progression of HLP into AS. Didang Decoction(DDD) is a potent prescription effective in activating blood circulation, removing blood stasis, resolving turbidity, lowering lipids, and dredging blood vessels, with the functions of dispelling stasis to promote regeneration, which has certain effects in the treatment of atherosclerotic diseases. This study employed high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS) to screen the main blood components of DDD, explored the targets and mechanisms of DDD against AS and HLP with network pharmacology, and verified the network pharmacological results by in vitro experiments. A total of 231 blood components of DDD were obtained, including 157 compounds with a composite score >60. There were 903 predicted targets obtained from SwissTargetPrediction and 279 disease targets from GeneCards, OMIM, and DisGeNET, and 79 potential target genes of DDD against AS and HLP were obtained by intersection. Gene Ontology(GO) analysis suggested that DDD presumably exerted regulation through biological processes such as cholesterol metabolism and inflammatory response, and Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis suggested that signaling pathways included lipid and atherosclerosis, insulin resistance, chemo-carcinogenesis-receptor activation, and AGE-RAGE signaling pathways in diabetic complications. In vitro experiments showed that DDD could reduce free fatty acid-induced lipid accumulation and cholesterol ester content in L02 cells and improve cellular activity, which might be related to the up-regulation of the expression of PPARα, LPL, PPARG, VEGFA, CETP, CYP1A1, and CYP3A4, and the down-regulation of the expression of TNF-α and IL-6. DDD may play a role in preventing and treating AS and HLP by improving lipid metabolism and inflammatory response, and inhibiting apoptosis with multi-component, multi-target, and multi-pathway characteristics.

Identification of the main flavonoids of Abelmoschus manihot (L.) medik and their metabolites in the treatment of diabetic nephropathy.

Introduction: Huangkui capsule (HKC) is made from the ethanol extract of Abelmoschus manihot (L.) Medik [Malvaceae; abelmoschi corolla] and received approval from the China Food and Drug Administration (Z19990040) in 1999. Currently, HKC is used for treatment of the patients with diabetic nephropathy (DN) in China. The bioactive chemical constituents in HKC are total flavonoids of A. manihot (L.) Medik (TFA). The present study aims to identify the primary flavonoid metabolites in HKC and TFA and their metabolism fates in db/db mice, the animal model for the study of type 2 diabetes and DN. Methods: HKC (0.84 g/kg/d) and TFA (0.076 g/kg/d) or vehicle were respectively administered daily via oral gavage in db/db mice for 4 weeks. The metabolism fate of the main metabolites of HKC in serum, liver, kidney, heart, jejunum, colon, jejunal contents, colonic contents, and urine of db/db mice were analyzed with a comprehensive metabolite identification strategy. Results and Discussion: In db/db mice administered with HKC and TFA, 7 flavonoid prototypes and 38 metabolites were identified. The related metabolic pathways at Phases I and II reactions included dehydroxylation, deglycosylation, hydrogenation, methylation, glucuronidation, sulphation, and corresponding recombined reactions. Quercetin, isorhamnetin, quercetin sulphate, quercetin monoglucuronide, and isorhamnetin monoglucuronide presented a high exposure in the serum and kidney of db/db mice. Thereby, the present study provides a pharmacodynamic substance basis for better understanding the mechanism of A. manihot (L.) Medik for medication of DN.

[Identification of Q-markers for Cistanches Herba based on HPLC-Q-TOF-MS/MS and network pharmacology].

This study aims to establish a method for analyzing the chemical constituents in Cistanches Herba by high performance liquid chromatography(HPLC) and quadrupole-time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS), and to reveal the pharmacological mechanism based on network pharmacology for mining the quality markers(Q-markers) of Cistanches Herba. The chemical constituents of Cistanche deserticola and C. tubulosa were analyzed via HPLC-Q-TOF-MS/MS. The potential targets and pathways of Cistanches Herba were predicted via SwissTargetPrediction and DAVID. The compound-target-pathway-pharmacological action-efficacy network was constructed via Cytoscape. A total of 47 chemical constituents were identified, involving 95 targets and 56 signaling pathways. We preliminarily elucidated the pharmacological mechanisms of echinacoside, acteoside, isoacteoside, cistanoside F, 2'-acetylacteoside, cistanoside A, campneoside Ⅱ, salidroside, tubuloside B, 6-deoxycatalpol, 8-epi-loganic acid, ajugol, bartsioside, geniposidic acid, and pinoresinol 4-O-ß-D-glucopyranoside, and predicted them to be the Q-markers of Cistanches Herba. This study identified the chemical constituents of Cistanches Herba, explained the pharmacological mechanism of the traditional efficacy of Cistanches Herba based on network pharmacology, and introduced the core concept of Q-markers to improve the quality evaluation of Cistanches Herba.

Identification of Q-markers for Cistanches Herba based on HPLC-Q-TOF-MS/MS and network pharmacology / 中国中药杂志

This study aims to establish a method for analyzing the chemical constituents in Cistanches Herba by high performance liquid chromatography(HPLC) and quadrupole-time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS), and to reveal the pharmacological mechanism based on network pharmacology for mining the quality markers(Q-markers) of Cistanches Herba. The chemical constituents of Cistanche deserticola and C. tubulosa were analyzed via HPLC-Q-TOF-MS/MS. The potential targets and pathways of Cistanches Herba were predicted via SwissTargetPrediction and DAVID. The compound-target-pathway-pharmacological action-efficacy network was constructed via Cytoscape. A total of 47 chemical constituents were identified, involving 95 targets and 56 signaling pathways. We preliminarily elucidated the pharmacological mechanisms of echinacoside, acteoside, isoacteoside, cistanoside F, 2'-acetylacteoside, cistanoside A, campneoside Ⅱ, salidroside, tubuloside B, 6-deoxycatalpol, 8-epi-loganic acid, ajugol, bartsioside, geniposidic acid, and pinoresinol 4-O-β-D-glucopyranoside, and predicted them to be the Q-markers of Cistanches Herba. This study identified the chemical constituents of Cistanches Herba, explained the pharmacological mechanism of the traditional efficacy of Cistanches Herba based on network pharmacology, and introduced the core concept of Q-markers to improve the quality evaluation of Cistanches Herba.

Analysis of Chemical Constituents in Daishenning by HPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

Objective:High performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry （HPLC-Q-TOF-MS/MS） was used to identify the main chemical constituents of Daishenning. Method:Cosmosil 5 C₁₈-AR-Ⅱ column （4.6 mm×250 mm， 5 μm） was employed for chromatographic separation with mobile phase of acetonitrile （A）-0.5% formic acid aqueous solution （B） for gradient elution （0-10 min， 5%A； 10-20 min， 5%-20%A； 20-30 min， 20%A； 30-55 min， 20%-35%A； 55-65 min， 35%-55%A； 65-75 min， 55%-100%A； 75-80 min， 100%A； 80-85 min， 100%-5%A； 85-90 min， 5%A）， the flow rate was 1 mL·min^-1， column temperature was 40 ℃， and injection volume was 10 μL. Electrospray ionization （ESI）， positive and negative ion detection modes and mass scanning range of <italic>m</italic>/<italic>z</italic> 100-2 000 were selected for mass spectrometry. The main chemical constituents in Daishenning were identified by MassHunter B.06.00 software in combination with PubChem， MassBank， ChemicalBook and other databases， and reference information. Result:A total of 96 components were identified from Daishenning， including 32 flavonoids， 19 organic acids， 6 glycosides， 6 terpenoids， 5 phenylpropanoids， 8 phenols， 14 other components and 6 unknown components. Conclusion:The established method can simultaneously analyze different types of compounds in Daishenning， it is helpful for further research on the extraction and separation of main chemical components and quality control of this preparation. In addition， through the rapid identification of the chemical constituents in Daishenning， it is speculated that the main effective substances of Daishenning may be flavonoids and organic acids.

Ameliorative effects of the traditional Chinese medicine formula Qing-Mai-Yin on arteriosclerosis obliterans in a rabbit model.

CONTEXT: Qing-Mai-Yin (QMY) is a clinically used herbal formula for treating arteriosclerosis obliterans (ASO). OBJECTIVE: To evaluate the chemical constituents and effects of QMY on ASO rabbit model. MATERIALS AND METHODS: Forty-eight New Zealand rabbits were divided into six groups (n = 8): normal (normal rabbits treated with 0.5% CMC-Na), vehicle (ASO rabbits treated with 0.5% CMC-Na), positive (simvastatin, 1.53 mg/kg), and QMY treatment (300, 600, and 1200 mg/kg). ASO rabbit model was prepared by high fatty feeding, roundly shortening artery, and bovine serum albumin immune injury. QMY (300, 600 and 1200 mg/kg) was orally administered for 8 weeks. The effects and possible mechanisms of QMY on ASO rabbits were evaluated by pathological examination, biochemical assays, and immunohistochemical assays. The compositions of QMY were analysed using HPLC-Q-TOF-MS/MS analysis. RESULTS: Compared to the vehicle rabbit, QMY treatment suppressed plaque formation and intima thickness in aorta, and decreased intima thickness, whereas increased lumen area of femoral artery. Additionally, QMY treatment decreased TC, TG and LDL, decreased CRP and ET, and increased NO and 6-K-PGF1α in serum. Furthermore, the potential mechanisms studied revealed that QMY treatment could suppress expression of TNF-α, IL-6, ICAM-1 and NF-κB in endothelial tissues, and increase IκB. In addition, HPLC analysis showed QMY had abundant anthraquinones, stilbenes, and flavonoids. CONCLUSION: QMY has ameliorative effects on ASO rabbit, and the potential mechanisms are correlated to reducing inflammation and down-regulating NF-κB. Our study provides a scientific basis for the future application and investigation of QMY.

Main chemical constituents of Changyanning Tablets based on HPLC-Q-TOF-MS/MS / 中草药

Objective: To study the chemical constituents of Changyanning Tablets, a high performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS/MS) was established to recognize and classify the ingredients accurately and rapidly. Methods: Agilent ZORBAX Eclipse XDB-C18 chromatographic column (250 mm × 4.6 mm, 5 μm) was employed and the separation was performed with the mobile phase consisting of methanol-0.05% acetic acid aqueous solution. The information of accurate mass and multistage fragment ions were obtained by the monitored simultaneously for positive and negative ions. The main chemical constituents of Changyanning Tablets were identified by high resolution mass spectrometry data, combining with Pubmed, Hmdb, Massbank network database, reference literature and comparing the reference. Results: Fifty-one chemical components were finally identified in this study, including two phenylpropanoids, eight iridoids, 12 flavonoids, four tannins, 23 organic acids, and two other classes. Conclusion: This study comprehensively studies the material basis in Changyanning Tablets, which provides a basis for improving the quality evaluation system of Changyanning Tablets and lays the foundation for elucidating the active components mechanism.

Screening and identification of potential active components in Ophiopogonis Radix against atherosclerosis by biospecific cell extraction.

Atherosclerosis is a chronic disease and an important pathological process associated with cardiovascular disease. Endothelial dysfunction, vascular smooth muscle cells (VSMCs) proliferation and neutrophil activation are involved in the development of atherosclerosis. Ophiopogonis Radix is a common traditional Chinese medicine use to treat cardiovascular diseases, however, its active constituents remain to be elucidated. In this study, primary vascular endothelial cells, primary VSMCs and neutrophils were prepared, and extract of Ophiopogonis Radix (EOR) was investigated to ameliorate H2O2 induced reactive oxygen species (ROS) and nitric oxide (NO) production. The results showed that EOR decreased levels of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, its protective effects against oxidative damage of endothelia and endothelial dysfunction. Additionally, EOR treatment inhibited oxidized low-density lipoprotein-induced VSMC proliferation, phorbol-12-myristate-13-acetate-mediated ROS production and neutrophil activation, malondialdehyde production, and decreased superoxide dismutase activity and myeloperoxidase release. By HPLC-Q-TOF-MS/MS analysis, 51 compounds in EOR were identified including 22 saponins and 24 homoisoflavonoids. Then biospecific cell extraction and LC-MS technique were employed to screening the antiatherosclerosis active components in Ophiopogonis Radix. After co-cultured with EOR, the multi-effective active constituents including four saponins and two homoisoflavonoids were acquired and subsequently verified to restore properties including endothelial injury, VSMC proliferation and neutrophil activation, indicating that these compounds may be multi-effective active constituents that were responsible for atherosclerosis and the cardiovascular protection of Ophiopogonis Radix.

Phenolic Profile and Antioxidant Activity of the Edible Tree Peony Flower and Underlying Mechanisms of Preventive Effect on H2O2-Induced Oxidative Damage in Caco-2 Cells.

The entire phenolic profiles and antioxidant activities of different organs of the edible tree peony flowers (Fengdan Bai (FDB)) were analyzed. HPLC-quadrupole time-of-flight mass spectrometer (Q-TOF-MS/MS) analyses of individual phenolic compounds revealed that the petal and stamen contained higher levels of flavonoid glycosides than other organs (p < 0.05). Kaempferol-3,7-di-O-glucoside was the dominant flavonoid in these two organs, however, the calyx and ovary contained higher contents of gallic acid derivatives than other organs (p < 0.05). Hexa-O-galloyl-glucose was the dominant species in the calyx and ovary. At the same concentration of total phenolic extract (TPE), the stamen had the highest protection effect on Caco-2 cell oxidative damage induced by H2O2. The antioxidant effect was attributed to potent antioxidant capability; restored redox state due to the increased expression of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD); and improved barrier function of Caco-2 cell owing to increased zonula occludens-1 (ZO-1), CLDN3 (Claudin 3), and occludin mRNA expression. As a new resource food, the edible tree peony flower is a potential functional food material and natural antioxidants resource.

Effects of Periploca forrestii Schltr on wound healing by Src meditated Mek/Erk and PI3K/Akt signals.

ETHNOPHARMACOLOGICAL RELEVANCE: Periploca forrestii Schltr. (PF) is a traditional folk medicine in China that has been used widely for treating rheumatoid arthritis and traumatic injuries for a long history. Previously, we have roughly demonstrated that the ethanol extract of PF possessed in vitro wound healing potential, and more in depth research deserves to be conducted. AIM OF THE STUDY: The present study is aiming to fully evaluate the wound healing activity of PF in vitro and in vivo, clarify the mechanism of actions and the primary constituents responsible for wound healing. MATERIALS AND METHODS: The total extract of Periploca forrestii Schltr. (EPF) and its fraction (65% ethanol fraction, EPFE65) were obtained and evaluated on in vitro wound healing properties using mouse dermal fibroblasts (L929). Cell proliferation was tested by MTT and EdU assay, confirmed by cell cycle analysis, cell migration was evaluated by scratch and transwell assay and collagen production was also determined. Then EPFE65 was tested on in vivo wound healing activity using the excision rat models. The wounded skin of rats was topically applied with 0.1% EPFE65 once daily for 6 days with hydrogel as the carrier and the recombinant bovine basic fibroblast growth factor hydrogel (rbFGF) as positive control. Histopathology of the wounded skin on day 6 and day 12 was studied via hematoxylin and eosin (HE) staining. The expression of phosphorylation of Src, Akt and Erk1/2 was determined after the treatment with EPFE65 by western blot. In order to figure out whether the activation of Src, Akt and Erk1/2 was directly in conjunction with wound healing process promoted by EPFE65, cell proliferation and migration were tested in the presence of three inhibitors of Src, Akt and Erk1/2. Finally, the chemical composition of the effective fraction EPFE65 was analyzed by HPLC-Q-TOF-MS/MS. RESULTS: In vitro experiments suggested that EPFE65 was comparable to EPF that had potent effect on promoting L929 fibroblasts proliferation, migration and increasing collagen production. 0.1% EPFE65 hydrogel also exhibited significant effect on promoting wound healing in rats. The wound closure was significantly faster in EPFE65 and positive rbFGF group than that in negative control group since the third day post wounding (p < 0.05). Specifically, on day10-12, the wounds in EPFE65 and rbFGF group were almost healed as the wound areas diminished into 13.3-5.3% and 7.7-4.0%, while the wound in control group was still apparent with 36.8-22.1% wound area. HE staining demonstrated that EPFE65 and rbFGF group could advance re-epithelialization in the early days and promote the transition of granulation tissue into complete dermis tissue with more skin appendages resembling those of normal skin in the last days. Western blot results suggested that the active fraction EPFE65 could increase the phosphorylation of Src, Akt and Erk1/2 in both dose-dependent and time-dependent manner, whereas Akt and Erk1/2 phosphorylation caused by EPFE65 could be abolished by Src inhibition. Inhibition experiments confirmed that the activation of Src, Akt and Erk1/2 were involved in cell proliferation and migration. All of these demonstrated that EPFE65 promoted wound healing at least in part via Src mediated Mek/Erk and PI3K/Akt signaling pathways. Analysis of chemical composition of EPFE65 revealed that cardiac glycosides were major components in EPFE65, among which periplocin showed effectiveness on promoting fibroblasts proliferation indicating that cardiac glycosides in EPFE65 maybe the active compounds responsible for wound healing. CONCLUSION: The present study confirmed that EPFE65, ethanol extract of Periploca forrestii Schltr. could accelerate wound healing in vitro and in vivo through Src meditated Mek/Erk and PI3K/Akt signaling pathways.

Rapid screening and identification of monoamine oxidase-A inhibitors from Corydalis Rhizome using enzyme-immobilized magnetic beads based method.

Monoamine oxidase-A (MAO-A) is considered an important therapeutic target in depression. In order to rapidly screen and identify novel MAO-A inhibitors from natural products, a magnetic bead (MB) based drug discovery tool was developed in this study. MAO-A was first immobilized onto the surface of MBs, and the resulting MAO-A-immobilized MBs (MAO-A-MBs) were then applied to ligand fishing by combining them with high-performance liquid chromatography (HPLC) coupled with quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS). The inherent catalytic activity and kinetic parameters of the immobilized-MAO-A were determined by measuring the peak area of the oxidation product. The immobilized MAO-A activity was found to remain over 80% after storage at 4 °C for about 7 days. Seven compounds (tetrahydrocolumbamine, protopine, jatrorrhizine, glaucine, tetrahydropalmatine, palmatine, dehydrocorydaline) with high binding affinity to MAO-A were fished out from the ethyl acetate fraction extract of Corydalis Rhizome. Their MAO-A inhibitory activity was further verified by enzymatic inhibition assay. These results show that the developed approach using MAO-A-MBs combined with HPLC-Q-TOF-MS/MS is suitable for the fast screening and identification of MAO-A inhibitors in complex mixtures.