Comparative toxicology of algal cell extracts and pure cyanotoxins: insights into toxic effects and mechanisms of harmful cyanobacteria Raphidiopsis raciborskii.

Ongoing research on cyanotoxins, driven by the socioeconomic impact of harmful algal blooms, emphasizes the critical necessity of elucidating the toxicological profiles of algal cell extracts and pure toxins. This study comprehensively compares Raphidiopsis raciborskii dissolved extract (RDE) and cylindrospermopsin (CYN) based on Daphnia magna assays. Both RDE and CYN target vital organs and disrupt reproduction, development, and digestion, thereby causing acute and chronic toxicity. Disturbances in locomotion, reduced behavioral activity, and weakened swimming capability in D. magna have also been reported for both RDE and CYN, indicating the insufficiency of conventional toxicity evaluation parameters for distinguishing between the toxic effects of algal extracts and pure cyanotoxins. Additionally, chemical profiling revealed the presence of highly active tryptophan-, humic acid-, and fulvic acid-like fluorescence compounds in the RDE, along with the active constituents of CYN, within a 15-day period, demonstrating the chemical complexity and dynamics of the RDE. Transcriptomics was used to further elucidate the distinct molecular mechanisms of RDE and CYN. They act diversely in terms of cytotoxicity, involving oxidative stress and response, protein content, and energy metabolism, and demonstrate distinct modes of action in neurofunctions. In essence, this study underscores the distinct toxicity mechanisms of RDE and CYN and emphasizes the necessity for context- and objective-specific toxicity assessments, advocating nuanced approaches to evaluate the ecological and health implications of cyanotoxins, thereby contributing to the precision of environmental risk assessments.

Anti-inflammatory effects of steroidal alkaloids of Solanum lyratum Thunb.

Solanum lyratum Thunb., a traditional Chinese herbal medicine, has a promising background. However, the anti-inflammatory effects of its component steroid alkaloid have not been explored. In this study, animal and cell experiments were performed to investigate the anti-inflammatory effects and mechanism of action of Solanum lyratum Thunb steroid alkaloid (SLTSA), in order to provide evidence for its potential utilization. SLTSA effectively inhibited ear swelling and acute abdominal inflammation of mice. We observed concentration-dependent inhibition of pro-inflammatory cytokines by SLTSA, as confirmed by the ELISA and RT-qPCR results. Flow cytometry, immunofluorescence and RT-qPCR analyses revealed that SLTSA suppressed TLR4 expression. Western blot results indicated that SLTSA inhibited the activation of the TLR4/MyD88/NF-κB signaling pathway. Our study demonstrated that SLTSA possesses anti-inflammatory properties.

Effect of pH and temperature on tropane alkaloids within a processing strategy to provide safe infant cereal-based food.

Tropane alkaloids (TAs) are secondary metabolites from weeds that can contaminate cereals and vegetables during harvest. Due to their toxicity, the Regulation (EC) 2023/915 sets maximum levels for atropine and scopolamine in cereal-based foods for infants containing millet, sorghum, buckwheat or their derived products. The aim of this study was to evaluate the effect of pH and temperature on the stability of TAs, as possible parameters in thermal processing to mitigate this chemical hazard in cereal-based infant food. The effect of pH (4 and 7) and temperature (80 °C and 100 °C) was assessed in buffer solutions. Also, treatment at 180 °C was performed in spiked and naturally incurred millet flour to assess the effect of high temperature, simulating cooking or drying, on the stability of TAs in the cereal matrix. The fate of 24 TAs was assessed by UHPLC-MS/MS. TAs showed high thermostability, although it was variable depending on the specific compound, pH, temperature and treatment time. In buffer solutions, higher degradation was found at 100 °C and pH 7. In spiked millet flour at 180 °C for 10 min, scopolamine and atropine contents decreased by 25 % and 22 %, similarly to other TAs which also showed a slow thermal degradation. Atropine, scopolamine, anisodamine, norscopolamine, scopine and scopoline were found in naturally contaminated millet flour. Interestingly, naturally incurred atropine was more thermostable than when spiked, showing a protective effect of the cereal matrix on TAs degradation. The present results highlight the need for an accurate monitorization of TAs in raw materials, as this chemical hazard may remain in infant cereal-based food even after intense thermal processing.

3-CMC, 4-CMC, and 4-BMC Human Metabolic Profiling: New Major Pathways to Document Consumption of Methcathinone Analogues?

Synthetic cathinones represent one of the largest and most abused new psychoactive substance classes, and have been involved in numerous intoxications and fatalities worldwide. Methcathinone analogues like 3-methylmethcathinone (3-MMC), 3-chloromethcathinone (3-CMC), and 4-CMC currently constitute most of synthetic cathinone seizures in Europe. Documenting their consumption in clinical/forensic casework is therefore essential to tackle this trend. Targeting metabolite markers is a go-to to document consumption in analytical toxicology, and metabolite profiling is crucial to support investigations. We sought to identify 3-CMC, 4-CMC, and 4-bromomethcathinone (4-BMC) human metabolites. The substances were incubated with human hepatocytes; incubates were screened by liquid chromatography-high-resolution tandem mass spectrometry and data were mined with Compound Discoverer (Themo Scientific). 3-CMC-positive blood, urine, and oral fluid and 4-CMC-positive urine and saliva from clinical/forensic casework were analyzed. Analyses were supported by metabolite predictions with GLORYx freeware. Twelve, ten, and ten metabolites were identified for 3-CMC, 4-CMC, and 4-BMC, respectively, with similar transformations occurring for the three cathinones. Major reactions included ketoreduction and N-demethylation. Surprisingly, predominant metabolites were produced by combination of N-demethylation and ω-carboxylation (main metabolite in 3-CMC-positive urine), and combination of ß-ketoreduction, oxidative deamination, and O-glucuronidation (main metabolite in 4-CMC-positive urine). These latter metabolites were detected in negative-ionization mode only and their non-conjugated form was not detected after glucuronide hydrolysis; this metabolic pathway was never reported for any methcathinone analogue susceptible to undergo the same transformations. These results support the need for comprehensive screening strategies in metabolite identification studies, to avoid overlooking significant metabolites and major markers of consumption.

Cytotoxicity of alkaloids isolated from Peganum harmala seeds on HCT116 human colon cancer cells.

BACKGROUND: The present study aimed to elucidate the potential anticancer activity and mechanism of P. harmala's alkaloid extract, harmine (HAR), and harmaline (HAL) in HCT-116 colorectal cancer cells. METHODS AND RESULTS: P. harmala's alkaloid was extracted from harmala seeds. HCT-116 cells were treated with P. harmala's alkaloid extract, HAR and HAL. Cytotoxicity was determined by MTT assay, apoptotic activity detected via flow cytometry and acridine orange (AO)/ethidium bromide (EB) dual staining, and cell cycle distribution analyzed with flow cytometry. The mRNA expression of Bcl-2-associated X protein (Bax) and glycogen synthase kinase-3 beta (GSK3ß) was measured by real-time PCR. Furthermore, the expression of Bax, Bcl-2, GSK3ß and p53 proteins, were determined by western blotting. The findings indicated that, P. harmala's alkaloids extract, HAR and HAL were significantly cytotoxic toward HCT116 cells after 24 and 48 h of treatment. We showed that P. harmala's alkaloid extract induce apoptosis and cell cycle arrest at G2 phase in the HCT116 cell line. Downregulation of GSK3ß and Bcl-2 and upregulation of Bax and p53 were observed. CONCLUSION: The findings of this study indicate that the P. harmala's alkaloid extract has anticancer activity and may be further investigated to develop future anticancer chemotherapeutic agents.

Reversal of gentamicin sulfate resistance in avian pathogenic Escherichia coli by matrine combined with berberine hydrochloride.

In recent years, the evolution of antibiotic resistance has led to the inefficacy of several antibiotics, and the reverse of resistance was a novel method to solve this problem. We previously demonstrated that matrine (Mat) and berberine hydrochloride (Ber) had a synergistic effect against multidrug-resistant Escherichia coli (MDREC). This study aimed to demonstrate the effect of Mat combined with Ber in reversing the resistance of MDREC. The MDREC was sequenced passaged in the presence of Mat, Ber, and a combination of Mat and Ber, which did not affect its growth. The reverse rate was up to 39.67% after MDREC exposed to Mat + Ber for 15 days. The strain that reversed resistance was named drug resistance reversed E. coli (DRREC) and its resistance to ampicillin, streptomycin, gentamicin, and tetracycline was reversed. The MIC of Gentamicin Sulfate (GS) against DRREC decreased 128-fold to 0.63 µg/mL, and it was stable within 20 generations. Furthermore, the susceptible phenotype of DRREC remained stable within 20 generations, as well. The LD50 of DRREC for chickens was 8.69 × 109 CFU/mL. qRT-PCR assays revealed that the transcript levels of antibiotic-resistant genes and virulence genes in the DRREC strain were significantly lower than that in the MDREC strain (P < 0.05). In addition, GS decreased the death, decreased the bacterial loading in organs, alleviated the injury of the spleen and liver, and decreased the cytokine levels in the chickens infected by the DRREC strain. In contrast, the therapeutic effect of GS in chickens infected with MDREC was not as evident. These findings suggest that the combination of Mat and Ber has potential for reversing resistance to MDREC.

Low-triggering-potential electrochemiluminescence based on mental-organic frameworks encapsulation of ruthenium for synthetic cathinone detection by coupling photonic crystal light-scattering signal amplification of covalent-organic frameworks.

Developing effective electrochemiluminescence (ECL) platforms is always an essential concern in highly sensitive bioanalysis. In this work, a low-triggering-potential ECL sensor was designed for detecting synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) based on a dual-signal amplification strategy. Initially, a probe was created by integrating Ruthenium into the hollow porphyrin-based MOF (PCN-222) structure to decrease the excitation potential and enhance ECL performance without external co-reaction accelerators. Additionally, for the first time, photonic crystals (PCs) assembled from covalent organic frameworks (COFs) were employed to amplify the ECL signal, thereby increasing the photon flux and the loading capacity of the ECL emitter to enhance sensitivity of the sensor. In the presence of the target MDPV, the aptamer labeled with Ferrocene (Fc) experienced conformational changes, causing Fc to approach the luminophore and resulting in ECL quenching. This effect was attributed to aptamer's conformational changes induced by the target, directly correlating with the target concentration. The constructed sensor showed good linearity with the target MDPV concentration, covering a dynamic range from 1.0 × 10-14 to 1.0 × 10-6 g/L and achieved an ultra-low detection limit of 4.79 × 10-15 g/L. This work employed dual amplification strategies to enhance ECL signals effectively, providing a novel method for developing highly responsive and bioactive sensors.

Chemical composition analysis of Qingyan dropping pills using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

RATIONALE: This study developed a method for the rapid classification and identification of the chemical composition of Qingyan dropping pills (QDP) to provide the theoretical basis and data foundation for further in-depth research on the pharmacological substance basis of the formula and the selection of quality control indexes. METHODS: Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) and data postprocessing technology were used to analyze the chemical composition of QDP. The fragmentation information on possible characteristic fragments and related neutral losses was summarized based on the literature and was compared with the MS data obtained from the assay, and thus a rapid classification and identification of chemical components in QDP could be achieved. RESULTS: A total of 73 compounds were identified, namely 24 flavonoids, 14 terpenoids, 30 organic acids and their esters, 3 alkaloids, and 2 phenylpropanoids. CONCLUSIONS: In this study, UHPLC-Q-TOF-MS and data postprocessing technology were used to realize the rapid classification and identification of the chemical constituents of QDP, which provided a comprehensive, efficient, and fast qualitative analysis method, a basis for further quality control and safe medication of QDP.

Novel Approaches for the Analysis and Isolation of Benzylisoquinoline Alkaloids in Chelidonium majus.

Benzylisoquinoline alkaloids are the major bioactive components in Chelidonium majus, a plant that has a long usage history for the treatment of gastrointestinal ailments in European and Asian phytomedicine. This study reports on the development and application of a supercritical fluid chromatography technique for the simultaneous qualitative and quantitative determination of seven benzylisoquinoline alkaloids in under six minutes using a Viridis BEH 2-EP column and a modifier comprising methanol with 30% acetonitrile and 20 mM ammonium formate. The method was fully validated according to ICH guidelines showing, e.g., excellent linearity (≥ 0.9997) and maximum deviations for intraday and inter-day precision of 2.99 and 2.76%, respectively. The new supercritical fluid chromatography assay was not only employed for the analysis of several C. majus samples but was also used for the subsequent development of a fast centrifugal partition chromatography technique, whereby five benzylisoquinoline alkaloids could be isolated within approximately 2.5 h, with only two of them, protopine and chelidonine, requiring an additional purification step. To achieve this, a solvent system composed of chloroform/methanol/0.3 M hydrochloric acid was used in descending mode. By injecting 500 mg of crude extract, stylopine (1.93 mg), sanguinarine (0.57 mg), chelidonine (1.29 mg), protopine (1.95 mg), and coptisine (7.13 mg) could be obtained. The purity of compounds was confirmed by supercritical fluid chromatography and MS.

Molecular modeling of multi-target analogs of huperzine A and applications in Alzheimer's disease.

CONTEXT: Given the diverse pathophysiological mechanisms underlying Alzheimer's disease, it is improbable that a single targeted drug will prove successful as a therapeutic strategy. Therefore, exploring various hypotheses in drug design is imperative. The sequestration of Fe(II) and Zn(II) cations stands out as a crucial mechanism based on the mitigation of reactive oxygen species. Moreover, inhibiting acetylcholinesterase represents a pivotal strategy to enhance acetylcholine levels in the synaptic cleft. This research aims to investigate the analogs of Huperzine A, documented in scientific literature, considering of these two hypotheses. Consequently, the speciation chemistry of these structures with Fe(II) and Zn(II) was scrutinized using quantum chemistry calculations, molecular docking simulations, and theoretical predictions of pharmacokinetics properties. From the pharmacokinetic properties, only two analogs, HupA-A1 and HupA-A2, exhibited a theoretical permeability across the blood-brain barrier; on the other hand, from a thermodynamic standpoint, the enantiomers of HupA-A2 showed negligible chelation values. The enantiomers with the most favorable interaction parameters were S'R'HupA-A1 (ΔGBIND = -40.0 kcal mol-1, fitness score = 35.5) and R'R'HupA-A1 (ΔGBIND = -35.5 kcal mol-1, fitness score = 22.61), being compared with HupA (ΔGBIND = -41.75 kcal mol-1, fitness score = 39.95). From this study, some prime candidates for promising drug were S'R'HupA-A1 and R'R'HupA-A1, primarily owing to their favorable thermodynamic chelating capability and potential anticholinesterase mechanism. METHODS: Quantum chemistry calculations were carried out at B3LYP/6-31G(d) level, considering the IEF-PCM(UFF) implicit solvent model for water. The coordination compounds were assessed using the Gibbs free energy variation and hard and soft acid theory. Molecular docking calculations were conducted using the GOLD program, based on the crystal structure of the acetylcholinesterase protein (PDB code = 4EY5), where the ChemScore function was employed with the active site defined as the region within a 15-Å radius around the centroid coordinates (X = -9.557583, Y = -43.910473, Z = 31.466687). Pharmacokinetic properties were predicted using SwissADME, focusing on Lipinski's rule of five.

Integrated metabolomic and transcriptomic analysis reveals the regulatory mechanisms of flavonoid and alkaloid biosynthesis in the new and old leaves of Murraya tetramera Huang.

BACKGROUND: Murraya tetramera Huang is a traditional Chinese woody medicine. Its leaves contain flavonoids, alkaloids, and other active compounds, which have anti-inflammatory and analgesic effects, as well as hypoglycemic and lipid-lowering effects, and anti-tumor effects. There are significant differences in the content of flavonoids and alkaloids in leaves during different growth cycles, but the synthesis mechanism is still unclear. RESULTS: In April 2021, new leaves (one month old) and old leaves (one and a half years old) of M. tetramera were used as experimental materials to systematically analyze the changes in differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) with transcriptomics and metabolomics technology. This was done to identify the signaling pathways of flavonoid and alkaloid synthesis. The results showed that the contents of total alkaloids and flavonoids in old leaves were significantly higher than those in new leaves. Thirteen flavonoid compounds, three isoflavone compounds, and nineteen alkaloid compounds were identified, and 125 and 48 DEGs related to flavonoid and alkaloid synthesis were found, respectively. By constructing the KEGG (Kyoto Encyclopedia of Genes and Genomes) network of DEGs and DAMs, it was shown that the molecular mechanism of flavonoid biosynthesis in M. tetramera mainly focuses on the "flavonoid biosynthetic pathway" and the "flavonoid and flavonol biosynthetic pathway". Among them, p-Coumaryl alcohol, Sinapyl alcohol, Phloretin, and Isoquercitrin were significantly accumulated in old leaves, the up-regulated expression of CCR (cinnamoyl-CoA reductase) might promote the accumulation of p-Coumaryl alcohol, upregulation of F5H (ferulate-5-hydroxylase) might promote Sinapyl alcohol accumulation. Alkaloids, including indole alkaloids, pyridine alkaloids, imidazole alkaloids, and quinoline alkaloids, were significantly accumulated in old leaves, and a total of 29 genes were associated with these substances. CONCLUSIONS: These data are helpful to better understand the biosynthesis of flavonoids and alkaloids in M. tetramera and provide a scientific basis for the development of medicinal components in M. tetramera.

Oxymatrine attenuates sepsis-induced inflammation and organ injury via inhibition of HMGB1/RAGE/NF-κB signaling pathway.

Sepsis is a life-threatening organ dysfunction that endangers patient lives and is caused by an imbalance in the host defense against infection. Sepsis continues to be a significant cause of morbidity and mortality in critically sick patients. Oxymatrine (OMT), a quinolizidine alkaloid derived from the traditional Chinese herb Sophora flavescens Aiton, has been shown to have anti-inflammatory effects on a number of inflammatory illnesses according to research. In this study, we aimed to evaluate the therapeutic effects of OMT on sepsis and explore the underlying mechanisms. We differentiated THP-1 cells into THP-1 macrophages and studied the anti-inflammatory mechanism of OMT in a lipopolysaccharide (LPS)-induced THP-1 macrophage sepsis model. Activation of the receptor for advanced glycation end products (RAGE), as well as NF-κB, was assessed by Western blot analysis and immunofluorescence staining. ELISA was used to measure the levels of inflammatory factors. We found that OMT significantly inhibited HMGB1-mediated RAGE/NF-κB activation and downstream inflammatory cytokine production in response to LPS stimulation. Finally, an in vivo experiment was performed on septic mice to further study the effect of OMT on injured organs. The animal experiments showed that OMT significantly inhibited HMGB1-mediated RAGE/NF-κB activation, protected against the inflammatory response and organ injury induced by CLP, and prolonged the survival rate of septic mice. Herein, we provide evidence that OMT exerts a significant therapeutic effect on sepsis by inhibiting the HMGB1/RAGE/NF-κB signaling pathway.

Soil conditioner improves soil properties, regulates microbial communities, and increases yield and quality of Uncaria rhynchophylla.

Uncaria rhynchophylla is an important traditional herbal medicine in China, and the yield and quality of Uncaria rhynchophylla can be improved by suitable soil conditioners because of changing the soil properties. In this paper, Uncaria rhynchophylla associated alkaloids and soil microbial  communities were investigated. The field experiment was set up with the following control group: (M1, no soil conditioner) and different soil conditioner treatment groups (M2, biomass ash; M3, water retention agent; M4, biochar; M5, lime powder and M6, malic acid). The results showed that M2 significantly increased the fresh and dry weight and the contents of isorhynchophylline, corynoxeine, isocorynoxeine, and total alkaloids. Acidobacteria, Proteobacteria, Actinobacteria, and Chloroflexi were major bacterial phyla. Correlation analysis showed that fresh and dry weight was significantly positively correlated with Acidobacteria, while alkali-hydrolyzable nitrogen, phosphatase activity, fresh and dry weight, corynoxeine, and isocorynoxeine were significantly negatively correlated with Chloroflexi. The application of soil conditioner M2 increased the abundance of Acidobacteria and decreased the abundance of Chloroflexi, which contributed to improving the soil nutrient content, yield, and quality of Uncaria rhynchophylla. In summary, biomass ash may be a better choice of soil conditioner in Uncaria rhynchophylla growing areas.

Effects of Combination of Curcumin and Piperine Supplementation on Glycemic Profile in Patients with Prediabetes and Type 2 Diabetes Mellitus: A Systematic Review and Meta-analysis.

Objective: This study aimed to evaluate the effects of the combination of curcumin and piperine supplementation on Fasting Plasma Glucose (FPG), Homeostatic Model of Insulin Resistance (HOMA-IR), and Body Mass Index (BMI) in patients with prediabetes and type 2 Diabetes Mellitus (T2DM). This review was done to identify potential herbal remedies that may help improve glycemic parameters, leading to better health outcomes in combination with current antidiabetic treatment. Methodology: This systematic review was based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). It was conducted in 2023 with sources and databases from MEDLINE, EBSCO-Host, ScienceDirect and ProQuest. This paper included randomized-controlled trials exploring the effects of the combination of curcumin and piperine on patients with prediabetes and T2DM. Systematic reviews, observational studies, case reports, case series, conference abstracts, book sections, commentaries/editorials, non-human studies and articles with unavailable full-text and written in non-English language, were excluded. The key terms for the literature search were "curcumin," "piperine," "prediabetes" and "Type 2 Diabetes Mellitus." We use Cochrane Risk of Bias (RoB) 2 for quality assessment of the included studies and Review Manager (RevMan) 5.4 to do the meta-analysis. Results: A total of three studies were included in this systematic review. Two studies from Neta et al., and Cicero et al., showed no significant difference in HOMA-IR, BMI and FPG levels between the curcumin, piperine and placebo groups. One study from Panahi et al. demonstrated a significant difference in BMI levels between the curcumin and piperine and placebo groups (p <0.01). The meta-analysis showed that FPG levels, HOMA-IR and BMI improved among patients with diabetes given in curcumin and piperine with reported mean differences (MD) of = -7.61, 95% CI [-15.26, 0.03], p = 0.05, MD = -0.36, 95% CI [-0.77 to 0.05], p = 0.09, and MD = -0.41, 95% CI [-0.85 to 0.03], p = 0.07, respectively). Conclusions: The supplementation of curcumin and piperine showed a numerical reduction in FPG, HOMA-IR and BMI, but were not statistically significant. Further research is needed as there is a paucity of studies included in the review.

Genome-wide association study revealed significant SNPs for anthracnose resistance, seed alkaloids and protein content in white lupin.

White lupin (Lupinus albus L.) is a high-protein grain legume alternative to soybean in Central Europe, but its cultivation is risky due to the fungal disease anthracnose that can cause severe yield damage. In addition, management of seed alkaloids is critical for human nutrition and animal feed. We report on a white lupin collection of genebank accessions, advanced breeding lines and cultivars that was genotyped and phenotypically characterized for anthracnose resistance and seed alkaloids and protein levels. Using genotyping by sequencing (GBS), SeqSNP-targeted GBS, BiomarkX genotyping and Sanger sequencing, a genetic resource of genome-wide SNPs for white lupin was established. We determined anthracnose resistance in two years field trials at four locations with infection rows and measured seed alkaloids and protein levels by near-infrared spectroscopy (NIRS). Few white lupin breeding lines showed anthracnose resistance comparable or better than Celina and Frieda, currently the best commercial cultivars in Germany. NIRS estimates for seed alkaloids and protein levels revealed variation in the white lupin collection. Using genome-wide association studies (GWAS), we identified SNPs significantly associated with anthracnose resistance in the field representing known and new genomic regions. We confirmed the pauper locus and detected new SNP markers significantly associated with seed alkaloids. For the first time, we present loci associated with total grain protein content. Finally, we tested the potential of genomic prediction (GP) in predicting the phenotype of these three quantitative traits. Application of results and resources are discussed in the context of fostering breeding programs for white lupin.

Product Selectivity in Baeyer-Villiger Monooxygenase-Catalyzed Bacterial Alkaloid Core Structure Maturation.

Baeyer-Villiger monooxygenases (BVMOs) play crucial roles in the core-structure modification of natural products. They catalyze lactone formation by selective oxygen insertion into a carbon-carbon bond adjacent to a carbonyl group (Baeyer-Villiger oxidation, BVO). The homologous bacterial BVMOs, BraC and PxaB, thereby process bicyclic dihydroindolizinone substrates originating from a bimodular nonribosomal peptide synthetase (BraB or PxaA). While both enzymes initially catalyze the formation of oxazepine-dione intermediates following the identical mechanism, the final natural product spectrum diverges. For the pathway involving BraC, the exclusive formation of lipocyclocarbamates, the brabantamides, was reported. The pathway utilizing PxaB solely produces pyrrolizidine alkaloids, the pyrrolizixenamides. Surprisingly, replacing pxaB within the pyrrolizixenamide biosynthetic pathway by braC does not change the product spectrum to brabantamides. Factors controlling this product selectivity have remained elusive. In this study, we set out to solve this puzzle by combining the total synthesis of crucial pathway intermediates and anticipated products with in-depth functional in vitro studies on both recombinant BVMOs. This work shows that the joint oxazepine-dione intermediate initially formed by both BVMOs leads to pyrrolizixenamides upon nonenzymatic hydrolysis, decarboxylative ring contraction, and dehydration. Brabantamide biosynthesis is enzyme-controlled, with BraC efficiently transforming all the accepted substrates into its cognate final product scaffold. PxaB, in contrast, shows only considerable activity toward brabantamide formation for the substrate analog with a natural brabantamide-type side chain structure, revealing substrate-controlled product selectivity.

Postmortem Diffusion of Aconitum Alkaloids and Their Metabolites in Rabbits.

OBJECTIVES: To explore the postmortem diffusion rule of Aconitum alkaloids and their metabolites in poisoned rabbits, and to provide a reference for identifying the antemortem poisoning or postmortem poisoning of Aconitum alkaloids. METHODS: Twenty-four rabbits were sacrificed by tracheal clamps. After 1 hour, the rabbits were administered with aconitine LD50 in decocting aconite root powder by intragastric administration. Then, they were placed supine and stored at 25 ℃. The biological samples from 3 randomly selected rabbits were collected including heart blood, peripheral blood, urine, heart, liver, spleen, lung and kidney tissues at 0 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h and 96 h after intragastric administration, respectively. Aconitum alkaloids and their metabolites in the biological samples were analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). RESULTS: At 4 h after intragastric administration, Aconitum alkaloids and their metabolites could be detected in heart blood, peripheral blood and major organs, and the contents of them changed dynamically with the preservation time. The contents of Aconitum alkaloids and their metabolites were higher in the spleen, liver and lung, especially in the spleen which was closer to the stomach. The average mass fraction of benzoylmesaconine metabolized in rabbit spleen was the highest at 48 h after intragastric administration. In contrast, the contents of Aconitum alkaloids and their metabolites in kidney were all lower. Aconitum alkaloids and their metabolites were not detected in urine. CONCLUSIONS: Aconitum alkaloids and their metabolites have postmortem diffusion in poisoned rabbits, diffusing from high-content organs (stomach) to other major organs and tissues as well as the heart blood. The main mechanism is the dispersion along the concentration gradient, while urine is not affected by postmortem diffusion, which can be used as the basis for the identification of antemortem and postmortem Aconitum alkaloids poisoning.

The efficacy and acceptability of pharmacological monotherapies and e-cigarette on smoking cessation: a systemic review and network meta-analysis.

Background and aims: Several pharmacological interventions, such as nicotine replacement therapy (NRT), varenicline, and bupropion, have been approved for clinical use of smoking cessation. E-cigarettes (EC) are increasingly explored by many RCTs for their potentiality in smoking cessation. In addition, some RCTs are attempting to explore new drugs for smoking cessation, such as cytisine. This network meta-analysis (NMA) aims to investigate how these drugs and e-cigarettes compare regarding their efficacy and acceptability. Materials and methods: This systematic review and NMA searched all clinical studies on smoking cessation using pharmacological monotherapies or e-cigarettes published from January 2011 to May 2022 using MEDLINE, COCHRANE Library, and PsychINFO databases. NRTs were divided into transdermal (TDN) and oronasal nicotine (ONN) by administrative routes, thus 7 network nodes were set up for direct and indirect comparison. Two different indicators measured the efficacy: prevalent and continuous smoking abstinence. The drop-out rates measured the acceptability. Results: The final 40 clinical studies included in this study comprised 77 study cohorts and 25,889 participants. Varenicline is more effective intervention to assist in smoking cessation during 16-32 weeks follow-up, and is very likely to prompt dropout. Cytisine shows more effectiveness in continuous smoking cessation but may also lead to dropout. E-cigarettes and oronasal nicotine are more effective than no treatment in encouraging prevalent abstinence, but least likely to prompt dropout. Finally, transdermal nicotine delivery is more effective than no treatment in continuous abstinence, with neither significant effect on prevalent abstinence nor dropout rate. Conclusion: This review suggested and agreed that Varenicline, Cytisine and transdermal nicotine delivery, as smoking cessation intervention, have advantages and disadvantages. However, we had to have reservations about e-cigarettes as a way to quit smoking in adolescents.

A simple and green offline-online capillary electrophoresis stacking strategy for the simultaneous determination of hydrophobic compounds in complicated samples using sodium dodecyl sulfate as the solubilizer and pseudophase.

BACKGROUND: Capillary electrophoresis is a powerful analytical method featured with high separation efficiency, minimal sample requirements, and reduced organic solvents consumption. However, its low sensitivity hinders its wide application in determination of trace analytes especially for the weakly ionized hydrophobic compounds. Offline and Online capillary electrophoresis stacking methods are more favored to enhance detection sensitivity of analytes. The determination of two sesquiterpenes and an alkaloid from the dried root of Lindera aggregata merged as an example for developing a simple, sensitive and green method for the simultaneous determination of two hydrophobic compounds in complicated matrix samples. RESULTS: An offline-online capillary electrophoresis stacking strategy by integrating micro matrix solid phase dispersion with field-amplified sample stacking and micelle to cyclodextrin stacking has been developed for the simultaneous determination of dehydrocostus lactone, linderane, norisoboldine in complex matrices. The optimized parameters were set at 65 mM sodium dihydrogen phosphate, 35 % methanol, 180 s for sample injection and 210 s for cyclodextrin injection, 20 mM sodium dodecyl sulfate of sample matrix for online stacking; 1:1 sample to MCM-48, 180 s grinding time, and 1000 µL of 20 mM sodium dodecyl sulfate elution for offline procedure. Under the optimum conditions, the method showed good linearity with correlation coefficients (R2 ≥ 0.9927), low limits of detection within the range of 25-50 ng mL-1, satisfactory repeatability and reproducibility below 3.98 %, and acceptable recoveries between 94 % and 97 %. The developed method was successfully applied to two real samples, the root of L. aggregata and rat feces. SIGNIFICANCE: Sodium dodecyl sulfate is firstly used as an eluent in micro matrix solid phase dispersion and plays a dual role throughout the analytical procedure, including extraction solvent in sample preparation and micelle pseudophase during online stacking. It brings great procedure convenience to the method. The sensitivity of this method can improve up to 1283-folds compared with the normal mode. Moreover, the overall strategy indicates satisfied green potential evaluated by greenness assessment tools.

Curcumin, Piperine and Taurine Combination Enhances the Efficacy of Transarterial Chemoembolization Therapy in patients with Intermediate Stage Hepatocellular Carcinoma: A Pilot Study.

INTRODUCTION: Diagnosis of the majority of hepatocellular carcinoma (HCC) patients occurs at intermediate to advanced stages, with a few curative therapeutic options being available. It is therefore strongly urgent to discover additional adjuvant therapy for this lethal malignancy. This study aimed to assess the effectiveness of curcumin (C), piperine (P) and taurine (T) combination as adjuvant agents on serum levels of IFN-γ, immunophenotypic and molecular characterization of mononuclear leukocytes (MNLs) in HCC patients treated with Transarterial chemoembolization (TACE). PATIENTS AND METHODS: Serum and MNLs were collected from 20 TACE-treated HCC patients before (baseline-control samples) and after treatment with 5 g curcumin capsules , 10 mg piperine and 0.5 mg taurine taken daily for three consecutive months. Immunophenotypic and molecular characterization of MNLs were determined by flow cytometry and quantitative real time PCR, respectively. In addition, serum IFN-γ level was quantified by ELISA. RESULTS: After receiving treatment with CPT combination, there was a highly significant increase in IFN- γ levels in the sera of patients when compared to basal line control samples. Additionally, the group receiving combined therapy demonstrated a downregulation in the expression levels of PD-1, in MNLs as compared to controls. MNLs' immunophenotyping revealed a significant decline in CD4+CD25+cells (regulatory T lymphocytes). Furthermore, clinicopathological characteristics revealed a highly significant impact of CPT combination on aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and alpha feto protein (AFP) levels. CONCLUSION: This study introduces a promising adjuvant CPT combined treatment as natural agents to enhance the management of HCC patients who are candidates to TACE treatment.