Dual-Stimuli-Responsive Gut Microbiota-Targeting Nitidine Chloride-CS/PT-NPs Improved Metabolic Status in NAFLD.

Background and Purpose: Nitidine chloride (NC) is a botanical drug renowned for its potent anti-inflammatory, antimalarial, and hepatocellular carcinoma-inhibiting properties; however, its limited solubility poses challenges to its development and application. To address this issue, we have devised a colon-targeted delivery system (NC-CS/PT-NPs) aimed at modulating the dysbiosis of the gut microbiota by augmenting the interaction between NC and the intestinal microbiota, thereby exerting an effect against nonalcoholic fatty liver disease. Methods: The NC-CS/PT-NPs were synthesized using the ion gel method. Subsequently, the particle size distribution, morphology, drug loading efficiency, and release behavior of the NC-CS/PT-NPs were characterized. Furthermore, the impact of NC-CS/PT-NPs on non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet (HFD) in mice was investigated through serum biochemical analysis, ELISA, and histochemical staining. Additionally, the influence of NC-CS/PT-NPs on intestinal microbiota was analyzed using 16S rDNA gene sequencing. Results: The nanoparticles prepared in this study have an average particle size of (255.9±5.10) nm, with an encapsulation rate of (72.83±2.13) % and a drug loading of (4.65±0.44) %. In vitro release experiments demonstrated that the cumulative release rate in the stomach and small intestine was lower than 22.0%, while it reached 66.75% in the colon. In vivo experiments conducted on HFD-induced NAFLD mice showed that treatment with NC-CS/PT-NPs inhibited weight gain, decreased serum aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and lipid levels, improved liver and intestinal inflammation, and altered the diversity of gut microbiota in mice. Conclusion: This study provides new evidence for the treatment of NAFLD through the regulation of gut microbiota using active ingredients from traditional Chinese medicine.

Characterization of CYP82 genes involved in the biosynthesis of structurally diverse benzylisoquinoline alkaloids in Corydalis yanhusuo.

Benzylisoquinoline alkaloids (BIAs) represent a significant class of secondary metabolites with crucial roles in plant physiology and substantial potential for clinical applications. CYP82 genes are involved in the formation and modification of various BIA skeletons, contributing to the structural diversity of compounds. In this study, Corydalis yanhusuo, a traditional Chinese medicine rich in BIAs, was investigated to identify the catalytic function of CYP82s during BIA formation. Specifically, 20 CyCYP82-encoding genes were cloned, and their functions were identified in vitro. Ten of these CyCYP82s were observed to catalyze hydroxylation, leading to the formation of protopine and benzophenanthridine scaffolds. Furthermore, the correlation between BIA accumulation and the expression of CyCYP82s in different tissues of C. yanhusuo was assessed their. The identification and characterization of CyCYP82s provide novel genetic elements that can advance the synthetic biology of BIA compounds such as protopine and benzophenanthridine, and offer insights into the biosynthesis of BIAs with diverse structures in C. yanhusuo.

Protopine ameliorates OVA-induced asthma through modulatingTLR4/MyD88/NF-κB pathway and NLRP3 inflammasome-mediated pyroptosis.

BACKGROUND: Chronic airway inflammation and hyperresponsiveness are characteristics of asthma. The isoquinoline alkaloid protopine (PRO) has been shown to exert anti-inflammatory effects, but its mechanism of action in asthma is not known. PURPOSE: Investigate the protective properties of PRO upon asthma and elucidate its mechanism. STUDY DESIGN AND METHODS: The effects of PRO in asthma treatment were assessed by histology, biochemical analysis, and real-time reverse transcription-quantitative polymerase chain reaction. Then, we integrated molecular docking, western blotting, cellular experiments, immunohistochemistry, immunofluorescence analysis, flow cytometry, and metabolomics analysis to reveal its mechanism. RESULTS: In vivo, PRO therapy reduced the number of inflammatory cells (eosinophils, leukocytes, monocytes) in bronchoalveolar lavage fluid (BALF), ameliorated pathologic alterations in lung tissues, and inhibited secretion of IgG and histamine. Molecular docking showed that PRO could dock with the proteins of TLR4, MyD88, TRAF6, TAK1, IKKα, and TNF-α. Western blotting displayed that PRO inhibited the TLR4/NF-κB signaling pathway. PRO regulated expression of the pyroptosis-related proteins NLR family pyrin domain containing 3 (NLRP3) inflammasome, gasdermin D, caspase-1, and drove caspase-1 inactivation to affect inflammatory responses by inhibiting the NLRP3 inflammasome. In vitro, 24 h after treatment with PRO, cell activity, as well as levels of reactive oxygen species (ROS) and interleukin (IL)-1ß and IL-18, decreased significantly. Immunofluorescence staining showed that PRO decreased expression of TLR4 and MyD88 in vitro. PRO decreased nuclear translocation of NF-κB p65. Twenty-one potential biomarkers in serum were identified using metabolomics analysis, and they predominantly controlled the metabolism of phenylalanine, tryptophan, glucose, and sphingolipids. CONCLUSION: PRO reduced OVA-induced asthma. The underlying mechanism was associated with the TLR4/MyD88/NF-κB pathway and NLRP3 inflammasome-mediated pyroptosis.

From genomics to metabolomics: Deciphering sanguinarine biosynthesis in Dicranostigma leptopodum.

Dicranostigma leptopodum (Maxim) Fedde (DLF) is a renowned medicinal plant in China, known to be rich in alkaloids. However, the unavailability of a reference genome has impeded investigation into its plant metabolism and genetic breeding potential. Here we present a high-quality chromosomal-level genome assembly for DLF, derived using a combination of Nanopore long-read sequencing, Illumina short-read sequencing and Hi-C technologies. Our assembly genome spans a size of 621.81 Mb with an impressive contig N50 of 93.04 Mb. We show that the species-specific whole-genome duplication (WGD) of DLF and Papaver somniferum corresponded to two rounds of WGDs of Papaver setigerum. Furthermore, we integrated comprehensive homology searching, gene family analyses and construction of a gene-to-metabolite network. These efforts led to the discovery of co-expressed transcription factors, including NAC and bZIP, alongside sanguinarine (SAN) pathway genes CYP719 (CFS and SPS). Notably, we identified P6H as a promising gene for enhancing SAN production. By providing the first reference genome for Dicranostigma, our study confirms the genomic underpinning of SAN biosynthesis and establishes a foundation for advancing functional genomic research on Papaveraceae species. Our findings underscore the pivotal role of high-quality genome assemblies in elucidating genetic variations underlying the evolutionary origin of secondary metabolites.

The induced and intrinsic resistance of Escherichia coli to sanguinarine is mediated by AcrB efflux pump.

IMPORTANCE: The use of plant extracts is increasing as an alternative to synthetic compounds, especially antibiotics. However, there is no sufficient knowledge on the mechanisms and potential risks of antibiotic resistance induced by these phytochemicals. In the present study, we found that stable drug resistant mutants of E. coli emerged after repetitive exposure to sanguinarine and demonstrated that the AcrB efflux pump contributed to the emerging of induced and intrinsic resistance of E. coli to this phytochemical. Our results offered some insights into comprehending and preventing the onset of drug-resistant strains when utilizing products containing sanguinarine.

Cytotoxicity and Genotoxicity Evaluation of Zanthoxylum rhoifolium Lam and In Silico Studies of Its Alkaloids.

The alkaloids isolated from Zanthoxylum rhoifolium have demonstrated great pharmacological potential; however, the toxic profiles of these extracts and fractions are still not well elucidated. This study evaluated the toxicity of the ethanol extract (EEZR) and neutral (FNZR) and alkaloid (FAZR) fractions. Chemical characterization was performed by chromatographic methods: thin-layer chromatography (TLC) and high-performance liquid chromatography coupled with diode array detection (HPLC-DAD). The cytotoxicity of the samples was evaluated in human hepatocellular carcinoma (HepG2) cells using the cell viability method (MTT) and mutagenicity by the Allium cepa assay (ACA). Alkaloids isolated from the species were selected for toxicity prediction using preADMET and PROTOX. The molecular docking of the topoisomerase II protein (TOPOII) was used to investigate the mechanism of cell damage. In the EEZR, FNZR, and FAZR, the presence of alkaloids was detected in TCL and HPLC-DAD analyses. These samples showed a 50% inhibitory concentration (IC50) greater than 400 µg/mL in HepG2 cells. In ACA, time- and concentration-dependent changes were observed, with a significant reduction in the mitotic index and an increase in chromosomal aberrations for all samples. Nuclear sprouts and a micronucleus of the positive control (PC) were observed at 10 µg/mL and in the FAZR at 30 µg/mL; a chromosomal bridge in FNZR was observed at 105 µg/mL, CP at a concentration of 40 µg/mL, and nuclear bud and mitotic abnormalities in the EEZR were observed at 170 µg/mL. The alkaloids with a benzophenanthridine were selected for the in silico study, as structural alterations demonstrated certain toxic effects. Molecular docking with topo II demonstrated that all alkaloids bind to the protein. In summary, the fractionation of Z. rhoifolium did not interfere with toxicity; it seems that alkaloids with a benzophenanthridine nucleus may be involved in this toxicity.

Targeting ABCB6 with nitidine chloride inhibits PI3K/AKT signaling pathway to promote ferroptosis in multiple myeloma.

Since multiple myeloma (MM) remains a cureless malignancy of plasma cells to date, it becomes imperative to develop novel drugs and therapeutic targets for MM. We screened a small molecule library comprising 3633 natural product drugs, which demonstrated that Nitidine Chloride (NC), an extract from traditional Chinese medicine Zanthoxylum nitidum. We used Surface Plasmon Resonance-High Performance Liquid Chromatography-Protein Mass Spectrometry (SPR-HPLC-MS), Cellular Thermal Shift Assay (CETSA), molecular docking, and SPR assay to identify the potential targets of NC, in which ABCB6 was the unique target of NC. The effects of ABCB6 on cellular proliferation and drug resistance were determined by CCK8, western blot, flow cytometry, site-mutation cells, transmission electron microscopy, immunohistochemistry staining and xenograft model in vitro and in vivo. NC induced MM cell death by promoting ferroptosis. ABCB6 is the direct target of NC. ABCB6 expression was increased in MM samples compared to normal controls, which was significantly associated with MM relapse and poor outcomes. VGSK was the inferred binding epitope of NC on the ABCB6 protein. In the ABCB6-mutated MM cells, NC did not display cancer resistance, implying the vital role of ABCB6 in NC's bioactivity. Moreover, the silencing of ABCB6 significantly inhibited MM cell growth. Mechanistically, the direct binding of NC to ABCB6 suppressed PI3K/AKT signaling pathway to promote ferroptosis. In conclusion, ABCB6 can be a potential therapeutic target and prognostic biomarker in MM, while NC can be considered a novel drug for MM treatment.

A new C-C linked benzophenathridine-2-quinoline dimer, and the antiplasmodial activity of alkaloids from Zanthoxylum holstzianum.

The CH2Cl2/MeOH (1:1) extract of Zanthoxylum holstzianum stem bark showed good antiplasmodial activity (IC50 2.5 ± 0.3 and 2.6 ± 0.3 µg/mL against the W2 and D6 strains of Plasmodium falciparum, respectively). From the extract five benzophenanthridine alkaloids [8-acetonyldihydrochelerythrine (1), nitidine (2), dihydrochelerythine (3), norchelerythrine (5), arnottianamide (8)]; a 2-quinolone alkaloid [N-methylflindersine (4)]; a lignan [4,4'-dihydroxy-3,3'-dimethoxylignan-9,9'-diyl diacetate (7)] and a dimer of a benzophenanthridine and 2-quinoline [holstzianoquinoline (6)] were isolated. The CH2Cl2/MeOH (1:1) extract of the root bark afforded 1, 3-6, 8, chelerythridimerine (9) and 9-demethyloxychelerythrine (10). Holstzianoquinoline (6) is new, and is the second dimer linked by a C-C bond of a benzophenanthridine and a 2-quinoline reported thus far. The compounds were identified based on spectroscopic evidence. Amongst five compounds (1-5) tested against two strains of P. falciparum, nitidine (IC50 0.11 ± 0.01 µg/mL against W2 and D6 strains) and norchelerythrine (IC50 value of 0.15 ± 0.01 µg/mL against D6 strain) were the most active.

6-Methoxydihydrosanguinarine induces apoptosis and autophagy in breast cancer MCF-7 cells by accumulating ROS to suppress the PI3K/AKT/mTOR signaling pathway.

6-Methoxydihydrosanguinarine (6-MDS) is a natural benzophenanthridine alkaloid extracted from Hylomecon japonica (Thunb.) Prantl. It is the first time to explore the effect and mechanism of 6-MDS in breast cancer. Network pharmacology, molecular docking, and molecular dynamics simulation technology were adopted to identify the potential targets and pathways of 6-MDS in breast cancer. Besides, cell proliferation, apoptosis, and western blotting assays were conducted to investigate the effect of 6-MDS on MCF-7 cells. Network pharmacology, molecular docking, and molecular dynamics simulation results confirmed the effect of 6-MDS on resisting breast cancer via the PI3K/AKT/mTOR signaling pathway. In addition, the functional experiments results demonstrated that 6-MDS inhibited proliferation and induced apoptosis and autophagy. The autophagy inhibitor chloroquine and the silence of Atg5 augmented the effect of 6-MDS on promoting apoptosis. Furthermore, 6-MDS suppressed the PI3K/AKT/mTOR signaling pathway, and the PI3K inhibitor LY294002 enhanced these changes and promoted the 6-MDS pro-apoptotic and autophagy effects. 6-MDS triggered the generation of reactive oxygen species. The pretreatment with antioxidant N-acetyl-L-cysteine reversed the changes induced by 6-MDS, including increases in apoptosis and autophagy and inhibition of the PI3K/AKT/mTOR pathway. In conclusion, 6-MDS induces the apoptosis and autophagy of MCF-7 cells by ROS accumulation to suppress the PI3K/AKT/mTOR signaling pathway.

(±)-Zanthonitidumines A and B: Two new benzophenanthridine alkaloids enantiomers from Zanthoxylum nitidum and their anti-inflammatory activity.

Two new benzophenanthridine alkaloids enantiomers (±)-zanthonitidumines A (1) and B (2), along with seven known analogues (3-9), were isolated from Zanthoxylum nitidium. Their structures were elucidated on the basis of extensive spectroscopic techniques and ECD data. Compound 2 exhibited the most significant inhibition of IL-6 generation as well as TNF-α release which suggest that it may be a potential anti-inflammatory agent.

Nitidine chloride induces cardiac hypertrophy in mice by targeting autophagy-related 4B cysteine peptidase.

Nitidine chloride (NC) is a standard active component from the traditional Chinese medicine Zanthoxylum nitidum (Roxb.) DC. (ZN). NC has shown a variety of pharmacological activities including anti-tumor activity. As a number of anti-tumor drugs cause cardiotoxicity, herein we investigated whether NC exerted a cardiotoxic effect and the underlying mechanism. Aqueous extract of ZN (ZNE) was intraperitoneally injected into rats, while NC was injected into beagles and mice once daily for 4 weeks. Cardiac function was assessed using echocardiography. We showed that both ZNE administered in rats and NC administered in mice induced dose-dependent cardiac hypertrophy and dysfunction, whereas administration of NC at the middle and high dose caused death in Beagles. Consistently, we observed a reduction of cardiac autophagy levels in NC-treated mice and neonatal mouse cardiomyocytes. Furthermore, we demonstrated that autophagy-related 4B cysteine peptidase (ATG4B) may be a potential target of NC, since overexpression of ATG4B reversed the cardiac hypertrophy and reduced autophagy levels observed in NC-treated mice. We conclude that NC induces cardiac hypertrophy via ATG4B-mediated downregulation of autophagy in mice. Thus, this study provides guidance for the safe clinical application of ZN and the use of NC as an anti-tumor drug.

Sanguinarine suppresses cell proliferation, migration and invasion in nasopharyngeal carcinoma inhibiting mTOR signaling.

OBJECTIVE: To confirm the anti-NPC effect of sanguinarine (SA) through a series of wet experiments. METHODS: NPC cell viability was determined by proliferation experiment. Cell clone formation experiment, cell scratch test, transwell migration and invasion experiment and flow cytometry-based cell apoptosis assay were further performed. In addition, Western blotting was performed to investigate the cell signaling pathway. All the relevant experimental data were statistically processed using SPSS 16.0 software. RESULTS: The results showed that sanguinarine represented a time and dose dependent inhibition effects on NPC cell proliferation including the low differentiated CNE2 cells and high metastatic 5-8F cells, along with the cell cloning ability reduction. In addition, sanguinarine has a certain inhibitory effect on the invasion and migration of NPC cells. Mechanistically, sanguinarine displayed the anti-NPC effects mainly involved into the suppression of mTOR signaling and cell apoptosis, which is closely associated with the tumor growth and metastatic malignancy. CONCLUSIONS: Collectively, we discover that sanguinarine is a new high-efficiency anti-NPC monomer of Chinese medicine, with a value for the follow-up pre-clinical research.

Understanding of black salve toxicity by multi-compound cytotoxicity assays.

BACKGROUND: Black salve is a controversial complementary and alternative medicine (CAM) associated with skin toxicity and skin cancer treatment failures. Black salve formulations vary between manufacturers and contain a number of botanical and synthetic constituents. The skin cancer cytotoxicity of a number of these constituents has not been assessed to date. The alkaloids from the rhizomes of Sanguinaria canadensis, a key black salve ingredient, have had their single compound cytotoxicity assessed; however, whether they possess synergistic cytotoxicity with other compounds has not been studied and is of direct clinical relevance. This research aimed to improve our understanding of the skin cancer cytotoxicity of black salve constituents. METHODS: The cytotoxicity of individual and combination black salve constituents were assessed against the A375 melanoma and A431 squamous cell carcinoma cell lines. Cytotoxicity was determined using the Resazurin assay with fluorescence measured using a Tecan Infinite 200 Pro Microplate reader, compound cytotoxicity being compared to that of the topical cancer therapeutic agent, 5- fluouracil. Docetaxal was used as a positive control. Dunnetts p value was used to determine whether significant synergistic cytotoxicity was present. RESULTS: Sanguinarine was the most cytotoxic compound tested with a 24-hour IC50 of 2.1 µM against the A375 Melanoma cell line and 3.14 µM against the A431 SCC cell line. All black salve constituents showed greater cytotoxicity against the two skin cancer cell lines tested than the skin cancer therapeutic 5-Fluouracil with 24 hours of compound exposure. Chelerythrine and minor Quaternary Benzophenanthridine Alkaloids (QBAs) present in black salve, at concentrations not having a cytotoxic effect by themselves, boosted the cytotoxic effects of sanguinarine. This could be a synergistic rather than additive cytotoxic effect although the synergistic effect was cell line and concentration dependent. CONCLUSIONS: Black salve contains several cytotoxic compounds, a number of which have been found to possess synergistic cytotoxicity for the first time against skin cancer cell lines. In addition, these compounds together increase the overall cytotoxic effect. Assessing multi-compound cytotoxicity in herbal medicine can provide additional information about both their therapeutic and toxicity potential. As black salve is currently being used by patients, further cytotoxicity work should be undertaken to assess whether synergistic cytotoxicity exists when tested in normal skin cells.

Sanguinarine ameliorates DSS induced ulcerative colitis by inhibiting NLRP3 inflammasome activation and modulating intestinal microbiota in C57BL/6 mice.

BACKGROUND: Sanguinarine (SAN) is an important natural anti-inflammatory constitutes and dietary supplementation with SAN could improve the relative length of the intestine, alter gut microbiota, and enhance growth performance of pigs, broiler chickens, and cattle. However, it is unclear whether it has the therapeutic effect on ulcerative colitis (UC). PURPOSE: This study aimed to investigate the therapeutic effect of SAN on UC and explore its mechanisms of action. STUDY DESIGN AND METHODS: Several efficacy indexes of SAN on dextran sulfate sodium (DSS)-induced C57BL/6 mice were evaluated. ELISA kit and western blot analysis were used to evaluate it's anti-inflammatory effect and the mechanism of action. 16S rDNA sequencing detection was used to determine the impact of SAN on gut microbiota. RESULTS: SAN and Sulfasalazine could significantly improve the colon length, the weight loss, the symptoms and the pathological injury of colon in DSS-induced mice. Meanwhile, SAN could decrease the levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1ß, IL-6, IL-13 and IL-18) and increase the levels of anti-inflammatory cytokines (IL-4 and IL-10) in colon, and suppress DSS-induced high expressions of NLRP3, caspase-1 and IL-1ß. In addition, SAN (0.5, 1 µM) could inhibit the expression level of NLRP3 and the activation of caspase-1 and IL-1ß in lipopolysaccharide-stimulated THP-1 cells in non-cytotoxic doses, which was similar to that of MCC950, a specific inhibitor of NLRP3 inflammasome activation. The abundance changes of many genera such as Muribaculaceae_unclassified, Escherichia-Shigella, Lachnospiraceae_NK4A136_group and Helicobacter were also closely related to the improvement of SAN on intestinal inflammatory response. CONCLUSION: SAN exhibited therapeutic effect on DSS-induced colitis by blocking NLRP3-(Caspase-1)/IL-1ß pathway and improving intestinal microbial dysbiosis. SAN might be developed to treat UC and other disorders associated with microbial dysbiosis.

Fluorescence, Absorption, Chromatography and Structural Transformation of Chelerythrine and Ethoxychelerythrine in Protic Solvents: A Comparative Study.

Chelerythrine (CH) and ethoxychelerythrine (ECH) are chemical reference substances for quality control of Chinese herbal medicines, and ECH is the dihydrogen derivative of CH. In this study, their fluorescence and absorption spectra, as well as their structural changes in different protic solvents were compared. It was observed that their emission fluorescence spectra in methanol were almost the same (both emitted at 400 nm), which may be attributed to the nucleophilic and exchange reactions of CH and ECH with methanol molecules with the common product of 6-methoxy-5,6-dihydrochelerythrine (MCH). When diluted with water, MCH was converted into CH, which mainly existed in the form of positively charged CH+ under acidic and near-neutral conditions with the fluorescence emission at 550 nm. With the increase of pH value of the aqueous solution, CH+ converted to 6-hydroxy-5,6-dihydrochelerythrine (CHOH) with the fluorescence emission at 410 nm. The fluorescence quantum yields of MCH and CHOH were 0.13 and 0.15, respectively, and both the fluorescence intensities were much stronger than that of CH+. It is concluded that CH and ECH can substitute each other in the same protic solvent, which was further verified by high-performance liquid chromatography. This study will help in the investigation of structural changes of benzophenanthridine alkaloids and will provide the possibility for the mutual substitution of standard substances in relevant drug testing.

Antinociceptive and gastroprotective activities of Bocconia arborea S. Watson and its bioactive metabolite dihydrosanguinarine in murine models.

ETHNOPHARMACOLOGICAL RELEVANCE: Bocconia arborea S. Watson (Papaveraceae) is known as "palo llora sangre" and is used in Mexican traditional medicine for the treatment of infections, it is also used as anxiolytic, analgesic, and antidiabetic, among others. AIM OF THE STUDY: to evaluate the antinociceptive and gastroprotective activities of extracts from B. arborea and dihydrosanguinarine (DHS) in murine models. MATERIALS AND METHODS: Organic extracts [hexane (HEX), dichloromethane (DCM) and methanol (MeOH)] were obtained by maceration. DHS was isolated and purified from HEX and DCM by precipitation and chromatographic column, respectively. Organic extracts and DHS were evaluated to determine their antinociceptive effect using formalin test in murine model. Also, the ambulatory effect of the HEX and DHS was determined in Open field test. The possible mechanism of action of DHS was explored in the presence of naltrexone (NTX, 1 mg/kg, i.p.), and picrotoxin (PTX, 1 mg/kg, i.p.). Gastric damage as possible adverse effect or gastroprotection were also investigated. Whereas DHS acute toxicological study was done, and 100 mg/kg of DHS was examined by electroencephalographic (EEG) analysis to discard neurotoxic effects. RESULTS: The B. arborea extracts significantly showed effects in both neurogenic and inflammatory phases of the formalin test, where the HEX extract reached the major antinociceptive effect. A significant and dose-response (10, 30, and 100 mg/kg) antinociceptive activity was observed with the HEX (ED50 = 69 mg/kg) and DHS (ED50 = 85 mg/kg) resembling the effect of the reference analgesic drug tramadol (30 mg/kg). The significant effect of DHS was inhibited in the presence of NTX and PTX. Neither the extracts or DHS produced sedative effects or gastric damage per se at antinociceptive doses. The EEG analysis demonstrated central depressant activity but not sedative or neurotoxic effects at the highest antinociceptive dosage tested, and LD50 is higher than 2000 mg/kg. CONCLUSIONS: HEX, DCM, and MeOH extracts showed significant antinociceptive activity, and DHS was identified as one of bioactive compounds without producing sedative, neurotoxic or gastric damage effects, as possible adverse effects reported for analgesic drugs. A role of opioid and GABAA neurotransmission appears to be involved as mechanisms of action of DHS, suggesting its potential for pain therapy and reinforcing the traditional use of B. arborea.

Chelerythrine ameliorates rheumatoid arthritis by modulating the AMPK/mTOR/ULK-1 signaling pathway.

BACKGROUND: Rheumatoid arthritis (RA) is a long-term, progressive, and disabling autoimmune disease. It causes inflammation, swelling and pain in and around the joints and other body organs. Currently, no cure is available for RA. Clinical interventions can only relieve the condition, and at least 30% of RA patients do not respond to first­line therapy. This means that the development of more effective therapies against RA is urgently needed. OBJECTIVE: This study aimed to assess the anti-rheumatoid arthritis effect of chelerythrine (CLT) and explore its mechanism of action. METHODS: The cytotoxic effect of CLT on human rheumatoid arthritis fibroblast-like synoviocyte (HFLS-RA) cells and HFLS-normal cells were measured by MTT assay. The growth and migration of HFLS-RA cells were determined by colony-formation and wound-healing assay. The level of intracellular reactive oxygen species (ROS) was detected using the DCFH-DA reagent. Cell apoptosis was measured by flow cytometry, TUNEL staining, caspase 3 activity, as well as the activation of apoptosis related proteins. In addition, the levels of autophagy related markers such as LC3B and P62 were determined by immunocytochemistry and western blotting. Lastly, the anti-RA effect of CLT was evaluated in an Adjuvant-Induced Arthritis(AIA) rat model and the severity of arthritis was detected and quantified using macroscopic inspection and X­ray imaging. RESULTS: We discovered that treatment with CLT effectively inhibited the migration and colony-formation of the HFLS-RA cells and resulted in cell death. Moreover, CLT increased the intracellular level of ROS and the apoptotic rate of HFLS-RA by activating the AMPK/mTOR/ULK-1 signaling pathways. In vivo study showed CLT effectively ameliorated AIA in rats, protecting them from inflammation and bone damage. CONCLUSION: Our study shows CLT is an effective agent for ameliorating RA in vitro and in vivo by modulation of the AMPK/mTOR/ULK-1 signaling pathway. These findings indicate that CLT is a great potential candidate for development as a therapeutic agent for the prevention and treatment of RA.

Sanguinarine, a major alkaloid from Zanthoxylum nitidum (Roxb.) DC., inhibits urease of Helicobacter pylori and jack bean: Susceptibility and mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum nitidum (Roxb.) DC. (Z. nitidum) is a traditional Chinese medicine and mainly adopted to treat gastric ulcer, gastritis and stomach cancer. Sanguinarine (SNG), a natural alkaloid isolated from Z. nitidum, possesses significant anti-Helicobacter pylori and gastric protection effects. However, the underlying mechanism is sparsely elucidated. AIM OF THIS STUDY: The present study aims to explore the inhibition effect, kinetics and potential mechanism of SNG against H. pylori urease (HPU) and jack bean urease (JBU). MATERIALS AND METHODS: The improved spectrophotometric berthelot method was applied to estimate the inhibitory effect of SNG against HPU and JBU. The Lineweaver-Burk plots were adopted for investigating the inhibitory pattern in enzymatic kinetics. Sulfydryl-containing compounds and competitive active-site Ni2+ binding depressors were used for mechanism research. RESULTS: SNG remarkably suppressed the activities of HPU and JBU in concentration-and time-dependent mode with IC50 of 0.48 ± 0.14 mM and 0.11 ± 0.02 mM, respectively, in comparison with urease retardant acetohydroxamic acid (0.06 ± 0.01 mM for HPU and 0.03 ± 0.00 mM for JBU, respectively). Kinetic analysis demonstrated that the inhibition of SNG against HPU and JBU were separately characterized by slow-binding, mixed-type and slow-binding, non-competitive type. Addition of sulfydryl-containing reagents (dithiothreitol, glutathione and L-cysteine) and competitive Ni2+ binding restrainers (boric acid and sodium fluoride) significantly abrogated the urease inhibitory effect of SNG, suggesting the significant role of the thiols and Ni2+ for the urease inhibition by SNG. By contrast, interaction with thiol groups possibly contributed to the repression of SNG on JBU. Furthermore, the urease suppression was proved to be partially reversible since the SNG-blocked enzyme could be partly reactivated by glutathione. CONCLUSION: SNG could observably inhibit H. pylori urease targeting the thiols and Ni2+, which indicated that SNG was a new urease suppressant with great promise. The present research also provided scientific evidence for the application of SNG and Z. nitidum treating H. pylori-associated gastrointestinal diseases.

Sanguinarine Represses the Growth and Metastasis of Non-small Cell Lung Cancer by Facilitating Ferroptosis.

OBJECTIVE: Ethnopharmacological relevance: Sanguinarine (SAG), a natural benzophenanthridine alkaloid derived from the root of Sanguinaria canadensis Linn. (Bloodroot), possesses a potential anticancer activity. Lung carcinoma is the chief cause of malignancy-related mortality in China. Non-small cell lung carcinoma (NSCLC) is the main subtype of lung carcinoma and accounts for about eighty-five percent of this disease. Current treatment in controlling and curing NSCLC remains deficient. AIM: The role and underlying mechanism of SAG in repressing the growth and metastasis of NSCLC were explored. MATERIALS AND METHODS: The role of SAG in regulating the proliferation and invasion of NSCLC cells was evaluated in vitro and in a xenograft model. After treatment with SAG, Fe2+ concentration, reactive oxygen species (ROS) levels, malondialdehyde (MDA), and glutathione (GSH) content in NSCLC cells were assessed to evaluate the effect of SAG on facilitating ferroptosis. RESULTS: SAG exhibited a dose- and time-dependent cytotoxicity in A549 and H3122 cells. SAG treatment effectively repressed the growth and metastasis of NSCLC in a xenograft model. We, for the first time, verified that SAG triggered ferroptosis of NSCLC cells, as evidenced by increased Fe2+ concentration, ROS level, and MDA content, and decreased GSH content. Mechanistically, SAG decreased the protein stability of glutathione peroxide 4 (GPX4) through E3 ligase STUB1-mediated ubiquitination and degradation of endogenous GPX4. GPX4 overexpression restored the proliferation and invasion of NSCLC cells treated with SAG through inhibiting ferroptosis. CONCLUSION: SAG inhibits the growth and metastasis of NSCLC by regulating STUB1/GPX4-dependent ferroptosis.

An Update of the Sanguinarine and Benzophenanthridine Alkaloids' Biosynthesis and Their Applications.

Benzophenanthridines belong to the benzylisoquinolic alkaloids, representing one of the main groups of this class. These alkaloids include over 120 different compounds, mostly in plants from the Fumariaceae, Papaveraceae, and Rutaceae families, which confer chemical protection against pathogens and herbivores. Industrial uses of BZD include the production of environmentally friendly agrochemicals and livestock food supplements. However, although mainly considered toxic compounds, plants bearing them have been used in traditional medicine and their medical applications as antimicrobials, antiprotozoals, and cytotoxic agents have been envisioned. The biosynthetic pathways for some BZD have been established in different species, allowing for the isolation of the genes and enzymes involved. This knowledge has resulted in a better understanding of the process controlling their synthesis and an opening of the gates towards their exploitation by applying modern biotechnological approaches, such as synthetic biology. This review presents the new advances on BDZ biosynthesis and physiological roles. Industrial applications, mainly with pharmacological approaches, are also revised.