Elucidating the substance basis and pharmacological mechanism of Fufang Qiling granules in modulating xanthine oxidase for intervention in hyperuricemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang Qiling granules (FQG), derived from the traditional Qiling Decoction with a longstanding clinical history, is utilized for the treatment of hyperuricemia (HUA). FQG is formulated with a combination of seven Chinese herbs based on the principles of traditional Chinese medicine (TCM) theories. Clinical evidence indicates that FQG exhibits favorable therapeutic effects in reducing uric acid (UA) levels and attenuating renal damage. AIM OF THIS STUDY: To elucidate the potential active components and pharmacological mechanism of FQG in the treatment of HUA, and to provide an experimental basis for the development of efficient and low-toxicity TCM for HUA treatment. MATERIALS AND METHODS: A HUA rat model induced by potassium oxonate and adenine was established to initially evaluate the hypouricemic effects of FQG. Chemical analyses were conducted using an ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Network pharmacology was used to investigate the active components and mechanism of FQG in the treatment of HUA. Potential Xanthine oxidase (XOD) inhibitors were screened from FQG based on ultrafiltration liquid chromatography and mass spectrometry (UF-LC-MS). Molecular docking, surface plasmon resonance (SPR) and circular dichroism (CD) spectroscopy were applied to validate the interactions between the active components and XOD. RESULTS: In comparison to the model group, treatment with FQG significantly decreased serum UA, serum creatinine (CREA), serum blood urea nitrogen (BUN), and liver XOD activity. Additionally, the FQG administration notably ameliorated HUA-induced renal injury in rats. Through the pharmacodynamics of the HUA rat models and network pharmacology, it was found that XOD was a key pathway enzyme in UA metabolism. 18 XOD inhibitors were screened from FQG by UF-LC-MS, and 11 compounds with strong affinity were verified by SPR, molecular docking and CD spectroscopy. CONCLUSION: In summary, flavonoids, organic acids and saponins may be the active components in FQG that alleviate HUA. The primary mechanism of FQG involves inhibiting XOD enzyme activity in the plasma to reduce UA production, alleviating renal tubular epithelial cell necrosis, tubulointerstitial injury, fibrosis, and urate deposition, ultimately exerting a therapeutic effect on HUA.

Network Pharmacology-Based Approach Uncovers the Mechanism of GuanXinNing Tablet for Treating Thrombus by MAPKs Signal Pathway.

BACKGROUND: GuanXinNing tablet (GXNT), a traditional Chinese patent medicine, has been found to have remarkable antithrombotic effects and can effectively inhibit pro-thrombotic factors in previous studies. However, the mechanism of its antithrombotic effects remains little known. METHODS: In this study, we first determined and identified the sources of each main compound in GXNT using liquid chromatography-mass spectrometry (LC-MS). Through the approach of network pharmacology, we predicted the action targets of the active components, mapped the target genes related to thrombus, and obtained potential antithrombotic targets for active ingredients. We then performed gene ontology (GO) enrichment analyses and KEGG signaling pathway analyses for the action targets, and constructed networks of active component-target and active component-target-pathway for GXNT. Additionally, we evaluated the pharmacodynamic effects of GXNT on thrombus using the rat thrombus model induced by FeCl3, observed the effects of antiplatelet aggregation via platelet assay, and further verified the results predicted by network pharmacology via Western blot. RESULTS: In total, 14 active ingredients were identified in GXNT, and 83 action targets were predicted, 17 of which are antithrombotic targets that potentially participate in processes including response to oxidative stress and positive regulation of blood vessel endothelial cell migration. KEGG pathway analyses revealed that the predicted action targets were involved in multiple signal pathways, such as MAPK, IL-17, and platelet activation. Pharmacodynamics study found that GXNT could significantly reduce the thrombus length and weight, lower platelet aggregation function, and decrease the levels of Fbg and PAI-1. In addition, GXNT could significantly increase 6-keto-PGF1α content and regulate the ratio of TXB2/6-keto-PGF1α, while not having dramatic effects on TXB2. GXNT was also observed to visibly inhibit maximum platelet aggregation. Herein, we further studied the thrombus-related MAPKs signaling pathway and found that GXNT could significantly reduce the phosphorylation levels of p38MAPK, ERK, and JNK proteins in platelet. CONCLUSIONS: This study revealed the pharmacodynamic material basis of GXNT and its potential multicomponent-multitarget-multipath pharmacological effects, confirmed the antithrombotic effects of GXNT, and showed that its mechanism may be related to inhibiting phosphorylation of p38, ERK, and JNK proteins in MAPKs signaling pathway, partially verifying the results from network pharmacology. The results from this study could provide a theoretical basis for the development and clinical application of GXNT.

De novo assembly and annotation of the Zhe-Maidong (Ophiopogon japonicus (L.f.) Ker-Gawl) transcriptome in different growth stages.

Zhe-Maidong (Ophiopogon japonicus (L.f.) Ker-Gawl) is a traditional medicinal herb in the family Liliaceae that has significant pharmacological effects on immunity and cardiovascular disease. In this study, three different growth stages of Zhe-Maidong were investigated using RNA-seq, and a total of 16.4 Gb of raw data was obtained. After filtering and assembling, 96,738 unigenes with an average length of 605.3 bp were ultimately generated. A total of 77,300 unigenes were annotated using information from five databases, including the NT, NR, SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases. Additionally, the mechanisms of flavonoid, saponin and polysaccharide biosynthesis and of accumulation at different stages of tuber development were also characterized. From the first to third years, the contents of flavonoids, saponins and polysaccharides all increased, whereas the expression levels of related genes decreased in the flavonoid and saponin pathways and first increased and then decreased in the polysaccharide pathway. The results of this study provide the most comprehensive expressed sequence resource for Zhe-Maidong and will expand the available O. japonicus gene library and facilitate further genome-wide research and analyses of this species.

Comparative evaluation of different cultivars of Flos Chrysanthemi by an anti-inflammatory-based NF-κB reporter gene assay coupled to UPLC-Q/TOF MS with PCA and ANN.

ETHNOPHARMACOLOGICAL RELEVANCE: Flos Chrysanthemi (FC), a commonly used traditional Chinese medicine, has five major cultivars ("Boju", "Chuju", "Gongju", "Hangbaiju" and "Huaiju") from different sources. However, the active constituents of these cultivars have not been studied or characterized with respect to their bioactivity, which is a serious problem when considering quality and safety. AIM OF THE STUDY: To evaluate the differences among the five cultivars of FC, and to establish a method for the standardization and quality control of FC related to its bioactivity. MATERIALS AND METHODS: In this study, the different ingredients in five cultivars of FC were identified by UPLC-Q/TOF and PCA, and the anti-inflammatory ingredients of FC were predicted and screened by artificial neural network (ANN) and an NF-κB luciferase reporter gene assay system. Using this comprehensive method, we successfully screened the anti-inflammatory markers of different cultivars of FC. RESULTS: Nineteen marker ingredients were confirmed to contribute strongly to the cluster, and eleven compounds in the five cultivars of FC were found to exert potential anti-inflammatory effects. Among these compounds, the NF-κB inhibitor activity of apigenin-7-O-6″-malonyl-glucoside, luteolin-7-O-rutinoside, quercetin-7-O-galactoside, quercetin-3-O-glucoside, apigenin-7-O-rutinoside and apigenin-7-O-glucoside were first reported here. Chlorogenic acid, luteolin-7-O-glucoside, 3,5-dicaffeoylquinic acid and luteolin were confirmed to be the most important anti-inflammatory marker ingredients useful for the quality control of FC. CONCLUSIONS: The proposed efficient and systematic method is helpful for the standardization and quality control of FC. Moreover, this comprehensive strategy may prove to be a powerful technique for the rapid establishment of quality control procedures related to bioactivity for other herbal samples and foods.

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.

BACKGROUND: Rhizoma 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. METHODS: In 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. RESULTS: Using this system, seven potential NF-κB inhibitors were detected, including sinomenine, norsinoacutin, N-norsinoacutin-ß-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-ß-D-glucopyranoside were revealed as new NF-κB inhibitors. CONCLUSION: This 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.

UPLC-DAD/Q-TOF-MS Based Ingredients Identification and Vasorelaxant Effect of Ethanol Extract of Jasmine Flower.

Chinese people commonly make jasmine tea for recreation and health care. Actually, its medicinal value needs more exploration. In this study, vasorelaxant effect of ethanol extract of jasmine flower (EEJ) on isolated rat thoracic aorta rings was investigated and [Ca(2+)] was determined in vascular smooth muscle cells by laser scanning confocal microscope (LSCM). The result of aorta rings showed that EEJ could cause concentration-dependent relaxation of endothelium-intact rings precontracted with phenylephrine or KCl which was attenuated after preincubation of the rings with L-NAME and three different K(+) channel inhibitors; however, indomethacin and glibenclamide did not affect the vasodilatation of EEJ. In addition, EEJ could inhibit contraction induced by PE on endothelium-denuded rings in Ca(2+)-free medium as well as by accumulation of Ca(2+) in Ca(2+)-free medium with high K(+). LSCM also showed that EEJ could lower the elevated level of [Ca(2+)] induced by KCl. These indicate that the vasodilation of EEJ is in part related to causing the release of nitric oxide, activation of K(+) channels, inhibition of influx of excalcium, and release of calcium from sarcoplasmic reticulum. A total of 20 main ingredients, were identified in EEJ by UPLC-DAD/Q-TOF-MS. The vasodilation activity should be attributed to the high content of flavonoid glycosides and iridoid glycosides found in EEJ.

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.

Identification and analysis of absorbed components and their metabolites in rat plasma and tissues after oral administration of 'Ershiwuwei Shanhu' pill extracts by UPLC-DAD/Q-TOF-MS.

ETHNOPHARMACOLOGICAL RELEVANCE: 'Ershiwuwei Shanhu' pill (ESP), a classical and famous prescription of traditional Tibetan medicine, has a long history of empirical clinical use for the treatment of cerebrovascular and neurological diseases, but the absence of scientific evidence for its effect restricted its clinical application and further development. MATERIALS AND METHODS: The methodology of plasma pharmacochemistry was adopted to analyze the potentially bioactive components in ESP extracts. A method based on UPLC-DAD/Q-TOF-MS was established to identify herb components in ESP extracts and analyze the absorbed components of ESP and their metabolites in rat plasma, brain, heart, liver and kidney samples after oral administration of ESP extracts. RESULTS: A total of 61 herb components were detected and identified in ESP extracts, while 35 absorbed components-including 19 prototype compounds and 16 metabolites-were discovered as potentially bioactive components in rat plasma and tissues by comparative analysis of the UV and MS chromatograms of ESP extracts, blank biosamples and dosed biosamples. CONCLUSIONS: The potentially bioactive components of ESP extracts identified from rat plasma and tissues provide useful information for further study of the pharmacology and mechanism of action of ESP.

Tanshinone IIA exhibits anticonvulsant activity in zebrafish and mouse seizure models.

Danshen or Chinese red sage (Salvia miltiorrhiza, Bunge) is used by traditional Chinese medicine (TCM) practitioners to treat neurological, cardiovascular, and cerebrovascular disorders and is included in some TCM formulations to control epileptic seizures. In this study, acetonic crude extracts of danshen inhibited pentylenetetrazol (PTZ)-induced seizure activity in zebrafish larvae. Subsequent zebrafish bioassay-guided fractionation of the extract resulted in the isolation of four major tanshinones, which suppressed PTZ-induced activity to varying degrees. One of the active tanshinones, tanshinone IIA, also reduced c-fos expression in the brains of PTZ-exposed zebrafish larvae. In rodent seizure models, tanshinone IIA showed anticonvulsive activity in the mouse 6-Hz psychomotor seizure test in a biphasic manner and modified seizure thresholds in a complex manner for the mouse i.v. PTZ seizure assay. Interestingly, tanshinone IIA is used as a prescription drug in China to address cerebral ischemia in patients. Here, we provide the first in vivo evidence demonstrating that tanshinone IIA has anticonvulsant properties as well.

Identification and analysis of the constituents in an aqueous extract of Tricholoma matsutake by HPLC coupled with diode array detection/electrospray ionization mass spectrometry.

The main constituents in an aqueous extract of Tricholoma matsutake (Tm) were identified by high-performance liquid chromatography coupled with diode array detection and electrospray ionization time-of-flight mass spectrometry (HPLC-DAD/TOF-MS) and ion trap mass spectrometry (HPLC-DAD/Trap-MSn). The main factors in the extraction process which affect the yields of nutrients were optimized by single-factor experiments and orthogonal experiment design. In total, 12 constituents were identified from the aqueous extract of Tm, including tyrosine, cytidine, uridine, eritadenine, phenylalanine, nicotinamide, inosine, guanosine, tryptophan, adenosine, 5'-deoxy-5'-methylthioadenosine and riboflavin. The optimized extraction conditions were: the ratio of water to sample was 10 : 1 (v/w), Tm was extracted by ultrasonic-assisted extraction for 10 min, followed by water bath heating at 60 °C for 1 h. Among these extraction factors, the heating temperature is significant based on analysis of variance (ANOVA). The yields of nutrients were affected dramatically at high temperature leading to the loss of nutrients, especially for nucleosides and some amino acids.