A novel nano-drug delivery system of glycyrrhetinic acid-mediated intracellular breakable brucine for enhanced anti-hepatitis B efficacy.

Background: Glycyrrhetinic acid-mediated brucine self-assembled nanomicelles enhance the anti-hepatitis B properties of brucine by improving its water solubility, short half-life, toxicity, and side effects. Brucine (B) is an indole alkaloid extracted from the seeds of Strychnos nux-vomica (Loganiaceae). Purpose: To assess the efficacy of the Brucine-Glycyrrhetnic acid-Polyethylene glycol-3,3'-dithiodipropionic acid-Glycerin monostearate (B-GPSG) in treating hepatitis B, its potential to protect against acute liver injury caused by d-galactosamine and its anti-hepatoma activities were studied. Research Design: The concentration of B-GPSG used in the in vivo and in vitro experiments was 0.63 mg/mL. The rats injected with d-GalN (450 mg/kg) were used as liver injury models. The rats were separated into normal, model, positive, positive control, B-PSG and B-GPSG groups. Hepatoma cells expressing HBV HepG2.2.15 were used for in vitro experiments. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, plate cloning, Hoechst staining and flow cytometry were conducted to explore the mechanism of B-GPSG against hepatitis B. Results: Compared with the model group, the liver coefficient of B-GPSG group decreased (4.59 ± 0.17 vs 5.88 ± 0.42), the content of MDA in rat liver homogenate decreased (12.54 ± 1.81 vs 23.05 ± 2.98), the activity of SOD increased, the activity of ALT and AST in rat serum decreased. In vitro, the IC50 values of B-GPSG group decreased. B-GPSG group effectively inhibited the proliferation and migration of HepG2.2.15 cells. Conclusions: The hepatoprotective effects of B-GPSG nanomicelles, which are attributed to their GA-mediated liver targeting and synergistic actions with brucine, suggest their therapeutic potential against hepatitis B. This development opens up new possibilities for the application of traditional Chinese medicine and nanomedicine in anti-hepatitis B.

Oral acute toxicity study and in vivo antimalarial activity of Strychnos lucida R. Br. tablet.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria eradication has been a major goal of the Indonesian government since 2020. Medicinal plants, such as Strychnos lucida R. Br., are empirically used to treat malaria through traditional preparation methods. However, the safety and efficacy of these plants have not yet been confirmed. Therefore, further investigations are necessary to confirm the safety and efficacy of S. lucida as an antimalarial agent. AIMS OF THE STUDY: To quantify the concentration of brucine in the S. lucida extract, determine the acute oral toxicity of the standardized extract, and evaluate the in vivo antimalarial potency of S. lucida tablet (SLT). MATERIALS AND METHODS: Acute oral toxicity of S.lucida extract was determined using the Organization for Economic Co-operation and Development 420 procedure, and the analytical method for brucine quantification was validated using high-performance liquid chromatography. In addition, antimalarial activity was determined using the Peter's four-day suppressive method. RESULTS: Acute toxicity analysis revealed S. lucida as a low-toxicity compound with a cut-off median lethal dose of 2000-5000 mg/kg body weight [BW], which was supported by the hematological and biochemical profiles of the kidneys, liver, and pancreas (p > 0.05). Extract standardization revealed that S. lucida contained 3.91 ± 0.074% w/w brucine, adhering to the limit specified in the Indonesian Herbal Pharmacopeia. Antimalarial test revealed that SLT inhibited the growth of Plasmodium berghei by 27.74-45.27%. Moreover, SLT improved the hemoglobin and hematocrit levels. White blood cell and lymphocyte counts were lower in the SLT-treated group than in the K (+) group (p < 0.05). CONCLUSION: Histopathological and biochemical evaluations revealed that S. lucida extract was safe at a dose of 2000 mg/kg BW with low toxicity. SLT inhibited Plasmodium growth and improved the hemoglobin, hematocrit, and red blood cell profiles. Additionally, SLT reduced the lymphocyte and WBC counts and increased the monocyte and thrombocyte counts as part of the immune system response against Plasmodium infection.

Brucine alleviates fibroblast-like synoviocytes dysfunction and inflammation by regulating YY1 during rheumatoid arthritis.

Brucine is a weak alkaline indole alkaloid with wide pharmacological activities and has been identified to protect against rheumatoid arthritis (RA) process. Circular RNAs (circRNAs) are also reported to be involved in the pathogenesis of RA. Here, we aimed to probe the role and mechanism of Brucine and circ_0139658 in RA progression. The fibroblast-like synoviocytes of RA (RA-FLSs) were isolated for functional analysis. Cell proliferation, apoptosis, invasion, migration, as well as inflammatory response were evaluated by CCK-8 assay, EdU assay, flow cytometry, transwell assay, and ELISA analysis, respectively. qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. The binding between miR-653-5p and circ_0139658 or Yin Yang 1 (YY1), was verified using dual-luciferase reporter and RNA pull-down assays. Brucine suppressed the proliferation, migration, and invasion of RA-FLSs, and alleviated inflammation by reducing the release of pro-inflammatory factors and macrophage M1 polarization. RA-FLSs showed increased circ_0139658 and YY1 levels and decreased miR-653-5p levels. Circ_0139658 is directly bound to miR-653-5p to regulate YY1 expression. Brucine treatment suppressed circ_0139658 and YY1 expression but increased YY1 expression in RA-FLSs. Functionally, circ_0139658 overexpression reversed the suppressing effects of Brucine on RA-FLS dysfunction and inflammation. Moreover, circ_0139658 silencing alleviated the dysfunction and inflammation in RA-FLSs, which were reverted by YY1 overexpression. Brucine suppressed the proliferation, migration, invasion, and inflammation in RA-FLSs by decreasing YY1 via circ_0139658/miR-653-5p axis.

Synthesis and Characterization of Graphene Oxide/Polyethylene Glycol/Folic Acid/Brucine Nanocomposites and Their Anticancer Activity on HepG2 Cells.

Background: Liver cancer is the sixth most prevalent form of cancer and the second major cause of cancer-associated mortalities worldwide. Cancer nanotechnology has the ability to fundamentally alter cancer treatment, diagnosis, and detection. Objective: In this study, we explained the development of graphene oxide/polyethylene glycol/folic acid/brucine nanocomposites (GO/PEG/Bru-FA NCs) and evaluated their antimicrobial and anticancer effect on the liver cancer HepG2 cells. Methodology: The GO/PEG/Bru-FA NCs were prepared using the co-precipitation technique and characterized using various techniques. The cytotoxicity of the GO/PEG/Bru-FA NCs was tested against both liver cancer HepG2 and non-malignant Vero cells using an MTT assay. The antimicrobial activity of the GO/PEG/Bru-FA NCs was tested against several pathogens using the well diffusion technique. The effects of GO/PEG/Bru-FA NCs on endogenous ROS accumulation, apoptosis, and MMP levels were examined using corresponding fluorescent staining assays, respectively. The apoptotic protein expressions, such as Bax, Bcl-2, and caspases, were studied using the corresponding kits. Results: The findings of various characterization assays revealed the development of GO/PEG/Bru-FA NCs with face-centered spherical morphology and an agglomerated appearance with an average size of 197.40 nm. The GO/PEG/Bru-FA NCs treatment remarkably inhibited the growth of the tested pathogens. The findings of the MTT assay evidenced that the GO/PEG/Bru-FA NCs effectively reduced the HepG2 cell growth while not showing toxicity to the Vero cells. The findings of the fluorescent assay proved that the GO/PEG/Bru-FA NCs increased ROS generation, reduced MMP levels, and promoted apoptosis in the HepG2 cells. The levels of Bax, caspase-9, and -3 were increased, and Bcl-2 was reduced in the GO/PEG/Bru-FA NCs-treated HepG2 cells. Conclusion: The results of this work demonstrate that GO/PEG/Bru-FA NCs suppress viability and induce apoptosis in HepG2 cells, indicating their potential as an anticancer candidate.

Brucine Inhibits Proliferation of Pancreatic Ductal Adenocarcinoma through PI3K/AKT Pathway-induced Mitochondrial Apoptosis.

INTRODUCTION: The purpose of this research was to settle the role of brucine in pancreatic ductal adenocarcinoma (PDAC) and the mechanisms involved. METHODS: The findings of this study suggest that brucine exerts inhibitory effects on cell growth, clonogenicity, and invasive potential of Panc02 and Mia Paca-2 cells. These effects may be linked to an increase in apoptotic-prone cell population. RESULTS: Gene sequencing data suggests that these effects are mediated through the induction of apoptosis. Experimental evidence further supports the notion that brucine reduces mitochondrial membrane potential and upregulates Bax expression while downregulating Bcl-2 expression. These effects are believed to be a result of brucine-mediated suppression of PI3K/Akt activity, which serves as a regulatory factor of mTOR, Bax, and Bcl-2. Suppression of PI3K activity enhances the tumor-suppressing effects of brucine. CONCLUSION: Overall, these findings suggest that brucine has therapeutic potential as a remedy option for PDAC.

Brucine D restrains colorectal cancer tumorigenesis and autophagy by downregulating circ_0068464.

Bruceine D (BD) from Brucea javanica (L) exerts an antitumor effect in several human cancers. At present, it has not been reported whether BD inhibits the malignancy of colorectal cancer (CRC) cells. Therefore, investigating the role and regulatory mechanisms of BD in CRC is the main thrust of this study. Effect of BD on CRC cell viability, proliferation, apoptosis, invasion, and autophagy was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, 5-ethynyl-2'-deoxyuridine, flow cytometry, transwell invasion, and western blotting assays. Expression changes of has_circ_0068464 (circ_0068464) were detected using real time quantitative polymerase chain reaction. The molecular mechanisms related to circ_0068464 were predicted through online prediction websites Starbase 2.0, circinteractome, and CircBank and validated using dual-luciferase reporter and RNA pull-down assays. The tumorigenic ability of BD and circ_0068464 on CRC was confirmed by xenograft experiments. The results showed that BD lessened CRC cell proliferation, invasion, autophagy, and prompted cell apoptosis. Circ_0068464 was overexpressed in CRC samples and cells. BD led to a significant reduction in circ_0068464 levels in cells of this carcinoma, but circ_0068464 overexpression partially rescued these effects urged by BD. Also, the combination of BD and circ_0068464 silencing decreased xenograft tumor growth compared to BD alone. Importantly, circ_0068464 could regulate ATG5 expression by functioning as a miR-520h molecular sponge. In conclusion, BD might suppress CRC growth by inhibiting the circ_0068464/miR-520h/ATG5 axis, providing a new perspective for the molecular pathogenesis of CRC and preliminarily indicating that BD may be a promising drug for CRC treatment.

Biosynthesis of strychnine.

Strychnine is a natural product that, through isolation, structural elucidation and synthetic efforts, shaped the field of organic chemistry. Currently, strychnine is used as a pesticide to control rodents1 because of its potent neurotoxicity2,3. The polycyclic architecture of strychnine has inspired chemists to develop new synthetic transformations and strategies to access this molecular scaffold4, yet it is still unknown how plants create this complex structure. Here we report the biosynthetic pathway of strychnine, along with the related molecules brucine and diaboline. Moreover, we successfully recapitulate strychnine, brucine and diaboline biosynthesis in Nicotiana benthamiana from an upstream intermediate, thus demonstrating that this complex, pharmacologically active class of compounds can now be harnessed through metabolic engineering approaches.

The Production and Evaluation of an Electrochemical Sensors for Strychnine and Its Main Metabolite Strychnine N-Oxide for Their Use in Biological Samples.

Strychnine (STN) and its major metabolite Strychnine N-Oxide (SNO) were examined electrochemically. Both parent compounds and its major metabolite showed electroactivity on glassy carbon electrodes using CV and DPV techniques. One oxidation peak at 1008 mV was observed for STN with the optimum peak intensity at pH 7. SNO produced two oxidation peaks, at 617 mV and 797 mV, at pH 5. The peaks demonstrated irreversible behaviour and the irreversibility of the system was confirmed at different scan rates. A calibration curve was produced for both CV and DPV measurements and the sensitivity of the proposed EC method was good compared with previous electrochemical and non-electrochemical methods. The precision of oxidation peak of STN using the STN-MIP method produced a maximum value of 11.5% and 2.32% for inter-day and intraday %RSD, respectively. The average% recovery was around 92%. The electrochemical method has been successfully applied to the determination of STN in spiked plasma and urine samples. For SNO, both anodic peaks of SNO demonstrated irreversible behaviour. A different sweep rate was used for calculating the number of 'transfer electrons' in the system; based on this, the mechanism of oxidation reaction was proposed. Calibration curves for both oxidative peaks were produced using DPV measurements. The second anodic peak demonstrated high linearity and precision with %RSD < 1.96%.

Brucine-Loaded Ethosomal Gel: Design, Optimization, and Anti-inflammatory Activity.

Brucine, one of the natural medications obtained from Nux vomica seeds, is used as an anti-inflammatory drug. Several investigations were performed to overcome its drawbacks, which will affect significantly its pharmaceutical formulation. The goal of the current investigation was to design, optimize, and evaluate the anti-inflammatory performance of BRU ethosomal gel. Brucineethosomal formulations were prepared using thin film hydration method and optimized by central composite design approach using three independent variables (lecithin concentration, cholesterol concentration, and ethanol percentage) and three response variables (vesicular size, encapsulation efficiency, and skin permeation). The optimized formulation was examined for its stability and then incorporated into HPMC gel to get BRU ethosomal gel. The obtained BRU-loaded ethosomal gel was evaluated for its physical properties, in vitro release, and ex vivo permeation and skin irritation. Finally, carrageenan-induced rat hind paw edema test was adopted for the anti-inflammatory activity. The developed BRU ethosomal gel exhibited good physical characteristics comparable with the conventional developed BRU gel. In vitro release of BRU from ethosomal gel was effectively extended for 6 h. Permeation of BRU from ethosomes was significantly higher than all formulations (p < 0.05), since it recorded steady state transdermal flux value 0.548 ± 0.03 µg/cm2 h with enhancement ratio 2.73 ± 0.23. Eventually, BRU ethosomal gel exhibited potent anti-inflammatory activity as manifested by a significant decrease in rat hind paw inflammation following 24 h. In conclusion, the study emphasized the prospective of ethosomal gel as a fortunate carrier for intensifying the anti-inflammatory effect of Brucine.

The phosphorylation status of eukaryotic elongation factor-2 indicates neural activity in the brain.

Assessment of neural activity in the specific brain area is critical for understanding the circuit mechanisms underlying altered brain function and behaviors. A number of immediate early genes (IEGs) that are rapidly transcribed in neuronal cells in response to synaptic activity have been used as markers for neuronal activity. However, protein detection of IEGs requires translation, and the amount of newly synthesized gene product is usually insufficient to detect using western blotting, limiting their utility in western blot analysis of brain tissues for comparison of basal activity between control and genetically modified animals. Here, we show that the phosphorylation status of eukaryotic elongation factor-2 (eEF2) rapidly changes in response to synaptic and neural activities. Intraperitoneal injections of the GABA A receptor (GABAAR) antagonist picrotoxin and the glycine receptor antagonist brucine rapidly dephosphorylated eEF2. Conversely, potentiation of GABAARs or inhibition of AMPA receptors (AMPARs) induced rapid phosphorylation of eEF2 in both the hippocampus and forebrain of mice. Chemogenetic suppression of hippocampal principal neuron activity promoted eEF2 phosphorylation. Novel context exploration and acute restraint stress rapidly modified the phosphorylation status of hippocampal eEF2. Furthermore, the hippocampal eEF2 phosphorylation levels under basal conditions were reduced in mice exhibiting epilepsy and abnormally enhanced excitability in CA3 pyramidal neurons. Collectively, the results indicated that eEF2 phosphorylation status is sensitive to neural activity and the ratio of phosphorylated eEF2 to total eEF2 could be a molecular signature for estimating neural activity in a specific brain area.

Quality by Design for Development, Optimization and Characterization of Brucine Ethosomal Gel for Skin Cancer Delivery.

Natural products have been extensively used for treating a wide variety of disorders. In recent times, Brucine (BRU) as one of the natural medications extracted from seeds of nux vomica, was investigated for its anticancer activity. As far as we know, this is the first study on BRU anticancer activity against skin cancer. Thus, the rational of this work was implemented to develop, optimize and characterize the anticancer activity of BRU loaded ethosomal gel. Basically, thin film hydration method was used to formulate BRU ethosomal preparations, by means of Central composite design (CCD), which were operated to construct (32) factorial design. Two independent variables were designated (phospholipid percentage and ethanol percentage) with three responses (vesicular size, encapsulation efficiency and flux). Based on the desirability function, one formula was selected and incorporated into HPMC gel base to develop BRU loaded ethosomal gel. The fabricated gel was assessed for all physical characterization. In-vitro release investigation, ex-vivo permeation and MTT calorimetric assay were performed. BRU loaded ethosomal gel exhibited acceptable values for the characterization parameters which stand proper for topical application. In-vitro release investigation was efficiently prolonged for 6 h. The flux from BRU loaded ethosome was enhanced screening optimum SSTF value. Finally, in-vitro cytotoxicity study proved that BRU loaded ethosomal gel significantly improved the anticancer activity of the drug against A375 human melanoma cell lines. Substantially, the investigation proposed a strong motivation for further study of the lately developed BRU loaded ethosomal gel as a prospective therapeutic strategy for melanoma treatment.

ATF3 contributes to brucine-triggered glioma cell ferroptosis via promotion of hydrogen peroxide and iron.

Ferroptotic cell death is characterized by iron-dependent lipid peroxidation that is initiated by ferrous iron and H2O2 via Fenton reaction, in which the role of activating transcription factor 3 (ATF3) remains elusive. Brucine is a weak alkaline indole alkaloid extracted from the seeds of Strychnos nux-vomica, which has shown potent antitumor activity against various tumors, including glioma. In this study, we showed that brucine inhibited glioma cell growth in vitro and in vivo, which was paralleled by nuclear translocation of ATF3, lipid peroxidation, and increases of iron and H2O2. Furthermore, brucine-induced lipid peroxidation was inhibited or exacerbated when intracellular iron was chelated by deferoxamine (500 µM) or improved by ferric ammonium citrate (500 µM). Suppression of lipid peroxidation with lipophilic antioxidants ferrostatin-1 (50 µM) or liproxstatin-1 (30 µM) rescued brucine-induced glioma cell death. Moreover, knockdown of ATF3 prevented brucine-induced accumulation of iron and H2O2 and glioma cell death. We revealed that brucine induced ATF3 upregulation and translocation into nuclei via activation of ER stress. ATF3 promoted brucine-induced H2O2 accumulation via upregulating NOX4 and SOD1 to generate H2O2 on one hand, and downregulating catalase and xCT to prevent H2O2 degradation on the other hand. H2O2 then contributed to brucine-triggered iron increase and transferrin receptor upregulation, as well as lipid peroxidation. This was further verified by treating glioma cells with exogenous H2O2 alone. Moreover, H2O2 reversely exacerbated brucine-induced ER stress. Taken together, ATF3 contributes to brucine-induced glioma cell ferroptosis via increasing H2O2 and iron.

Brucine promotes apoptosis in cervical cancer cells (ME-180) via suppression of inflammation and cell proliferation by regulating PI3K/AKT/mTOR signaling pathway.

Brucine are the main constituents of Strychnos nux-vomica. Earlier reports have determined brucine shows anti-inflammatory, analgesic and excellent anti-tumor drug. Even though its anticervical cancer cells remains not clearly evaluated. So that, we hypothesized the anti-cervical cancer activity of brucine against the cervical (ME-180) cells. Brucine inhibited the inflammation, cell proliferation and promoted rate of apoptotic cell death ad reduced the mitochondrial potential, which is evidenced by respective (AO/EB, Rh-123, and PI) staining. Furthermore ELISA and real time PCR reaction determined that brucine were down regulated inflammatory (TNF-α, NF-kB, IL-6 & COX-2) cell proliferation (Cyclin D1) and apoptotic marker Bax, caspase-3, PI3K (phosphoinosital 3 kinase), AKT, mTOR (mammalian target of rapamycin) and over expression Bcl-2, associated death promoter. These findings were confirmed and finally suggested that brucine inhibited inflammation, cell proliferation and promoted the apoptosis through the down-regulation of PI3K/AKT/mTOR pathway. Taken together, these data were exhibited brucine as a good therapeutic agents for the prevention of anticancer cervical cancer drugs.

Preparation, characterization and evaluation of anti-inflammatory and anti-nociceptive effects of brucine-loaded nanoemulgel.

Brucine (BRU) is a natural product derived from nux-vomica seeds. It is commonly used as an anti-inflammatory and anti-nociceptive drug to relieve arthritis and traumatic pain. Nevertheless, its use is significantly limited by its low aqueous solubility, as well as the gastrointestinal problems and systemic toxicity that may occur following oral administration. The goal of this study, therefore, was to formulate and evaluate a nanoemulgel formulation of BRU for enhanced topical anti-inflammatory and anti-nociceptive activities. Different formulations were developed (BRU gel, emulgel and nanoemulgel) using 1% w/w NaCMC as a gelling agent. The formulated preparations were assessed for their physical appearance, spreadability, viscosity, particle size, in vitro drug release and ex vivo permeation studies. In addition, the carrageenan-induced rat hind paw edema method was adopted to scrutinize the anti-inflammatory activity, while the hot plate method and acetic acid-induced writhing test were used to assess the anti-nociceptive activity of different formulations in male BALB/c mice. The formulated BRU-loaded preparations showed good physical characteristics. Cumulative drug release from BRU-loaded nanoemulgel was remarkably higher than that of the other formulations. Ex vivo drug permeation of the nanoemulgel formulation across rat skin showed enhanced drug permeation and higher transdermal flux as compared to BRU-loaded gel or emulgel. Most importantly, the carrageenan-induced rat hind paw edema model verified the efficient anti-inflammatory potential of BRU-loaded nanoemulgel. In addition, BRU-loaded nanoemulgel exhibited significant protective effects against thermal stimulation in the hot plate test and remarkably inhibited acetic acid-induced abdominal writhing in mice. Furthermore, a skin irritation test indicated that BRU-loaded nanoemulgel elicited neither edema nor erythema upon application to rat skin. Collectively, our results suggest that myrrh oil-based nanoemulgel might represent a promising delivery vehicle for potentiating the anti-inflammatory and anti-nociceptive actions of brucine.

Chronotoxicity of Semen Strychni is associated with circadian metabolism and transport in mice.

OBJECTIVES: We aimed to determine the circadian responses of mice to Semen Strychni and to investigate the role of pharmacokinetics in generating chronotoxicity. METHODS: Total extract of Semen Strychni was administered by oral gavage to wild-type (WT) and Bmal1-/- (a circadian clock-deficient model) mice at different circadian time points for toxicity (including survival) and pharmacokinetic characterization. Nephrotoxicity and neurotoxicity were evaluated by measuring plasma creatinine and creatine kinase BB (CK-BB), respectively. Drug metabolism and transport assays were performed using liver/intestine microsomes and everted gut sacs, respectively. KEY FINDINGS: Semen Strychni nephrotoxicity and neurotoxicity as well as animal survival displayed significant circadian rhythms (the highest level of toxicity was observed at ZT18 and the lowest level at ZT2 to ZT6). According to pharmacokinetic experiments, herb dosing at ZT18 generated higher plasma concentrations (and systemic exposure) of strychnine and brucine (two toxic constituents) compared with ZT6 dosing. This was accompanied by reduced formation of both dihydroxystrychnine and strychnine glucuronide (two strychnine metabolites) at ZT18. Bmal1 ablation sensitized mice to Semen Strychni-induced toxicity (with increased levels of plasma creatinine and CK-BB) and abolished the time dependency of toxicity. Metabolism of Semen Strychni (strychnine and brucine) in the liver and intestine microsomes of WT mice was more extensive at ZT6 than at ZT18. These time differences in hepatic and intestinal metabolism were lost in Bmal1-/- mice. Additionally, the intestinal efflux transport of Semen Strychni (strychnine and brucine) was more extensive at ZT6 than ZT18 in WT mice. However, the time-varying transport difference was abolished in Bmal1-/- mice. CONCLUSIONS: Circadian responses of mice to Semen Strychni are associated with time-varying efflux transport and metabolism regulated by the circadian clock (Bmal1). Our findings may have implications for optimizing phytotherapy with Semen Strychni via timed delivery.

Alleviation of isoprenaline hydrochloride induced myocardial ischemia injury by brucine through the inhibition of Na+/K+-ATPase.

Myocardial infarction (MI) is the most extensive manifestations of cardiovascular disease (CVD), associated with prolonged supply and demand blood oxygen imbalance to the heart muscle. The treatment of MI includes several conventional medicines which are beta-blockers and calcium antagonists. Though, these were reported to be either not efficient or associated with life threatening adverse effects. Brucine, the main alkaloid bioactive compound from Strychnos nux-vomica seeds, offers unique compatibility advantages in inflammatory diseases associated clinical practices. Thus, the present investigation was projected to explore the activity of brucine towards MI provoked by isoprenaline hydrochloride (ISO) in rats. The cardioprotective properties of brucine were evaluated via detecting the infarct size, serum cardiac marker enzymes (CK, CK-MB, cTnT, and cTnI), endogenous antioxidants (CAT, SOD, GPx), and lipid peroxidation (TBARS and LOOH), inflammatory mediators (NF-κB, TNF-α and IL-6) and histopathological analysis. The results demonstrated, brucine effectively restored the infarct size by increasing the endogenous antioxidants and decreasing the status of TBARS and LOOH, marker enzymes and ameliorated the histopathological injuries. Brucine's cardioprotective effect might be associated with TNF-α, IL-6 signaling molecules activation, revealing its pharmacological actions.

C-2-Linked Dimeric Strychnine Analogues as Bivalent Ligands Targeting Glycine Receptors.

Strychnine is the prototypic antagonist of glycine receptors, a family of pentameric ligand-gated ion channels. Recent high-resolution structures of homomeric glycine receptors have confirmed the presence of five orthosteric binding sites located in the extracellular subunit interfaces of the receptor complex that are targeted by strychnine. Here, we report the synthesis and extensive pharmacological evaluation of bivalent ligands composed of two strychnine pharmacophores connected by appropriate spacers optimized toward simultaneous binding to two adjacent orthosteric sites of homomeric α1 glycine receptors. In all bivalent ligands, the two strychnine units were linked through C-2 by amide spacers of various lengths ranging from 6 to 69 atoms. Characterization of the compounds in two functional assays and in a radioligand binding assay indicated that compound 11a, with a spacer consisting of 57 atoms, may be capable of bridging the homomeric α1 GlyRs by simultaneous occupation of two adjacent strychnine-binding sites. The findings are supported by docking experiments to the crystal structure of the homomeric glycine receptor. Based on its unique binding mode, its relatively high binding affinity and antagonist potency, and its slow binding kinetics, the bivalent strychnine analogue 11a could be a valuable tool to study the functional properties of glycine receptors.

Autophagy Impairment through Lysosome Dysfunction by Brucine Induces Immunogenic Cell Death (ICD).

Autophagy is an important tightly controlled cellular process that regulates cellular homeostasis and is involved in deciding cell fate such as cell survival and death. The role of autophagy in many intracellular signaling pathways explains its interaction with other different types of cell death, including apoptosis and immunogenic cell death (ICD). The reports showed the complex and intriguing relationship existing between autophagy and immune system signaling pathways. However, the role of autophagy in ICD remains to be clearly elucidated. In this study, we demonstrated that Brucine, a clinically-used small molecule in traditional Chinese medicine, elicited autophagy inhibition. Brucine also triggered cell stress and induced features of ICD, including calreticulin (CRT) exposure and high-mobility group box 1 (HMGB1) release in MDA-MB-231 and CT26 cancer cells. Brucine impaired autolysosomal degradation and exerted a feedback regulation of ERK1/2-mTOR-p70S6K signaling cascade. Brucine-elicited ICD was confirmed by the rejection of CT26 tumor cells, implanted in the mice after vaccination with Brucine-treated CT26 cells. The impaired autophagy contributed to Brucine-induced ICD, as knock-down of Atg5 significantly reduced Brucine-elicited CRT exposure and HMGB1 release. Our results revealed Brucine as a novel autophagy regulator, ICD inducer and hitherto undocumented role of autophagy in ICD. Thus, these results imply the importance of Brucine in cancer immunotherapy. Therefore, Brucine may be used as an ICD inducer and improve its application in cancer treatment with minimized toxicity.

Effect of Baizhu (Rhizoma Atractylodis Macrocephalae) extract on intestinal absorption of brucine and strychnine in vitro and in situ.

OBJECTIVE: To investigate the antagonistic effect of the extract of Baizhu (Rhizoma Atractylodis Macrocephalae) (RAM) on the intestinal absorption of brucine and strychnine in Strychnos nux-vomica (NUX) and propose the mechanism of these effects. METHODS: The apparent permeability value (Papp) and absorption rate constant (Ka) were chosen as indices. The everted intestinal sac model and in situ single-pass intestinal perfusion model were used to study the effects of the RAM extract on the absorption of brucine and strychnine. To confirm the results, the brucine and strychnine concentrations in hepatic portal venous blood were determined. Western blotting was used to study P-glycoprotein (P-gp) expression in the Caco-2 cell line. RESULTS: Papp and Ka of brucine and strychnine were significantly increased in the presence of a P-gp inhibitor, but no significant increase was noted in the presence of a tight junction regulator. The RAM extract inhibited the absorption of brucine and strychnine and enhanced P-gp expression. CONCLUSION: The primary absorption mechanism for brucine and strychnine is passive transport, which is affected by P-gp.

Development, optimization, and evaluation of PEGylated brucine-loaded PLGA nanoparticles.

The application of nanotechnology to drug delivery systems for cancer therapy has progressively received great attention. The most heavily investigated approach is the development of nanoparticles (NPs) utilizing biodegradable and biocompatible polymers such as poly (lactic-co-glycolic acid) (PLGA). These NPs could be further improved by surface modification utilizing a hydrophilic biodegradable polymer such as polyethylene glycol (PEG) to achieve passive targeting. Modified NPs can deliver drugs such as brucine (BRU), which has shown its potential in cancer therapy. The objective of the current investigation was to develop and evaluate the passive targeting of long-circulating PLGA NPs loaded with BRU. NPs were characterized in terms of drug-excipient compatibility studies, including FTIR and DSC; physicochemical evaluations including particle size, zeta potential, morphological evaluation, entrapment efficiency and percentage yield; total serum protein adsorbed onto NP surfaces; and in vitro release of the loaded drug. Factorial design was employed to attain optimal PLGA-loaded NPs. Finally, the in vivo anti-tumor activity of BRU-loaded PLGA NPs was evaluated in tumor-bearing mice. The NPs obtained had smooth surfaces with particle sizes ranged from 94 ± 3.05 to 253 ± 8.7 nm with slightly positive surface charge ranged from 1.09 ± 0.15 to 3.71 ± 0.44 mV. Entrapment of BRU ranged between 37.5 ± 1.8% and 77 ± 1.3% with yields not less than 70.8%. Total protein adsorbed was less than 25.5 µg total protein/1 mg NP. In vitro drug release was less than 99.1% at 168 h. Finally, significant reductions in tumor growth rate and mortality rate were observed for PEG PLGA NP formulations compared to both BRU solution and naked NPs.