Mengge Zhou

Identification of NF-κB Inhibitors in Xuebijing injection for sepsis treatment based on bioactivity-integrated UPLC-Q/TOF

ETHNOPHARMACOLOGICAL EVIDENCE Inflammation plays an important role in sepsis, and NF-κB is a key mediator of inflammation. Xuebijing (XBJ) injection is a traditional Chinese medicine injection that was widely used in the treatment of sepsis in China. However, the active constituents and mechanism responsible for its actions have not been investigated experimentally. AIM OF THE STUDY To screen the anti-inflammatory components in XBJ injection and investigate the modulation of NF-κB in the treatment of sepsis. MATERIALS AND METHODS In this study, the effect of XBJ was assessed in the cecal ligation and puncture (CLP) -induced sepsis model. Subsequently, a bioactivity-integrated ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF) assay system was established for screening potential anti-inflammatory ingredients in XBJ. Ultimately, six active ingredients were confirmed through an in vitro test. RESULTS XBJ significantly reduced the mortality rate, anal temperature and expression of TNF-α, IL-1β and IL-6 induced by CLP. Nine potential anti-inflammatory ingredients, including gallic acid, danshensu, protocatechualdehyde, hydroxysafflor yellow A, oxypaeoniflorin, paeoniflorin, safflor yellow A, senkyunolide I and benzoylpaeoniflorin, were found based on the bioactivity-integrated UPLC-Q/TOF assay system. Among these compounds, the NF-κB inhibitor activity of senkyunolide I, safflor yellow A, oxypaeoniflorin, and benzoylpaeoniflorin are first reported here. CONCLUSIONS XBJ showed significant efficacy in the treatment of sepsis induced by CLP. Multiple targets of the different components were related to these anti-inflammatory effects, which contributed to the treatment of sepsis by XBJ in a clinical setting.

Identification and Comparison of Anti-Inflammatory Ingredients from Different Organs of Lotus Nelumbo by UPLC/Q-TOF and PCA Coupled with a NF-κB Reporter Gene Assay

Lotus nelumbo (LN) (Nelumbo nucifera Gaertn.) is an aquatic crop that is widely distributed throughout Asia and India, and various parts of this plant are edible and medicinal. It is noteworthy that different organs of this plant are used in traditional herbal medicine or folk recipes to cure different diseases and to relieve their corresponding symptoms. The compounds that are contained in each organ, which are named based on their chemical compositions, have led to their respective usages. In this work, a strategy was used to identify the difference ingredients and screen for Nuclear-factor-kappaB (NF-κB) inhibitors with anti-inflammatory ability in LN. Seventeen main difference ingredients were compared and identified from 64 samples of 4 different organs by ultra-performance liquid chromatography that was coupled with quadrupole/time of flight mass spectrometry (UPLC/Q-TOF-MS) with principal component analysis (PCA). A luciferase reporter assay system combined with the UPLC/Q-TOF-MS information was applied to screen biologically active substances. Ten NF-κB inhibitors from Lotus plumule (LP) extracts, most of which were isoquinoline alkaloids or flavone C-glycosides, were screened. Heat map results showed that eight of these compounds were abundant in the LP. In conclusion, the LP extracts were considered to have the best anti-inflammatory ability of the four LN organs, and the chemical material basis (CMB) of this biological activity was successfully validated by multivariate statistical analysis and biological research methods.

The Screening Research of Anti-Inflammatory Bioactive Markers from Different Flowering Phases of Flos Lonicerae Japonicae

Flos Lonicerae Japonicae (FLJ) is an important cash crop in eastern Asia, and it is an anti-inflammatory Traditional Chinese Medicine. There are large variations in the quality of the marketed FLJ products. To find marker ingredients useful for quality control, a tandem technology integrating ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF), principal component analysis (PCA), heat map analysis and hierarchical cluster analysis coupled with a NF-κB luciferase reporter gene assay were used to identify the different ingredients from the green bud, white bud, flowering stage and leaf stages, as well as to screen the anti-inflammatory activity of FLJ compositions. As flowering progressed, the anti-inflammatory effects of FLJ gradually decreased; however, chlorogenic acid, swertiamarin and sweroside should be used to evaluate the quality of FLJ products.

Searching for Synergistic Bronchodilators and Novel Therapeutic Regimens for Chronic Lung Diseases from a Traditional Chinese Medicine, Qingfei Xiaoyan Wan

Classical Chinese pharmacopeias describe numerous excellent herbal formulations, and each prescription is an outstanding pool of effective compounds for drug discovery. Clarifying the bioactivity of the combined mechanisms of the ingredients in complex traditional Chinese medicine formulas is challenging. A classical formula known as Qingfei Xiaoyan Wan, used clinically as a treatment for prevalent chronic lung disease, was investigated in this work. A mutually enhanced bioactivity-guided ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) characterization system was proposed, coupled with a dual-luciferase reporter assay for β2AR-agonist cofactor screening. Arctiin, arctigenin, descurainoside and descurainolide B, four lignin compounds that showed synergistic bronchodilation effects with ephedrine, were revealed. The synergistic mechanism of arctigenin with the β2ARagonist involved with the reduction of free Ca2+ was clarified by a dual-luciferase reporter assay for intracellular calcium and the Ca2+ indicator fluo-4/AM to monitor changes in the fluorescence. The relaxant and contractile responses of airway smooth muscle are regulated by crosstalk between the intracellular cAMP and calcium signaling pathways. Our data indicated the non-selective βAR agonist ephedrine as the principal bronchodilator of the formula, whereas the lignin ingredients served as adjuvant ingredients. A greater understanding of the mechanisms governing the control of these pathways, based on conventional wisdom, could lead to the identification of novel therapeutic targets or new agents for the treatment of asthma and COPD.

Comparison and evaluation of antimuscarinic and anti-inflammatory effects of five Bulbus fritillariae species based on UPLC-Q/TOF integrated dual-luciferase reporter assay, PCA and ANN analysis.

Many species of Bulbus fritillariae are used as traditional medicines for thousands of years; however, their application is not standardized. To clarify the differences and homologies, the antimuscarinic and anti-inflammatory effects of five BM species were firstly tested and compared at cellular level. With an integrated strategy combining UPLC-Q/TOF MS, PCA and ANN analysis, the active ingredients among 28 different chemical markers were predicted and identified. SB and QB extracts showed the best antimuscarinic effects and several steroidal alkaloids, such as solanidine, contributed to this effects. However, ZB was superior to reduce the inflammatory response. Another five components were responsible by decreasing the expression of NF-κB, including puqiedine, zhepeiresinol, 2-monopalmitin, N-demethylpuqietinone, and isoverticine. More novelty, a new cluster of five BM species based on active ingredients as potential quality markers was depicted to illustrate their functions. These results of the study could make a reference for the medicinal application of BM species in clinic; and the integrated strategy provided an effective method to obtain the quality markers from medical herbs, which was helpful for the quality control of traditional medicinal products.

Identification of nuclear factor-κB inhibitors in the folk herb Rhizoma Menispermi via bioactivity-based ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analysis

BackgroundRhizoma Menispermi (RM) is the dried root of Menispermum dauricum DC, which is traditionally used to treat swelling and pain for sore throat, enteritis and rheumatic arthralgia in the clinic, but its bioactive compounds remain unclear.MethodsIn this study, RM extract was administered orally to ICR mice followed by challenging with an intratracheal Pseudomonas aeruginosa suspension. Then mortality, histological features of lung, and inflammatory cytokines were evaluated. RM treatment significantly ameliorated Pseudomonas aeruginosa-induced acute lung inflammation and reduced levels of inflammatory mediators. To screen for potential anti-inflammatory constituents of the RM extract, a simple and rapid method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) coupled with a luciferase reporter assay system to detect nuclear factor-κB (NF-κB) activity was established.ResultsUsing this system, seven potential NF-κB inhibitors were detected, including sinomenine, norsinoacutin, N-norsinoacutin-β-D-glucopyranoside, 6-O-methyl-laudanosoline-13-O-glucopyranoside, magnoflorine, laurifloline and dauricinoline. Furthermore, IL-6 and IL-8 assays confirmed the anti-inflammatory effects of these potential NF-κB inhibitors, in which norsinoacutin, 6-O-methyl-laudanosoline-13-O-glucopyranoside laurifloline, dauricinoline and N-norsinoacutin-β-D-glucopyranoside were revealed as new NF-κB inhibitors.ConclusionThis method of UPLC-Q/TOF coupled with the luciferase reporter assay system was initially applied to the study of RM and was demonstrated to represent a simple, rapid and practical approach to screen for anti-inflammatory compounds. This study provided useful results for further investigation on the anti-inflammatory mechanism of RM.

Anti-inflammatory effects of Shufengjiedu capsule for upper respiratory infection via the ERK pathway

BACKGROUND Shufengjiedu Capsule (SFJD) is a type of Chinese traditional medicine compound for the treatment of acute upper respiratory tract infection. The present work aims to decipher the mechanism of SFJD. METHODS In this study, we used target prediction and RNA sequence (RNA-Seq) based on transcriptome analysis to clarify the inflammation-eliminating mechanism of SFJD. Firstly, Pseudomonas aeruginosa (PAK) was used to induce acute lung injury in KM mice. After being treated by SFJD, the differently expressed genes were analyzed by RNA-Seq. Secondly, the chemical constituents of SFJD were identified by ultra-performance liquid chromatography quadrupole/time of flight mass spectrometry (UPLC/Q-TOF-MS) and submitted to PharmMapper to predict targets. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and String 9.1 websites were employed to establish the interaction network of inflammation of these targets. RESULTS The results indicated that SFJD alleviated PAK induced lung injury in KM mice. We infer that the mechanism is a complex network containing 15 pathways related to inflammation regulated by 16 types of components from six types of herbs via 29 proteins. The ERK signaling pathway was a key pathway among them, which was predicted to be regulated by 14 types of components in SFJD. Phillyrin, emodin, and verbenalin were screened out by binding capacity, and the synergistic effect of them was further confirmed. CONCLUSIONS Various components of SFJD ameliorated PAK induced upper respiratory tract infection via multiple targets, of which ERK phosphorylation might be the key event regulated specifically by verbenalin, phillyrin and emodin.

Active fragments-guided drug discovery and design of selective tropane alkaloids using ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry coupled with virtual calculation and biological evaluation

AbstractTropane alkaloids (TAs), rich in the plant of Physochlaina infundibularis Kuang, which is named Huashanshen (HSS) in China, showed good effects on types of spasms. However, no data were collected to explore the relationship between the specificity for muscarinic receptor subtypes and the structures of these TAs. To address this issue, an extracted ion chromatogram (EIC) strategy using ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) based on the fragmentation behavior of the TA standards was established to rapidly capture the varied TAs from HSS. Based on the provided structural information of diagnostic ions or neutral loss, 29 TAs were efficiently profiled, especially some trace ingredients. In additional, via virtual validation combined with molecular dynamic simulation, approximately a dozen alkaloids were found with high selectivity for muscarinic receptors. In additional, N-acetyl convolicine was chosen for selectivity evaluation of M2 or M3 receptors through the use of a dual-luciferase reporter assay system at the cellular level and an ACh-induced constricted strip test in vitro. After summarizing the active fragments and the structure-activity relationship (SAR) information, a new modified TA that takes advantage of both the high affinity and high selectivity for M3 receptors was proposed and evaluated successfully. This study provided an effective approach for the discovery and design of natural products based on highly selective drugs by UPLC-Q/TOF-MS coupled with virtual calculation and biological evaluation. Graphical AbstractActive fragments-guided strategy for selective inhibitors from HSS

Identification of NF‐κB inhibitors in Qishenyiqi dropping pills for myocardial infarction treatment based on bioactivity‐integrated UPLC‐Q/TOF MS

Qishenyiqi dropping pills (QSYQ) are a type of standardized cardiovascular multiherb medicine for the treatment of myocardial infarction (MI). Knowledge concerning the systemic identification of nuclear factor-kappa B (NF-κB) inhibitors of QSYQ is generally lacking. Therefore, it is necessary to establish an effective method to screen the bioactive components of NF-κB inhibition. In the present study, a rat model of coronary artery ligation was used to assess the cardioprotective effects of QSYQ. The electrocardiograms, histopathology of heart tissues and serum biochemical indicators, such as brain natriuretic peptide, cardiac troponin I and inflammatory cytokines, were measured. Subsequently, ultra-performance liquid chromatography quadrupole/time-of-flight mass spectrometry (UPLC-Q/TOF MS) combined with the NF-κB luciferase reporter assay system was applied to screen the potential anti-inflammatory compounds in QSYQ. The results revealed that the administration of QSYQ could improve heart function, ameliorate neutrophil infiltration and diminish the levels of inflammatory cytokines in MI rats. Furthermore, 22 compounds were determined to be potential NF-κB inhibitors. In conclusion, NF-κB inactivation and cytokine suppression might be the main mechanisms of QSYQ for MI treatment. The method of UPLC-Q/TOF MS combined with a bioactive human cell functional evaluation system was proved to be a simple and effective strategy for screening bioactive compounds in traditional Chinese medicine prescriptions.

Mahuannin B an adenylate cyclase inhibitor attenuates hyperhidrosis via suppressing β2-adrenoceptor/cAMP signaling pathway

BACKGROUND Based on the traditional application of traditional Chinese Medicines (TCMs), Ephedra Herba (EH) is used to cure cold fever by inducing sweating, whereas Ephedra Radix (ER) is used to treat hyperhidrosis. Although they come from the same plant, Ephedra sinica Stapf, but have play opposing roles in clinical applications. EH is known to contain ephedrine alkaloids, which is the driver of the physiological changes in sweating, heart rate and blood pressure. However, the active pharmacological ingredients (APIs) of ER and the mechanisms by which it restricts sweating remain unknown. PURPOSE The current work aims to discover the hidroschesis APIs from ER, as well as to establish its action mechanism. METHODS UPLC-Q/TOF-MS, PCA, and heat map were utilized for identifying the differences between EH and ER. HPLC integrated with a β2-adrenoceptor (β2-AR) activity luciferase reporter assay system was used to screen active inhibitors; molecular docking and a series of biological assays centered on β2-AR-related signaling pathways were evaluated to understand the roles of APIs. RESULTS The opposite effect on sweating of EH and ER can be attributed to the APIs of amphetamine-type alkaloids and flavonoid derivatives. Mahuannin B is an effective anti-hydrotic agent, inhibiting the production of cAMP via suppression of adenylate cyclase (AC) activity. CONCLUSION The effects of EH and ER on sweat and β2-AR-related signaling pathway are opposite due to different alkaloids and flavonoids of APIs in EH and ER. The present work not only sheds light on the hidroschesis action of mahuannin B, but also presents a potential target of AC in the treatment of hyperhidrosis.