Proteomics and UHPLC-DAD-Q/Orbitrap-MS used to identify impurities in andrographolide.

INTRODUCTION: Andrographolide active pharmaceutical ingredient (API) is a semidiurnal diterpene lactone with significant antipyretic, antiviral, anti-inflammatory and anticancer activities. A large amount of andrographolide API could only be obtained by extraction from Andrographis paniculata. Therefore, there may be related compounds, plant proteins and other impurities in andrographolide API. OBJECTIVE: In order to improve the safety of andrographolide related preparations, it was necessary to clarify the impurities and improve the quality standard of andrographolide API. METHODS: The related compounds were identified by ultrahigh-performance liquid chromatography with diode-array detector quadrupole Orbitrap mass spectrometry (UHPLC-DAD-Q/Orbitrap-MS), and the residual proteins were determined by ultrafiltration combined with proteomics. The proteomics method included protein extraction, content determination, digestion, desalination and nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) analysis. Then, MS results were compared with Andrographis paniculata protein database by Peaks Studio. RESULTS: The results showed that 32 related compounds were putatively identified, of which 30 impurities were identified for the first time. Seven residual proteins together with 11 highly suspected proteins were uniquely identified, including the T1UNN5_ANDPA protein with the highest intensity. CONCLUSIONS: This study will provide useful information on the composition of andrographolide API, which is important for the quality control and clinical safety assurance of API and related prescriptions. Reasonable guidance will also be provided on the necessity of ultrafiltration in the production process of related injections.

Two New Isomeric Andrographolides with Anti-Inflammatory and Cytotoxic Activity.

Two novel epimerized andrographolides, 8,17-dihydro-7,8-dehydroandrographolide and 10ß-8,17-dihydro-7,8-dehydroandrographolide, were isolated from andrographolide sulfonates. Their structures were elucidated by detailed NMR analysis, single-crystal X-ray diffraction and quantum chemical ECD calculations. In addition, these compounds exhibited suppression of NO production in LPS-stimulated RAW264.7 cells over the range of 1.564 to 25.000 µg/mL.

[Chemical profiling for bile acid derivatives in yak bile].

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

Salvianolic acids from Salvia miltiorrhiza Bunge and their anti-inflammatory effects through the activation of α7nAchR signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiovascular disease (CVD) is a serious disease with a high incidence rate and mortality. Inflammation is closely related to the occurrence of CVDs. As an essential medicine of promoting blood circulation and removing blood stasis in China, Salvia miltiorrhiza Bunge (Danshen) is widely used to treat CVDs due to its anti-inflammatory and cardiovascular protective effects. Salvianolic acids are the most abundant component in the water extract of S. miltiorrhiza, which has a significant effect on the treatment of CVDs. However, due to the complex composition of salvianolic acids, the active molecules and their underlying mechanisms have not been fully explored. AIM OF THIS STUDY: The present study aims to isolate and identify salvianolic acids from Danshen with anti-inflammatory activity and explore the potential mechanisms of isolates. METHODS: The structures of isolated salvianolic acids were elucidated by UV, IR, NMR, MS and electronic circular dichroism (ECD) calculations. Then anti-inflammatory activities of isolates were screened out by the zebrafish inflammation models. The most active compound was further used to explore the anti-inflammatory mechanisms on LPS-stimulated RAW 264.7 cells. The key inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of STAT3, p-STAT3 (Tyr705), NF-κB p65, IκBα, p-IκBα (Ser32) and α7nAchR were determined by Western blotting. The nuclear translocation of p-STAT3 (Tyr705) and NF-κB p65 was evaluated by immunofluorescence assays. Finally, the in vivo anti-inflammatory mechanisms were investigated by observation of neutrophil migration, H&E staining, survival analysis and quantitative PCR (Q-PCR) in LPS-microinjected zebrafish. RESULTS: Two new and four known compounds were isolated from Danshen. Among them, isosalvianolic acid A-1 (C1) and ethyl lithospermate (C5) inhibited neutrophil migrations in three zebrafish inflammation models and C1 with the best activities decreased the secretion of IL-6 and TNF-α and inhibited the expression level of p-IκBα (Ser32) in LPS stimulated RAW 264.7 cells. In addition, C1 also reduced the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Moreover, C1 significantly upregulated the protein expression of α7nAchR, and the knockdown of α7nAchR counteracted the effects of C1 on the production of IL-6 and TNF-α and the expression levels of p-STAT3 (Tyr705), NF-κB p65 and p-IκBα (Ser32). In vivo experiments, C1 decreased the migration and infiltration of inflammatory cells, increased the survival ratio and inhibited the mRNA level of IL-6, TNF-α, STAT3, NF-κB and IκBα in LPS-microinjected zebrafish. CONCLUSION: Two new and four known compounds were isolated from Danshen. Among them, C1 exerted anti-inflammatory activities by activating α7nAchR signaling and subsequently inhibiting STAT3 and NF-κB pathways. This study provided evidence for the clinical application of Danshen and contributed to the development of C1 as a novel in the treatment of cardiovascular disease.

A new coumarin isolated from the roots of Heracleum dissectum Ledeb.

A new coumarin named (9 R, 10 R)-9, 10-dihydro-10-hydroxy-9-methoxy-bergapten (1) and 13 known compounds (2-14) were isolated from the roots of Heracleum dissectum Ledeb., in which compounds (2-13) were obtained from H. dissectum for the first time. Their structures were illuminated by HR-ESI MS, 1 D and 2 D NMR, optical rotation and comparison with literatures. All compounds were evaluated against hepatocellular carcinoma HepG2 cell lines and the results showed that candinol C (8) had moderate cytotoxic activity against HepG2 cells with IC50 value at 57.6 ± 1.1 µM.

Bufotenines-loaded liposome exerts anti-inflammatory, analgesic effects and reduce gastrointestinal toxicity through altering lipid and bufotenines metabolism.

Bufotenines, a natural component from toad venom, showed great potential for development as a novel anti-inflammation and analgesia agent, but the potential toxicity limited its clinic use. In this paper, bufotenines-loaded liposome was prepared and optimized. Then, the therapeutic effects and drug safety of bufotenines-liposome were investigated against inflammation and pain on animal models, with a focus on gastrointestinal toxicity. Bufotenines and its liposome significantly increased paw withdrawal mechanical threshold (PWMT) in Von Frey test and hot paw withdrawal latency (HPWL) in hot-plate test. Moreover, intestinal absorption in vitro and pathological analysis in vivo showed that total bufotenines-loaded liposome significantly reduced the gastrointestinal irritation through reducing exposure of total bufotenines on intestinal tissue. High-sensitivity lipidomics analysis revealed the effect of total bufotenines-loaded liposome were be related to the down-regulation of inflammatory mediators from cyclooxygenase (COX) and lipoxygenase (LOX), the up-regulation of cytochrome P450 (CYP450), and other pathways, thus regulating lipid metabolism pathway and ultimately reducing gastrointestinal irritation. This study shows that liposome-loaded bufotenines has anti-inflammatory, analgesic effects and achieves toxicity reduction. These results provide systematic evidences for efficacy and safety of toad venom active ingredients.

Serial hyphenation of dried spot, reversed phase liquid chromatography, hydrophilic interaction liquid chromatography, and tandem mass spectrometry towards direct chemical profiling of herbal medicine-derived liquid matrices, an application in Cistanche sinensis.

Inspired by dried blood spots (DBS), "dried spots of herbal medicines" (DSHM) concept was proposed here. In response to this superior sampling means, a new platform integrating dried spot, serially coupled reversed phase liquid chromatography and hydrophilic interaction liquid chromatography (RPLC-HILIC), and tandem mass spectrometry (MS/MS), was configured for directly, comprehensively chemical profiling of HM-derived liquid matrices. As an important original source of Cistanches Herba (Chinese name: Roucongrong) that is a well-known tonic HM, Cistanche sinensis (Csi) was employed to illustrate and validate the applicability. Dried spots (I.D.  3.0 mm) were prepared by loading 2 µL aliquots of Csi extract onto filter paper. Each dried spot was packed into an in-line filter holder (I.D. 3.0 mm × 4.0 mm) and then inserted behind the auto-sampler of a well-defined instrumentation named RPLC-HILIC-MS/MS. Hybrid ion trap-time of flight MS and hybrid triple quadrupole-linear ion trap MS were deployed in combination for the in-depth MS/MS data acquisition of diverse chemical families, such as phenylethanoid glycosides, lignans, iridoids, amino acids, and so forth. To assist chemical profiling, an in-house chemical library was built by collecting as much prior knowledge as possible. A total of 88 components were detected and tentatively annotated in Csi by matching their multi-stage MS spectra with those of authentic standards and literature data. Collectively, DSHM carried all merits of DBS, and the integrative dried spot-RPLC-HILIC-MS/MS platform was a promising analytical tool for direct chemical analysis and rapid quality evaluation of HMs, in particular those traditional Chinese medicine injections.