Pharmacological Studies Research Articles

Cotinine influences the effect of high and low nicotine concentrations on planarian motility differently

Previous work from our laboratory showed that cotinine, a nicotine metabolite, reverses three nicotine-induced behavioral effects in freshwater planarians: motility decrease, seizure-like movements, and withdrawal-like behaviors. The present work explored whether cotinine, a nicotine metabolite, antagonized the nicotine-induced effects on planarian motility in a concentration-dependent manner. We found that nicotine decreased planarian motility at nicotine concentrations above 60 μM but increased planarian velocity at concentrations equal to or below 50 μM, in agreement with previous data. Cotinine did not affect planarian motility at a concentration range between 250 and 2750 μM. Furthermore, we found that cotinine alleviated the 100 μM nicotine-induced motility decrease in a concentration-dependent manner and reversed the low nicotine concentration motility increase, albeit in a concentration-independent manner. The apparent concentration-dependent alleviation of >60 μM nicotine-induced motility decrease by cotinine suggests an orthosteric relationship between nicotine and cotinine. On the other hand, the evident concentration-independent cotinine alleviation of the increase in motility induced by 50 μM nicotine suggests an allosteric relationship. Our data is consistent with the existing literature about the relationship between nicotine and cotinine in various models, reinforcing the case for the usefulness of the planarian model in pharmacological studies.

Poweromin X Ten, a polyherbal formulation improves male sexual function: In vivo and network pharmacology study.

Poweromin X Ten (PXT) is a polyherbal formulation, traditionally used to enhance male sexual function. However, the safety and benefits of PXT have not been scientifically evaluated. Therefore, the present study investigated the toxicity and aphrodisiac potential of PXT in male rats and explored its principal mechanisms of action. Male Wistar rats were orally administered PXT (50 or 100 mg/kg) for 28 days, and sexual activity parameters, including latency and frequency of mounting and intromissions, were studied. The reproductive toxicity and spermatogenic potential were also examined. Furthermore, dopamine and serotonin levels in brain regions associated with sexual activity were assessed. Network analysis was used to identify the key bioactive compounds and their core targets involved in their beneficial actions. Treatment with PXT improved sexual activity in male rats, as evidenced by reduced mounting and intromission latency and a significant increase in mount frequency. Moreover, PXT exhibited spermatogenic potential and did not induce reproductive toxicity. Notably, treatment with 50 mg/kg PXT elevated dopamine levels in median preoptic area and hypothalamus. Pathway analysis indicated that PXT primarily modulated the PI3K-Akt, calcium, and MAPK signalling pathways to enhance male sexual function. Network analysis identified macelignan, β-estradiol, testosterone, and paniculatine as key bioactive components of PXT, which likely act through core targets, such as androgen receptor (AR), Mitogen-activated protein kinase 3 (MAPK3), epidermal growth factor receptor (EGFR), estrogen receptor 1 (ESR1), and vascular endothelial growth factor (VEGF) to facilitate the improvement of male sexual function. Study results suggest that PXT is a safer alternative with aphrodisiac and spermatogenic potential. These effects are partly attributed to the enhanced dopamine levels in the brain. Furthermore, this study provides insights into the specific signalling pathways and bioactive compounds that underlie the improvements in male sexual function associated with PXT.

Daphnetin modulates GLP-1R to alleviate cognitive dysfunction in diabetes: implications for inflammation and oxidative stress.

Daphnetin exerts certain pharmacological function on a variety of diseases, but its role in diabetic cognitive dysfunction has not been elucidated. In this study, we carried a series of pharmacological studies of GLP-1R with daphnetin. In rats and PC12 cells, we found that daphnetin could alleviate diabetic cognitive dysfunction and increase the expression level of GLP-1R. Additionally, the anti-diabetic cognitive dysfunction effect of DAP was accompanied by the inhibition of inflammation and oxidative stress. Further in-depth studies demonstrated that the inhibition GLP-1R enhanced the protective effect of daphnetin, whilst, the overexpression of GLP-1R weakened the protective effect of daphnetin. These results indicated that daphnetin protects diabetes cognitive dysfunction by regulating GLP-1R-mediated inflammation and oxidative stress, act as a GLP-1R agonist. The study further demonstrated that daphnetin has great value in preventing cognitive dysfunction in type 2 diabetes, and GLP-1R is a key potential target for the treatment of related diseases.

Toward Model-Informed Precision Dosing for Remimazolam: A Population Pharmacokinetic–Pharmacodynamic Analysis

Remimazolam, widely used for procedural sedation and general anesthesia, is a new ultra short-acting benzodiazepine for intravenous sedation and anesthesia. We aim to characterize the pharmacokinetics/pharmacodynamics (PK/PD) of remimazolam and its metabolite CNS 7054 in healthy Chinese volunteers using population analysis and suggest an optimal dosing regimen for sedation therapy. Data were collected from a single-center, placebo-controlled, randomized, and dose–escalation clinical pharmacology study. Forty-six healthy volunteers received a single infusion dose of remimazolam, while nine healthy subjects received a continuous infusion of remimazolam. A population PK/PD model was established and RxODE and Shiny in R were used to design the remimazolam dosing regimens. A three-compartment model best described the PK of remimazolam and a two-compartment model with one transit compartment was adopted for CNS 7054. The relationship between exposure and the bispectral index was best described using an effect compartment model with an inhibitory sigmoid model. Additionally, a web-based dashboard was developed to provide individualized dosing regimens, complemented by a graphical illustration of the PK/PD profiles of the proposed dosing regimen. The established population PK/PD model characterized the dose–exposure–response relationship of remimazolam well, which could be applied to optimize individual dosing regimens.

Large animal model species in pluripotent stem cell therapy research and development for retinal diseases: a systematic review.

Retinal cell therapy modalities, in the category of advanced therapy medicinal products (ATMPs), are being developed to target several retinal diseases. Testing in large animal models (LAMs) is a crucial step in translating retinal ATMPs into clinical practice. However, challenges including budgetary and infrastructure constraints can hinder LAM research design and execution. Here, to facilitate the comparison of the various LAMs in pluripotent retinal cell therapy research, we aimed to systematically evaluate the species distribution, reported scientific utility, and methodology of a range of LAMs. A systematic search using the words retina, stem cell, transplantation, large animal, pig, rabbit, dog, and nonhuman primate was conducted in the PubMed, Embase, Science Direct and GoogleScholar databases in February 2023. We included 22 studies involving pluripotent stem cells (induced pluripotent stem cells or human embryonic stem cells) in LAMs, including non-human primates (NHP), pigs, dogs, and rabbits. Nearly half of the studies utilized wild-type animal models. In other studies, retinal degeneration features were simulated via laser, chemical, or genetic insult. Transplants were delivered subretinally, either as cell suspensions or pre-formed monolayers (with or without biodegradable scaffolding). The transplanted cells dose per eye varied widely (40,000 - 4,000,000 per dose). Cells were delivered via vitrectomy surgery in 15 studies and by an "ab externo" approach in one study. Structural outcomes were assessed using confocal scanning laser ophthalmoscopy imaging. Functional outcomes included multifocal electroretinogram and, in one case, a measure of visual acuity. Generally, cell suspension transplants exhibited low intraretinal incorporation, while monolayer transplants incorporated more efficiently. Immune responses posed challenges for allogeneic transplants, suggesting that autologous iPSC-derived transplants may be required to decrease the likelihood of rejection. The use of appropriate LAMs helps to advance the development of retinal ATMPs. The anatomical similarity of LAM and human eyes allows the implementation of clinically-relevant surgical techniques. While the FDA Modernization Act 2.0 has provided a framework to consider alternative methods including tissue-on-a-chip and human cell culture models for pharmacologic studies, LAM testing remains useful for cell and tissue replacement studies to inform the development of clinical trial protocols.

Exploration of Potential Anti-inflammatory cum Anti-Rheumatoid-arthritis Phyto-molecule through Integrated Green Approach: Network Pharmacology, Molecular Docking, Molecular dynamics, In-vitro and Ex-vivo study.

Rheumatoid arthritis (RA) and associated inflammatory complications are the most prevalent illnesses and can turn into fatal conditions if left untreated. Allopathic medicine is not satisfactory for curing RA. Scientific literature reports reveal that several phyto-compounds viz. flavonoids, saponins, and terpenoids, can heal joints and organs from auto-inflammatory rheumatoid arthritis and pain. Gene ontology, gene network analysis, molecular clustering, and literature review were used to optimise RA-specific highly expressed genes. In-silico molecular docking was performed to short-out potential phytomolecules (Neohesperidin dihydrochalcone (NHDC)) from 1000 datasets-library against RA and validate using MD simulation running at 100 ns. In-vitro anti-inflammatory assays of NHDC inhibited egg-albumin denaturation, IC50 of 47.739 ± 0.51 µg/ml. The ex-vivo MTT assay with NHDC rendered 67.209% inhibition at 100 µM against fd-FLS-cells. NHDC downregulated pro-inflammatory cytokine IL-17A production by 61.11% and 50% at 300 and 200 μM, respectively. Thus, this Studies recommend that NHDC may be highlighted as a novel multi-target PADI4 and JAK3 inhibitor with better efficacy and minimal toxicity in RA warranted to In-Vivo and clinical investigation. The current findings have uncovered remarkable genes and signalling pathways linked to RA, which could enhance our existing comprehension of the molecular mechanisms that drive its development and progression.

Uncovering the mechanisms of Zhubi decoction against rheumatoid arthritis through an integrated study of network pharmacology, metabolomics, and intestinal flora

Ethnopharmacological relevanceZhubi Decoction (ZBD) is a modified formulation derived from the classic traditional Chinese medicine prescription “Er-Xian Decoction” documented in the esteemed “Clinical Manual of Chinese Medical Prescription”. While the utilization of ZBD has exhibited promising clinical outcomes in treating rheumatoid arthritis (RA), the precise bioactive chemical constituents and the underlying mechanisms involved in its therapeutic efficacy remain to be comprehensively determined. Aim of the studyThis study aims to systematically examine ZBD's pharmacological effects and molecular mechanisms for RA alleviation. Materials and methodsUtilizing the collagen-induced arthritis (CIA) rat model, we comprehensively evaluated the anti-rheumatoid arthritis effects of ZBD in vivo through various indices, such as paw edema, arthritis index, ankle diameter, inflammatory cytokine levels, pathological conditions, and micro-CT analysis. The UPLC-MS/MS technique was utilized to analyze the compounds of ZBD. The potential therapeutic targets and signaling pathways of ZBD in the management of RA were predicted using network pharmacology. To analyze comprehensive metabolic profiles and identify underlying metabolic pathways, we conducted a serum-based widely targeted metabolomics analysis utilizing LC-MS technology. Key targets and predicted pathways were further validated using immunofluorescent staining, which integrated findings from serum metabolomics and network pharmacology analysis. Additionally, we analyzed the gut microbiota composition in rats employing 16 S rDNA sequencing and investigated the effects of ZBD on the microbiota of CIA rats through bioinformatics and statistical methods. ResultsZBD exhibited remarkable efficacy in alleviating RA symptoms in CIA rats without notable side effects. This included reduced paw redness and swelling, minimized joint damage, improved the histopathology of cartilage and synovium, mitigated the inflammatory state, and lowered serum concentrations of cytokines TNF-α, IL-1β and IL-6. Notably, the effectiveness of ZBD was comparable to MTX. Network pharmacology analysis revealed inflammation and immunity-related signaling pathways, such as PI3K/AKT, MAPK, IL-17, and TNF signaling pathways, as vital mediators in the effectual mechanisms of ZBD. Immunofluorescence analysis validated ZBD's ability to inhibit PI3K/AKT pathway proteins. Serum metabolomics studies revealed that ZBD modulates 170 differential metabolites, partially restored disrupted metabolic profiles in CIA rats. With a notable impact on amino acids and their metabolites, and lipids and lipid-like molecules. Integrated analysis of metabolomics and network pharmacology identified 6 pivotal metabolite pathways and 3 crucial targets: PTGS2, GSTP1, and ALDH2. Additionally, 16 S rDNA sequencing illuminated that ZBD mitigated gut microbiota dysbiosis in the CIA group, highlighting key genera such as Ligilactobacillus, Prevotella_9, unclassified_Bacilli, and unclassified_rumen_bacterium_JW32. Correlation analysis disclosed a significant link between 47 distinct metabolites and specific bacterial species. ConclusionZBD is a safe and efficacious TCM formulation, demonstrates efficacy in treating RA through its multi-component, multi-target, and multi-pathway mechanisms. The regulation of inflammation and immunity-related signaling pathways constitutes a crucial mechanism of ZBD's efficacy. Furthermore, ZBD modulates host metabolism and intestinal flora. The integrated analysis presents experimental evidence of ZBD for the management of RA.

Preparation of kakkatin derivatives and their anti-tumor activity.

Modern pharmacological studies have confirmed that plant-derived compounds from Puerariae flos (PF) has significant biological activities against liver damage, tumors and inflammation. Kakkatin is an isoflavone polyphenolic compound isolated from PF flower. However, the effect of kakkatin and its derivatives on anti-tumor has not been well explored. To design and synthesize a kakkatin derivative [6-(hept-6-yn-1-yloxy)-3-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one (HK)] to explore its anti-tumor biological activity. Hept-6-yn-1-yl ethanesulfonate was introduced to replace hydrogen at the hydroxyl position of kakkatin phenol, and the derivative of kakkatin was prepared; the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to detect cell viability, a clone formation assay was adopted to detect cell proliferation, apoptosis, necrosis, and cell cycles were analyzed by Annexin V/propidium iodide staining and flow cytometry. Cell migration and invasion ability were evaluated by cell scratch assay and transwell assay. The potential mechanism of HK on hepatocellular carcinoma (HCC) SMMC-7721 cells was explored through network pharmacology and molecular docking, and finally real-time PCR assays was used to verify the potential targets and evaluate the biological activity of HK. Compared with kakkatin, the modified HK did not significantly increase the inhibitory activity of gastric cancer MGC803 cells, but the inhibitory activity of HCC SMMC-7721 cells was increased by about 30 times, with an IC50 value of 2.5 μM, and the tumor inhibition effect was better than cisplatin, which could significantly inhibit the cloning, invasion and metastasis of HCC SMMC-7721 cells, and induce apoptosis and G2/M cycle arrest. Its mechanism of action is mainly related to the upregulation of PDE3B and NFKB1 target proteins in the cAMP pathway. HK have a significant inhibitory effect on HCC SMMC-7721 cells, and the targets of their action may be PDE3B and NFKB1 proteins in the cAMP pathway, making it a good lead drug for the treatment of HCC.

Lathyrane and ent-isopimarane diterpenoids from Euphorbia wallichii and target prediction of active ingredient

Fourteen undescribed diterpenoids, including eleven lathyrane diterpenoids wallathyanes A-K (1–11) and three ent-isopimarane diterpenoids wallisopiranes A-C (12–14), together with fourteen known analogues 15–28, were obtained from the whole plant of Euphorbia wallichii. Their chemical structures were elucidated by spectroscopic data analysis, experimental electronic circular dichroism measurements, and X-ray crystallography. Bioactivity screening indicated that compound 2 exhibited an inhibitory effect on NO generation in LPS-stimulated RAW264.7 macrophage cells with an IC50 value of 4.76 ± 1.08 μM. The network pharmacology and molecular docking studies also revealed that compound 2 can bind with the potential targets GRB, AKT1, MAPK1, MAPK14, HSP90AA1, PIK3R1, PIK3CB, PRKACA, SRC, CASP3, RXRA, PTPNA11, ZAP70, and PRKC of inflammation.

Bisphenol A-induced ovarian damage countered by luteolin: Experiments in in vitro CHO cells and in vivo PCOS phenotype zebrafish

Bisphenol-A (BPA) is a widely used chemical that can harm the human body, including the reproductive system. BPA accumulates in the body and is found in 95 % of individuals due to everyday exposure through food, water, and skin absorption. BPA can impair female fertility by interfering with ovarian folliculogenesis, inhibiting follicular growth, and inducing atresia, leading to polycystic ovary syndrome (PCOS). PCOS is a prevalent endocrine disorder that affects many reproductive-aged women. While current treatments can help manage symptoms, they do not entirely prevent complications. Luteolin, a natural flavonoid with medicinal properties, is commonly used to treat metabolic and inflammatory disorders. Therefore, we evaluated Luteolin's properties against PCOS in Network pharmacology and molecular docking studies; further, the antioxidant and anti-inflammatory properties in protecting the Chinese Hamster ovarian (CHO) cells from Reactive Oxygen Species, cellular damage, and negative mitochondrial membrane potential were evaluated. Additionally, an in-vivo PCOS-like model was developed using zebrafish, and the localization of Luteolin was identified using fluorescein isothiocyanate (FITC). Luteolin protected the CHO cells from cellular damage, ROS, and negative mitochondrial membrane potential. Luteolin alleviated the total SOD levels in the Zebrafish ovary, induced follicular maturation, and altered the key genes in ovarian proliferation and pro-inflammatory cytokines TNF-α and IL-1β expression. Natural Phyto-oxidants such as Luteolin may protect follicular development and early PCOS in adult zebrafish to prevent oxidative stress and inflammation. This study suggests using Luteolin as a phytomedicine to alleviate ovarian function decline.

Pancreatic lipase immobilization on cellulose filter paper for inhibitors screening and network pharmacology study of anti-obesity mechanism

The discovery of pancreatic lipase (PL) inhibitors is an essential route to develop new anti-obesity drugs. In this experiment, chitosan was used to add amino groups to cellulose filter paper (CFP) and then glutaraldehyde was used to covalently combine PL with amino-modified CFP through the Schiff base reaction. Under optimal immobilization conditions, CFP immobilized PL has a wide range of pH and temperature tolerance, as well as excellent reproducibility, reusability and storage stability. Subsequently, 26 natural products (NPs) were screened by immobilized PL with black tea extract having the highest inhibition rate. Three compounds with binding effects on PL (epigallocatechin gallate, theaflavin-3-gallate and theaflavin-3,3′-digallate) were captured. Molecular docking proved that these three compounds have a strong binding affinity for PL. Fluorescence spectra further revealed that theaflavin-3,3′-digallate could statically quench the intrinsic fluorescence of pancreatic lipase. The molecular docking and thermodynamic parameters indicated that electrostatic interaction was considered as the main interaction force between PL and theaflavin-3,3′-digallate. Finally, the potential anti-obesity targets and pathways of the three compounds were discussed through network pharmacology. This study not only proposes a simple and efficient method for screening PL inhibitors, but also sheds light on the anti-obesity mechanism of active compounds in black tea.

Clinical Properties and Non-Clinical Testing of Mineralocorticoid Receptor Antagonists in In Vitro Cell Models.

Mineralocorticoid receptor antagonists (MRAs) are one of the renin-angiotensin-aldosterone system inhibitors widely used in clinical practice. While spironolactone and eplerenone have a long-standing profile in clinical medicine, finerenone is a novel agent within the MRA class. It has a higher specificity for mineralocorticoid receptors, eliciting less pronounced adverse effects. Although approved for clinical use in patients with chronic kidney disease and heart failure, intensive non-clinical research aims to further elucidate its mechanism of action, including dose-related selectivity. Within the field, animal models remain the gold standard for non-clinical testing of drug pharmacological and toxicological properties. Their role, however, has been challenged by recent advances in in vitro models, mainly through sophisticated analytical tools and developments in data analysis. Currently, in vitro models are gaining momentum as possible platforms for advanced pharmacological and pathophysiological studies. This article focuses on past, current, and possibly future in vitro cell models research with clinically relevant MRAs.

Si-Ni-San alleviates intestinal and liver damage in ulcerative colitis mice by regulating cholesterol metabolism

Ethnopharmacological relevanceSi-Ni-San (SNS), a traditional Chinese medicinal formula derived from Treatise on Febrile Diseases, is considered effective in the treatment of inflammatory bowel diseases based upon thousands of years of clinical practice. However, the bioactive ingredients and underlying mechanisms are still unclear and need further investigation. Aim of the studyThis study aimed to evaluate the effect, explore the bioactive ingredients and the underlying mechanisms of SNS in ameliorating ulcerative colitis (UC) and associated liver injury in dextran sodium sulphate (DSS)-induced mouse colitis models. Materials and methodsThe effect of SNS (1.5, 3, 6 g/kg) on 3% DSS-induced acute murine colitis was evaluated by disease activity index (DAI), colon length, inflammatory cytokines, hematoxylin-eosin (H&E) staining, tight junction proteins expression, ALT, AST, and oxidative stress indicators. HPLC-ESI-IT/TOF MS was used to analyze the chemical components of SNS and the main xenobiotics in the colon of UC mice after oral administration of SNS. Network pharmacological study was then conducted based on the main xenobiotics. Flow cytometry and immunohistochemistry techniques were used to demonstrate the inhibitory effect of SNS on Th17 cells differentiation and the amelioration of Th17/Treg cell imbalance. LC-MS/MS, Real-time quantitative polymerase chain reaction (RT-qPCR), and western blotting techniques were performed to investigate the oxysterol-Liver X receptor (LXRs) signaling activity in colon. Targeted bile acids metabolomics was conducted to reveal the change of the two major pathways of bile acid synthesis in the liver, and the expression of key metabolic enzymes of bile acids synthesis was characterized by RT-qPCR and western blotting techniques. ResultsSNS (1.5, 3, 6 g/kg) decreased the DAI scores, protected intestinal mucosa barrier, suppressed the production of pro-inflammatory cytokines, improved hepatic and splenic enlargement and alleviated liver injury in a dose-dependent manner. A total of 22 components were identified in the colon of SNS (6 g/kg) treated colitis mice, and the top 10 components ranked by relative content were regarded as the potential effective chemical components of SNS, and used to conduct network pharmacology research. The efficacy of SNS was mediated by a reduction of Th17 cell differentiation, restoration of Th17/Treg cell homeostasis in the colon and spleen, and the experimental results were consistent with our hypothesis and the biological mechanism predicted by network pharmacology. Mechanistically, SNS regulated the concentration of 25-OHC and 27-OHC by up-regulated CH25H, CYP27A1 protein expression in colon, thus affected the expression and activity of LXR, ultimately impacted Th17 differentiation and Th17/Treg balance. It was also found that SNS repressed the increase of hepatic cholesterol and reversed the shift of BA synthesis to the acidic pathway in UC mice, which decreased the proportion of non-12-OH BAs in total bile acids (TBAs) and further ameliorated colitis and concomitant liver injury. ConclusionsThis study set the stage for considering SNS as a multi-organ benefited anti-colitis prescription based on the significant effect of ameliorating intestinal and liver damage, and revealed that derivatives of cholesterol, namely oxysterols and bile acids, were closely involved in the mechanism of SNS anti-colitis effect.

Epigenetic regulation of noncanonical menin targets modulates menin inhibitor response in acute myeloid leukemia

AbstractMenin inhibitors that disrupt the menin-MLL interaction hold promise for treating specific acute myeloid leukemia (AML) subtypes, including those with KMT2A rearrangements (KMT2A-r), yet resistance remains a challenge. Here, through systematic chromatin-focused CRISPR screens, along with genetic, epigenetic, and pharmacologic studies in a variety of human and mouse KMT2A-r AML models, we uncovered a potential resistance mechanism independent of canonical menin-MLL targets. We show that a group of noncanonical menin targets, which are bivalently cooccupied by active menin and repressive H2AK119ub marks, are typically downregulated after menin inhibition. Loss of polycomb repressive complex 1.1 (PRC1.1) subunits, such as polycomb group ring finger 1 (PCGF1) or BCL6 corepressor (BCOR), leads to menin inhibitor resistance by epigenetic reactivation of these noncanonical targets, including MYC. Genetic and pharmacological inhibition of MYC can resensitize PRC1.1-deficient leukemia cells to menin inhibition. Moreover, we demonstrate that leukemia cells with the loss of PRC1.1 subunits exhibit reduced monocytic gene signatures and are susceptible to BCL2 inhibition, and that combinational treatment with venetoclax overcomes the resistance to menin inhibition in PRC1.1-deficient leukemia cells. These findings highlight the important roles of PRC1.1 and its regulated noncanonical menin targets in modulating the menin inhibitor response and provide potential strategies to treat leukemia with compromised PRC1.1 function.

Metabolic profiling of triptophenolide in rat plasma, urine, bile and faeces after intragastric administration

Triptophenolide, a major diterpenoid extracted from Tripterygium wilfordii Hook. f., has been reported to possess significant anti-tumour, anti-androgen and anti-inflammatory activities. However, the metabolic fate of triptophenolide remains unknown. Therefore, this study focused on the metabolic profiling of triptophenolide in rat plasma, urine, bile and faeces following intragastric administration. An ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry with combination of extracted ion chromatogram strategy based on 71 typical metabolic reactions was established to comprehensively profile the metabolites of triptophenolide. This strategy allowed for the identification of 17 metabolites from the biosamples. Reduction, oxidation, glucuronide conjugation, and hydroxylation were considered as its main metabolic pathways in vivo. The present study will be greatly helpful for the further pharmacological studies on triptophenolide and would provide valuable information for its clinical application.

Pharmacodynamics of Qingxin Jieyu Granules for treatment of atherosclerosis and its regulatory mechanism for lipid metabolism

To elucidate the therapeutic mechanism of Qingxin Jieyu Granule (QXJYG) against atherosclerosis (AS) based on network pharmacology. The major targets and pathways of QXJYG against AS were analyzed using network pharmacology. Rat models of AS established by high-fat feeding combined with intraperitoneal vitamin D3 injection were treated daily with normal saline, atorvastatin (13.15 mg/kg), or QXJYG at 0.99, 1.98, and 3.96 g/kg for 8 weeks (n=6). Ultrasound and HE staining were used to assess the function and pathologies of the abdominal aorta. Blood lipids and serum levels of Ang Ⅱ, ET-1, TXA2, PGI2, and ox-LDL of the rats were detected using an automatic biochemical analyzer or ELISA. The expressions of LOX-1, PPARγ, RXRα, p-P65, VCAM-1 and ICAM-1 in the abdominal aorta were detected with immunohistochemistry. The rat models of AS showed obvious abdominal aorta wall thickening, increased pulse wave velocity and pulse index, decreased inner diameter of the abdominal aorta, elevated levels of TC, LDL-C, Ang Ⅱ, ET-1 and TXA2, and lowered levels of HDL-C and PGI2. QXJYG and atorvastatin treatment of the rat models significantly alleviated histopathological changes of the abdominal aorta, decreased serum levels of TC, LDL-C, Ang Ⅱ, ET-1 and TXA2, and increased the levels of HDL-C and PGI2. Network pharmacology study suggested the therapeutic effect of QXJYG against AS was mediated by regulating lipid metabolism, PPAR and NF-κB pathways. Consistently, treatments with QXJYG were found to significantly decrease ox-LDL level and LOX-1, P-P65, VCAM-1 and ICAM-1 protein expressions while increasing PPARγ and RXRα expressions in the aorta of AS rats. QXJYG alleviates lipid metabolism disorder and improves histopathological changes of the abdominal aorta of AS rats possibly by lowering ox-LDL level, reducing LOX-1 expression, activating PPARγ and RXRα, and inhibiting P65 phosphorylation to reduce VCAM-1 and ICAM-1 expression in the aorta.

Cloning and Direct Evolution of a Novel 7-O-Glycosyltransferase from Cucurbita moschata and Its Application in the Efficient Biocatalytic Synthesis of Luteolin-7-O-glucoside.

Luteolin-7-O-glucoside(L7G), a glycosylation product of luteolin, is present in a variety of foods, vegetables, and medicinal herbs and is commonly used in dietary supplements due to its health benefits. Meanwhile, luteolin-7-O-glucoside is an indicator component for the quality control of honeysuckle in the pharmacopoeia. However, its low content in plants has hindered its use in animal pharmacological studies and clinical practice. In this study, a novel 7-O-glycosyltransferase CmGT from Cucurbita moschata was cloned, which could efficiently convert luteolin into luteolin-7-O-glucoside under optimal conditions (40 °C and pH 8.5). To further improve the catalytic efficiency of CmGT, a 3D structure of CmGT was constructed, and directed evolution was performed. The mutant CmGT-S16A-T80W was obtained by using alanine scanning and iterative saturation mutagenesis. This mutant exhibited a kcat/Km value of 772 s-1·M-1, which was 3.16-fold of the wild-type enzyme CmGT. Finally, by introducing a soluble tag and UDPG synthesis pathway, the strain BXC was able to convert 1.25 g/L of luteolin into 1.91 g/L of luteolin-7-O-glucoside under optimal conditions, achieving a molar conversion rate of 96% and a space-time yield of 27.08 mg/L/h. This study provides an efficient method for the biosynthesis of luteolin-7-O-glucoside, which holds broad application prospects in the food and pharmaceutical industry.

Immobilised artificial membrane liquid chromatography vs liposome electrokinetic capillary chromatography: Suitability in drug/bio membrane partitioning studies and effectiveness in the assessment of the passage of drugs through the respiratory mucosa

This study pioneers a comparison of the application of biomimetic techniques, immobilised artificial membrane liquid chromatography (IAM LC) and liposome electrokinetic capillary chromatography (LEKC), for the prediction of pulmonary drug permeability. The pulmonary absorption profiles of 26 structurally unrelated drug-like molecules were evaluated using their IAM hydrophobicity index (CHI IAM) measured in IAM LC, and the logarithm of distribution constants (log KLEKC) derived from the LEKC experiments. Lipophilicity (phospholipids) parameters obtained from IAM LC and most LEKC analyses were linearly related to the n-octanol/water partitioning coefficients of the neutral forms (i.e., log Po/w values) to a moderate extent. However, the relationship with distribution coefficients at the experimental pH (7.4) (i.e., log D7.4) were weaker overall for IAM LC data and sigmoidal for some liposome compositions (phosphatidyl choline (PC): phosphatidyl inositol (PI) 85:15 mol% and 90:10 mol%) and concentrations (4 mM) in LEKC. This suggests that phospholipid partitioning supports both hydrophobic and electrostatic interactions occurring between ionised drugs and charged phospholipid moieties. The latter interactions are original when compared to those taking place in the more established n-octanol/water partitioning systems.A stronger correlation (R2 > 0.65) was identified between the LEKC retention parameters, and the experimental apparent lung permeability (i.e., log Papp values) as opposed to the values obtained by IAM LC. Therefore, LEKC offers unprecedented advantages over IAM LC in simulating cell membrane partitioning processes in the pulmonary delivery of drugs. Although LEKC has the advantage of more effectively simulating the electrostatic and hydrophobic forces in drug/pulmonary membrane interactions in vitro, the technique is unsuitable for analysing highly hydrophilic neutral or anionic compounds at the experimental pH. Conversely, IAM LC is useful for analysing compounds spanning a wider range of lipophilicity. Its simpler and more robust implementation, and propensity for high-throughput automation make it a favourable choice for researchers in drug development and pharmacological studies.

Low concentration dimethyl sulfoxide (DMSO) modulates epileptiform synchronization in the 4-aminopyridine in vitro model

Dimethyl sulfoxide (DMSO) is commonly used to dissolve water-insoluble drugs due to its dipolar and aprotic properties. It also serves as a vehicle in many pharmacological studies. However, it has been reported that DMSO can induce seizures in human patients, lower seizure threshold in vivo, and modulate ion receptors activities in vitro. Therefore, we investigated here the effect of 0.03 % and 0.06 % DMSO, which are 10–50 times lower than what usually employed in previous studies, in the 4-aminopyridine (4AP) model of epileptiform synchronization in male mouse brain slices. We found that 0.03 % and 0.06 % DMSO increase 4AP-induced ictal discharge rate, while 0.06 % DMSO decreases ictal discharge duration. Our results suggest that the effects of DMSO on neuronal excitability deserve further analysis and that investigators need to be aware of its confounding effect as a solvent, even at very low concentrations.

Anti-biofilm and anti-quorum sensing activities of extract, fractions and compounds from the leaves of Cassia alata L. against yeast pathogens

BackgroundCassia alata or Senna alata, also known as “ringworm bush” because of its very effective fungicidal properties, is commonly used in African traditional medicine to treat fungal infections. Despite extensive phytochemical and pharmacological studies previously reported on C. alata, the antibiofilm activity against pathogenic yeast as well as the related anti-quorum sensing mechanism of some active constituents has not yet been elucidated. The aim of the study was to isolate the bioactive constituents from the methanol extract of the leaves of C. alata (CAExt) using antibiofilm-guided fractionation against yeast fungal pathogens and then to investigate the anti-quorum sensing activity of the active constituents by assessing their ability to inhibit violacein production in Chromobacterium violaceum. MethodsChromatographic methods were used to isolate the constituents of CAExt, and spectroscopic methods were used to elucidate the chemical structures of the isolated compounds. The broth microdilution assay was used to evaluate the antifungal activity against Candida albicans and C. parapsilosis, while crystal violet staining was used for the inhibition of biofilm formation and the disruption of preformed biofilm. The biosensor strain C. violaceum ATCC 12472 was used to investigate the anti-quorum sensing activity of the most active constituents. ResultsThe crude extract exhibited biofilm inhibition and eradication activities against the tested pathogenic yeast. The biofilm inhibition percentages ranged from 53.22 % to 75.38 %, while the biofilm eradication percentages ranged from 23.21 % to 64.25 %. The ethyl acetate fraction demonstrated high biofilm inhibition and eradication activities against the tested microorganisms. The biofilm inhibition percentages ranged from 58.19 % to 79.30 %, while the biofilm eradication percentages ranged from 34.105 % to 69.54 %. The purification of subfractions led to the identification of six compounds: stigmasterol (1), sitosterol (2), lupeol (3), emodin (4), kaempferol (5) and stigmasterol glycoside (6), two of which (4 and 5) showed potent biofilm inhibition and eradication activities. Both compounds demonstrated significantly lower MBIC50 values of 70.81 µg/mL and 65.65 µg/mL against Candida albicans and MBEC50 values of 63.65 µg/mL and 82.66, respectively, against C. albicans and C. parapsilosis. The crude extract and compounds (4) and (5) also demonstrated quorum sensing inhibitory activity, as indicated by the MQSIC value of 1024 µg/mL for the crude extract and 128 µg/mL for the two compounds. Moreover, compounds (4) and (5) displayed significant inhibitory effects on violacein production, as indicated by their low IC50 values of 28.08 µg/mL and 26.44 µg/mL, respectively. ConclusionsData obtained in this study not only support the traditional use of C. alata in the treatment of fungal infections but also reveal C. alata extract, as well as the two isolated bioactive compounds emodin (4) and kaempferol (5), as a potential source for developing antibiofilm alternative agents against biofilm-associated yeast infections. List of compounds studiedstigmasterol (1), sitosterol (2), lupeol (3), emodin (4), kaempferol (5), stigmasterol glycoside (6)