Brucine N-oxide Research Articles

A Combination of In Silico ADMET Prediction, In Vivo Toxicity Evaluation, and Potential Mechanism Exploration of Brucine and Brucine N-oxide-A Comparative Study.

Brucine (BRU) and brucine N-oxide (BNO) are prominent, bioactive, and toxic alkaloids in crude and processed Semen Strychni. Studies have demonstrated that BRU and BNO possess comprehensive pharmacological activities, such as anti-inflammatory and analgesic. In this context, a comparative study of BRU and BNO was performed by combination analysis of in silico ADMET prediction, in vivo toxicity evaluation, and potential action mechanism exploration. ADMET prediction showed that BRU and BNO might induce liver injury, and BRU may have a stronger hepatoxic effect. The prediction was experimentally verified using the zebrafish model. The BRU-induced hepatotoxicity of zebrafish larvae had a dose-response relationship. The mechanism of BRU-induced hepatotoxicity might relate to phosphorylation, kinase activity, and signal transduction. By comparison, signal transduction and gap junctions might involve BNO-induced hepatotoxicity. Our results provided a better understanding of BRU- and BNO-induced hepatotoxicity. We also built a foundation to elucidate the material base of the hepatotoxicity of traditional Chinese medicine Semen Strychni.

Molecular docking-based virtual screening of antidiabetic agents from Songga (Strychnos lucida R.Br.): an Indonesian native plant

This study was carried out to predict the compounds derived from Songga that have potential as antidiabetic and predict their mechanism of action on various pathways of glucose regulation in diabetes mellitus by molecular docking. Molecular docking-based virtual screening was done by using AutoDock Vina software assisted by AutoDockTools. The test compounds used for virtual screening were obtained from literature studies and were combined with Lipinski’s rule to select the compounds for the prediction of lead candidates that can be used in oral administration. The receptors used in this study were human aldose reductase, human maltase-glucoamylase, PPAR-gamma, pancreatic beta-cell SUR1, and human DPP-IV. The validation of the molecular docking method of five target receptors showed that RMSD values of human aldose reductase, human maltase-glucoamylase, PPAR-gamma, pancreatic beta-cell SUR1, and human DPP-IV were 0.6446 Å, 1.8668 Å, 0.2527 Å, 1.7452 Å, and 1.7439 Å, respectively. From the molecular docking-based virtual screening, we discovered that for each target protein, there were one to three optimal compounds that have the best interaction in our investigation. Those compounds were chlorogenic acid on human aldose reductase, phyllamycin A, chlorogenic acid, and brucine N-oxide on human maltase-glucoamylase, phyllamycin A on PPAR-gamma, strychnine N-oxide on pancreatic beta-cell SUR1 and strychnine on human DPP-IV with binding affinity value of -9.9 kcal/mol, -7.6 kcal/mol, -9.9 kcal/mol, -8.8 kcal/mol, and -6.2 kcal/mol, respectively. Several compounds are predicted to have potential to be developed as antidiabetic agents. However, further laboratory investigations like in vitro and in vivo assays need to be conducted.

Brucine Diol-Catalyzed Enantioselective Morita-Baylis-Hillman Reaction in the Presence of Brucine N-Oxide

Brucine diol (BD) catalyzed asymmetric Morita–Baylis–Hillman (MBH) reaction is observed for the first time. Brucine N-oxide (BNO) was found to not have an effective chiral catalyst. Faster reaction rate was obtained using unsaturated ester or aromatic aldehydes in the presence of BNO. 4-Nitrobenzaldehyde and α,β-unsaturated ketone/ester were converted to the MBH adduct in moderate yields (up to 74%) with 70% ee value by this catalytic system. The mechanism of BD catalysis is probably initiated by conjugating the vicinal diol of BD to the carbonyl group of the aromatic aldehyde through hydrogen bonding. The tertiary amine of BD acts as a nucleophile to activate vinyl ketone for coupling with the carbonyl of aldehyde through an intramolecular carbonylated reaction. Finally, the breakdown of the complex caused the formation of the MBH adduct (a benzyl-allyl alcohol). The chirality of the benzyl-allyl alcohol is likely affected by the interaction of the bulky asymmetric plane of BD.

Brucine N-Oxide Reduces Ethanol Intake and Preference in Alcohol-Preferring Male Fawn-Hooded Rats.

Alcohol use disorder places a heavy burden on global public health systems and thus is in urgent need of improved pharmacotherapies. Previously, our group has demonstrated that 30mg/kg of the indole alkaloid brucine significantly attenuates alcohol-drinking behavior; however, the high toxicity, poor water solubility, short half-life, and limited therapeutic window of brucine restrain its clinical application as an antialcoholism medication. We subsequently hypothesized that the oxide of brucine (brucine N-oxide) would produce a similar behavioral effect without the risk profile associated with brucine. Male Fawn-Hooded rats with high innate alcohol preference underwent 2-bottle choice procedures (Experiments 1 to 3). Experiment 1 examined the effects of 7 daily BNO injections of 0, 30, 50, or 70mg/kg (s.c.) on voluntary alcohol consumption (n=9/group). Experiment 2 evaluated the impact of a single dose of 0 or 70mg/kg BNO on the increased alcohol intake induced by a 4-day alcohol deprivation (n=8/group). Experiment 3 tested the effect of 7 daily BNO injections of 0 or 70mg/kg (s.c.) on sucrose preference (n=6/group). Experiment 4 measured the median lethal dose (LD50) values of BNO and brucine to compare their acute toxicity in rats. Experiment 5 tested whether BNO (0, 30, 50, and 70mg/kg, s.c.) affected locomotor activity using an open-field paradigm (n=8/group). Finally, Experiment 6 evaluated the possible conditioned rewarding effects of 0, 30, 50, and 70mg/kg BNO using the conditioned place preference paradigm (n=6/group). BNO administration dose-dependently attenuated alcohol consumption without affecting food intake, total fluid consumption, or the natural preference for a sucrose solution, with 70mg/kg BNO reducing consumption by 22.8%. A single dose of 70mg/kg BNO significantly inhibited the alcohol deprivation effect. The LD50 values of BNO and brucine in rats were determined to be 1,103.5±177.0mg/kg and 264.6±17.7mg/kg, respectively. Finally, BNO administration did not affect spontaneous locomotor activity or induce a place preference. BNO may help to control excessive alcohol use and should be considered a treatment strategy for future study and development.

Removal of hERG potassium channel affinity through introduction of an oxygen atom: Molecular insights from structure-activity relationships of strychnine and its analogs

Nux vomica has been effectively used in Traditional Chinese Medicine. The processing of Nux vomica is necessary to reduce toxicity before it can be used in clinical practice. However, the mechanism for processing detoxification is unclear. hERG channels have been subjected to a routine test for compound cardiac toxicity in the drug development process. Therefore, we examined the effects and mechanisms of strychnine and brucine, two main ingredients of Nux vomica, and their N-oxides on hERG channels. Strychnine and brucine exhibited concentration-dependent inhibition of hERG channels with IC50 values of 25.9 μM and 44.18 μM, respectively. However, their nitrogen oxidative derivatives produced by processing of Nux vomica, strychnine N-oxide and brucine N-oxide, lost their activity on hERG channels. Compared to their parent compounds, only an oxygen atom was introduced in the nitrogen oxidative isoforms to compensate for the N+ − charge, suggesting that the protonated nitrogen is the key group for strychnine and brucine binding to hERG channel. Alanine-mutagenesis identified Y652 is the most important residue for strychnine and brucine binding to hERG channel. Y652A mutation increased the IC50 for strychnine and brucine by 21.64-fold and 29.78-fold that of WT IhERG, respectively. Docking simulations suggested that the protonated nitrogen of strychnine and brucine formed a cation–π interaction with the aromatic ring of Y652. This study suggests that introduction of an oxygen to compensate for the N+ − charge could be a useful strategy for reducing hERG potency and increasing the safety margin of alkaloid-type compounds in drug development.

Determination of strychnine, brucine, strychnine N-oxide, and brucine N-oxide in plasma samples after the oral administration of processed semen strychni extract by high-performance liquid chromatography with ultrasound-assisted mixed cloud point extraction.

A sensitive and efficient mixed cloud point extraction combined with high-performance liquid chromatography was developed for the simultaneous separation and determination of four alkaloids (strychnine, strychnine N-oxide, brucine, and brucine N-oxide) in plasma after the oral administration of processed semen strychni extract. Tergitol TMN-6 and cetyl-trimethyl ammonium bromide were chosen as the mixed surfactants, and ultrasound was employed to enhance the extraction efficiency. Some important parameters affecting the mixed cloud point extraction efficiency, such as the content of Tergitol TMN-6 and cetyl-trimethyl ammonium bromide, pH, salt effect, extraction temperature, and ultrasound time were studied and optimized. Under optimum conditions, the linear range of four alkaloids was from 1.0 to 1000 ng/mL. All correlation coefficients of the calibration curves were higher than 0.9993. The intraday and interday precision were below 8.65% and the limits of detection for the four alkaloids were less than 1.0 ng/mL (S/N = 3).

Ultra-performance liquid chromatography-tandem mass spectrometric assay for the simultaneous determination of brucine, strychnine and brucine N-oxide in rat plasma: application to a pharmacokinetic study.

A rapid, simple and sensitive UHPLC-MS/MS method was developed and validated for the simultaneous determination of brucine, strychnine and brucine N-oxide in rat plasma using huperzine A as an internal standard (IS) after protein precipitation with methanol. The analytes were separated on a Purospher® STAR RP18 UHPLC column (2 µm, 2.1 × 100 mm) by gradient elution using a mobile phase composed of methanol and water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. Brucine, strychnine, brucine N-oxide and IS were detected in positive ion multiple reaction monitoring mode by means of an electrospray ionization interface (m/z 395.2 → 324.1, m/z 335.2 → 184.1, m/z 411.2 → 394.2, m/z 243.1 → 226.1). The calibration curve was linear over the range of 1-500 ng/mL for brucine and strychnine and 0.2-50 ng/mL for brucine N-oxide. The intra- and inter-day precisions of these analytes were all within 15% and the accuracy ranged from 85 to 115%. The stability experiment indicated that the plasma samples at three concentration levels were stable under different conditions. The developed method was successfully applied for the first time to pharmacokinetic studies of brucine, strychnine and brucine N-oxide following a single oral and intravenous administration of modified total alkaloid fraction in rats. Copyright © 2016 John Wiley & Sons, Ltd.

LC-MS/MS determination and comparative pharmacokinetics of strychnine, brucine and their metabolites in rat plasma after intragastric administration of each monomer and the total alkaloids from Semen Strychni.

A rapid, specific and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous determination of strychnine, brucine, strychnine N-oxide and brucine N-oxide in rat plasma. Plasma samples were pretreated via simple protein precipitation with methanol and ephedrine hydrochloride was used as internal standard. Chromatographic separation was carried out on an ZORBAX Eclipse XDB-C18 column (2.1×150mm, 3.5μm) by gradient elution with methanol and 10mM ammonium acetate (adjusted to pH 4.0 with formic acid). The quantification of the analytes was performed by mass spectrometry with TurboIonSpray ionization (ESI) inlet in the positive ion multiple reaction monitoring (MRM) mode. The results showed that the calibration curve was linear in the concentration range of 0.510∼306.3ngmL(-1) for strychnine, brucine and 0.102∼306.0ngmL(-1) for strychnine N-oxide and brucine N-oxide, respectively. The intra- and inter-day precisions were less than 14.9%, and the accuracy ranged from 89.4 to 113% at three QC levels for the 4 analytes. The validated method was successfully applied to the pharmacokinetic study of strychnine, brucine, strychnine N-oxide and brucine N-oxide in rat plasma after oral administration of each monomer and the total alkaloids from Semen Strychni. After single oral administration of the total alkaloids from Semen Strychni at 4 dose levels, Cmax, AUC0-t of strychnine and brucine increased and were proportional to the oral doses. In comparative pharmacokinetics studies, no significant difference was found between each monomer and the total strychnos alkaloids on the pharmacokinetic parameters such as Cmax and AUC. Mean Cmax and AUC of strychnine and brucine were slight increased in the monomer groups in comparison to the total strychnos alkaloids groups, which suggested that some other alkaloids in the Semen Strychni may decrease the absorption of strychnine and brucine in body.

Functional Activation of G-Proteins Coupled With Muscarinic Acetylcholine Receptors in Rat Brain Membranes

The functional activation of Gi/o proteins coupled to muscarinic acetylcholine receptors (mAChRs) was investigated with the conventional guanosine-5'-O-(3-[(35)S]thio) triphosphate ([(35)S]GTPγS) binding assay in rat brain membranes. The most efficacious stimulation elicited by acetylcholine or carbachol (CCh) was obtained in striatal membranes. The pharmacological properties of mAChR-mediated [(35)S]GTPγS binding determined with a series of muscarinic agonists and antagonists were almost identical among the three brain regions investigated, i.e., cerebral cortex, hippocampus, and striatum, except for the apparent partial agonist effects of (αR)-α-cyclopentyl-α-hydroxy-N-[1-(4-methyl-3-pentenyl)-4-piperidinyl]benzeneacetamide fumarate (J 104129) observed only in the hippocampus, but not in the other two regions. Among the muscarinic toxins investigated, only MT3 attenuated CCh-stimulated [(35)S] GTPγS binding. The highly selective allosteric potentiator at the M4 mAChR subtype, 3-amino-N-[(4-chlorophenyl)methyl]-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide (VU 10010), shifted the concentration-response curve for CCh leftwards as well as upwards. On the other hand, neither thiochrome nor brucine N-oxide was effective. The increases induced by CCh and 5-HT were essentially additive, though not completely, indicating that the mAChRs and 5-HT1A receptors were coupled independently to distinct pools of Gi/o proteins. Collectively, all of the data suggest that functional activation of Gi/o proteins coupled to mAChRs, especially the M4 subtype, is detectable by means of CCh-stimulated [(35)S]GTPγS binding assay in rat discrete brain regions.

HPLC–DAD method for comprehensive quality control of Semen Strychni

Context: Semen Strychni is the seed of Strychnos nux-vomica L. (Loganiaceae). Its quality control procedure remains an issue since previous reports only focused on Strychnos alkaloids. To the best of our knowledge, chlorogenic acid (a phenolic acid) and loganin (an iridoid glycoside) are selected for the first time as marker constituents of quality control for Semen Strychni because of their bioactive activity correlating with therapeutic effects.Objective: This study aimed to develop a simple and comprehensive quantity control method for Semen Strychni.Materials and methods: The optimal ultrasonic extraction procedure was carried out for 45 min using 50% aqueous methanol with 1% formic acid. The satisfactory chromatographic separation was achieved on an Ultimate LP-C18 column with gradient elution using acetonitrile and water containing 30 mmol/L ammonium acetate and 1% formic acid. The high performance liquid chromatography method with diode array detector was validated for linearity, limit of detection and quantification (LOQ), precision, repeatability, accuracy and stability.Results: All the calibration curves showed good linearity (r2 ≥ 0.999). The LOQ values for chlorogenic acid, loganin, strychnine, brucine, strychnine N-oxide and brucine N-oxide were 0.54, 0.83, 0.48, 0.50, 0.52 and 0.54 μg/mL, respectively. The method was reproducible with good accuracy in the range 95.6–104.4% and relative standard deviation (RSD) values less than 4.55%. The method was then applied to determine the components of the seed coat, seed leaf, endosperm and whole seed of Semen Strychni.Conclusion: This newly established method is validated as a simple and practical tool for authentication and quality control of Semen Strychni.

Virtual screening of Indonesian herbal database as HIV-1 reverse transcriptase inhibitor.

HIV-1 (Human immunodeficiency virus type 1) is a member of retrovirus family that could infect human and causing AIDS disease. AIDS epidemic is one of most destructive diseases in modern era. There were more than 33 million people infected by HIV until 2010. Various studies have been widely employed to design drugs that target the essential enzymes of HIV-1 that is, reverse transcriptase, protease and integrase. In this study, in silico virtual screening approach is used to find lead molecules from the library or database of natural compounds as HIV-1 reverse transcriptase inhibitor. Virtual screening against Indonesian Herbal Database using AutoDock4 performed on HIV-1 reverse transcriptase. From the virtual screening, top ten compounds were mulberrin, plucheoside A, vitexilactone, brucine N-oxide, cyanidin 3-arabinoside, alpha-mangostin, guaijaverin, erycristagallin, morusin and sanggenol N.

Simultaneous Analysis of Strychnine and Brucine and their Major Metabolites by Liquid Chromatography-Electrospray Ion Trap Mass Spectrometry

A liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-ITMS) method was developed for the simultaneous analysis of strychnine, brucine and their major metabolites. Strychnine and brucine were individually incubated with rat liver S9 fraction. The incubation samples were pooled together and analyzed with LC-ESI-ITMS in positive ion and full-scan detection mode. The calibration curves of strychnine and brucine in rat liver showed good linearity in ranges of 0.020 to 8.0 µg/mL for strychnine and 0.020 to 8.5 µg/mL for brucine. The limits of detections were both 0.008 µg/mL and the recoveries were 88.3 and 83.2% for strychnine and brucine, respectively. Two metabolites were identified as strychnine N-oxide and brucine N-oxide by comparing the molecular mass, retention time, full-scan mass spectra, tandem MS and MS(3) spectra with those of strychnine and brucine. The developed method provided high sensitivity and selectivity for the determination of poisonous alkaloids and their major metabolites and can be applied in the determination of samples in forensic and clinically toxicological cases.

The Apoptotic Effect of Brucine from the Seed of Strychnos nux-vomica on Human Hepatoma Cells is Mediated via Bcl-2 and Ca 2+ Involved Mitochondrial Pathway

In an attempt to dissect the mechanism of Strychnos nuxvomica, a commonly used Chinese folk medicine in the therapy of liver cancer, the cytotoxic effects of four alkaloids in Strychnos nux-vomica, brucine, brucine N-oxide, strychnine, and isostrychnine, on human hepatoma cells (HepG2) were screened by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrasolium bromide (MTT) assay. Brucine, among the four alkaloids, exhibited the strongest toxic effect, the mechanism of which was found to cause HepG2 cell apoptosis, since brucine caused HepG2 cell shrinkage, the formation of apoptotic bodies, DNA fragmentation, cell cycle arrest, as well as phosphatidylserine externalization, all of which are typical characteristics of apoptotic programmed cell death. Brucine-induced HepG2 cell apoptosis was caspase dependent, with caspase-3 activated by caspase-9. Brucine also caused the proteolytic processing of caspase-9. In addition, brucine caused depolarization of the mitochondrial membrane of HepG2 cells, the inhibition of which by cyclosporine A completely abrogated the activation of casapses and release of cytochrome c in brucinetreated HepG2 cells. These findings suggested a pivotal role of mitochondrial membrane depolarization in HepG2 cell apoptosis elicited by brucine. Furthermore, brucine induced a rapid and sustained elevation of intracellular [Ca 2+ ], which compromised the mitochondrial membrane potential and triggered the process of HepG2 cell apoptosis. Finally, Bcl-2 was found to predominately control the whole event of cell apoptosis induced by brucine. The elevation of [Ca 2+ ]i caused by brucine was also suppressed by overexpression of Bcl-2 protein in HepG2 cells. From the facts given above, Ca 2+ and Bcl-2 mediated mitochondrial pathway

A Computational Study on the Role of Chiral N‐Oxides in Enantioselective Pauson–Khand Reactions

Density functional calculations were carried out to ascertain the origin of enantioselectivity in the brucine N-oxide (BNO)-assisted enantioselective Pauson-Khand reaction (PKR) of norbornene with 2-methyl-3-butyn-2-ol. The computed ee value in acetone is 68 % (R), which compares well to the previously reported experimental value of 58 % (R). In DME the computed ee value of 76 % (R) is in excellent agreement with the experimentally determined value of 78 % (R). The mechanism of enantioselectivity consists of several steps. First, the dicobalt complex is activated by BNO with chirality transfer from enantiopure BNO to the dicobalt complex. Second, competition occurs between a racemization process and complexation with the olefin reagent, which leads to the products. The lower ee value in acetone is due to the lower energy barrier of the racemization process. Calculations show that replacement of BNO by a hypothetical more enantioselective chiral N-oxide will hardly increase the ee value beyond 90 %.

A Cooperative Catalysis Approach to the Morita-Baylis-Hillman Reaction of Aryl Vinyl Ketones

The Morita-Baylis-Hillman (MBH) reaction of aryl vinyl ketones has been achieved for the first time through cooperative catalysis of proline and brucine N-oxide. A unique mode of activation of the aldehydes through proline iminium intermediates has been identified as a key reaction parameter for the successful MBH reaction of aryl vinyl ketones. The reaction pathways leading to 1:2 MBH products under typical nucleophilic catalysis conditions were investigated. A systematic study revealed two different reaction pathways leading to 1:2 MBH products: (1) the second reaction of normal MBH products with aryl vinyl ketones as a dominating reaction pathway at the initial stage of the reaction, and (2) the aldol reaction pathway of Rauhut-Currier products with aldehydes (auto-catalysis by alcohol products) as a major reaction pathway during the later stage of reaction.

Simultaneous Determination of Strychnine, Brucine, Strychnine N-Oxide and Brucine N-Oxide in Crude and Fermented Nux Vomica by CZE with Ephedrine Hydrochloride as an Internal Standard

A rapid capillary zone electrophoresis (CZE) method with ephedrine hydrochloride as an internal standard was developed and validated for separating and quantifying the alkaloids strychnine (1), brucine (2), strychnine N-oxide (3), and brucine N-oxide (4) in the seed of Strychnos nux-vomica L. (crude nux vomica) and its fermented products by fungus Trametes cinnabarina (Jacq.: Fr.) Fr. (fermented nux vomica). The contents of alkaloids (1 and 2) in crude nux vomica were higher than those in fermented drugs, whereas alkaloids (3 and 4) were only detected in fermented ones. The method developed is sensitive, specific and accurate, as well as effective and easy, which can be used to assess the qualities of crude and fermented nux vomica.

ChemInform Abstract: The Brucine N-Oxide Promoted Asymmetric Pauson-Khand Reaction.

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Brucine N-oxide-catalyzed Morita–Baylis–Hillman reaction of vinyl ketones: a mechanistic implication of dual catalyst system with proline

The brucine N-oxide promoted Morita-Baylis-Hillman (MBH) reaction of vinyl ketones with aldehydes has been achieved. The corresponding asymmetric version of MBH reaction was also investigated, and the electron-deficient aryl aldehydes have emerged as suitable reaction partners for vinyl ketones; where proline was employed as a co-catalyst. In this dual catalyst system, proline is believed to form iminium intermediates with electron-deficient aryl aldehydes, while the N-oxide activates vinyl ketones to provide enolates through conjugate addition. Upon the combination of these two intermediates, the MBH products with high enantioselectivities are obtained by controlling of the rate-determining step through H-bridged chair-like transition state. Intrinsically, the resulting MBH products, alcohols, are found to interfere with the formation of both intermediates, enolates and proline iminium intermediates, thus the observed enantioselectivity of products attenuates upon further reaction conversion, possibly due to autocatalysis. This current study sheds lights on the synthetic utility of iminium species, derived from electron-deficient aryl aldehydes and proline.

Intestinal Permeability of Antitumor Alkaloids from the Processed Seeds ofStrychnos nux-vomicain a Caco-2 Cell Model

The uptake and intestinal permeability of the seven alkaloids strychnine (Str), brucine (Bru), beta-colubrine (Col), strychnine N-oxide (S-N), brucine N-oxide (B-N), pseudostrychnine (Psd), and icajine (Ica), which were isolated from the processed seeds of Strychnos nux-vomica L., were investigated in the human intestinal Caco-2 model. Determination of compounds was carried out by HPLC. The apparent permeability coefficients ( P(app)) for Str, Bru, Col, S-N, B-N, Psd, and Ica in the apical-to-basolateral direction were (3.11 +/- 0.17) x 10(-5), (1.67 +/- 0.65) x 10(-5), (2.67 +/- 0.30) x 10(-5), (0.17 +/- 0.01) x 10(-5), (0.35 +/- 0.02) x 10(-5), (2.51 +/- 0.33) x 10(-5), and (2.61 +/- 0.34) x 10(-5) cm/s, respectively. In the concentration range of 10-200 microM, Str, Bru, Col, and Psd showed substantial concentration-dependent transport across the monolayers. The transports of all seven alkaloids were linear with time and showed moderate to high permeabilities. In the presence of 2,4-dinitrophenol or sodium azide, the P(app) of Ica was reduced significantly in both the apical-to-basolateral and basolateral-to-apical directions. The dominant mechanism of the intestinal absorption for Str, Bru, Col, S-N, B-N, and Psd was passive diffusion, while it was partially ATP dependent for Ica.

The cytotoxicity induced by brucine from the seed of Strychnos nux-vomica proceeds via apoptosis and is mediated by cyclooxygenase 2 and caspase 3 in SMMC 7221 cells

To study the cytotoxicity of four alkaloids: brucine, strychnine, brucine N-oxide and isostrychnine from nux vomica on SMMC 7721 cells and their possible mechanisms, MET assay was used to examine the growth inhibitory effects of these alkaloids. Brucine revealed the strongest growth inhibitory effect on SMMC-7721 cells. Furthermore, as directly observed under an inverted microscope, fluorescent microscope and transmission electronic microscope, brucine caused SMMC-7721 cell shrinkage, membrane blobbing, formation of apoptotic body as well as nucleus condensation, all of which are typical characteristics of apoptotic programmed cell death. In addition, brucine dose-dependently caused SMMC-7721 cells apoptosis via formation of subdipolid DNA and phosphatidylserine externalization, as evidenced by flow cytometry analysis. The brucine-induced apoptosis was partially attributed to the activation of caspase 3 as well as cyclooxygenase 2 inhibition, since neither caspase 3 specific inhibitor, z-DEVD-fmk nor was exogenous addition of prostaglandin E 2 able to completely abrogate the brucine-induced SMMC 7721 cell apoptosis. In sum, this paper indicate that the major alkaloids present in the seed of Strychnos nux-vomica are effective against SMMC-7721 cells proliferation, among which brucine proceeds SMMC-7721 cells death via apoptosis, probably through the participation of caspase 3 and cyclooxygenase 2.