Concise Syntheses of bis-Strychnos Alkaloids (-)-Sungucine, (-)-Isosungucine, and (-)-Strychnogucine†B from (-)-Strychnine.

The first chemical syntheses of complex, bis-Strychnos alkaloids (-)-sungucine (1), (-)-isosungucine (2), and (-)-strychnogucine B (3) from (-)-strychnine (4) is reported. Key steps included (1) the Polonovski-Potier activation of strychnine N-oxide; (2) a biomimetic Mannich coupling to forge the signature C23-C5' bond that joins two monoterpene indole monomers; and (3) a sequential HBr/NaBH3 CN-mediated reduction to fashion the ethylidene moieties in 1-3. DFT calculations were employed to rationalize the regiochemical course of reactions involving strychnine congeners.

Sweeping of hydrophobic amines under inhomogeneous electric field and low surfactant concentration in micellar electrokinetic chromatography.

The influence of sample matrix on sample sweeping in MEKC was examined in the presented manuscript. Significant focusing effect was observed for relatively hydrophobic cationic compounds (emetine, strychnine and quinine) using high ionic strength sample matrix (900 mM H3 PO4 /720 mM Tris) which conductivity was about ninefold higher than utilized BGE. Moreover, the results were obtained using BGE composed of comparatively low surfactant concentration (10 mM SDS) and 40 mM H3 PO4 /32 mM Tris buffer solution. About 200 to 300-fold preconcentration of analytes was reached with the presented method. Basing on experimental results and computer simulation using Simul5 software, hypothetical mechanism of observed phenomenon was proposed.

A potent antibacterial indole alkaloid from Psychotria pilifera.

A new strychnine alkaloid, 16,17,19,20-tetrahydro-2,16-dehydro-18-deoxyisostrychnine (1), and fourteen known alkaloids were isolated from the leaves of Psychotria pilifera. Their structures were identified on the basis of extensive spectroscopic analysis, as well as by comparison with the reported spectroscopic data. The new alkaloid (1) exhibited potent antibacterial activity against Escherichia coli, equivalent to cefotaxime with MIC value of 0.781 µg/ml.

Preparation of molecularly imprinted polymers for strychnine by precipitation polymerization and multistep swelling and polymerization and their application for the selective extraction of strychnine from nux-vomica extract powder.

Monodisperse molecularly imprinted polymers for strychnine were prepared by precipitation polymerization and multistep swelling and polymerization, respectively. In precipitation polymerization, methacrylic acid and divinylbenzene were used as a functional monomer and crosslinker, respectively, while in multistep swelling and polymerization, methacrylic acid and ethylene glycol dimethacrylate were used as a functional monomer and crosslinker, respectively. The retention and molecular recognition properties of the molecularly imprinted polymers prepared by both methods for strychnine were evaluated using a mixture of sodium phosphate buffer and acetonitrile as a mobile phase by liquid chromatography. In addition to shape recognition, ionic and hydrophobic interactions could affect the retention of strychnine in low acetonitrile content. Furthermore, molecularly imprinted polymers prepared by both methods could selectively recognize strychnine among solutes tested. The retention factors and imprinting factors of strychnine on the molecularly imprinted polymer prepared by precipitation polymerization were 220 and 58, respectively, using 20 mM sodium phosphate buffer (pH 6.0)/acetonitrile (50:50, v/v) as a mobile phase, and those on the molecularly imprinted polymer prepared by multistep swelling and polymerization were 73 and 4.5. These results indicate that precipitation polymerization is suitable for the preparation of a molecularly imprinted polymer for strychnine. Furthermore, the molecularly imprinted polymer could be successfully applied for selective extraction of strychnine in nux-vomica extract powder.

Strychnobaillonine, an unsymmetrical bisindole alkaloid with an unprecedented skeleton from Strychnos icaja roots.

A reinvestigation of the roots of Strychnos icaja resulted in the isolation of a new bisindole alkaloid named strychnobaillonine (1) with original C-17-N-1' and C-23-C-17' junctions, in addition to sungucine, bisnordihydrotoxiferine, and strychnohexamine (2). Compound 1 showed potent activity against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum in vitro with an IC50 value of 1.1 µM. The structures of the compounds were defined by detailed spectroscopic analyses, especially 1H and 13C NMR, DEPT, HSQC, COSY, NOESY, HMBC, and HRESIMS. The proposed absolute configuration was based on biosynthetic considerations and spectroscopic data (CD, NMR) supported by molecular modeling.

On-line two-step stacking in capillary zone electrophoresis for the preconcentration of strychnine and brucine.

An on-line sample preconcentration method by two-step stacking i.e., sweeping and micelle to solvent stacking, in capillary zone electrophoresis (CZE) has been developed for the determination of strychnine and brucine in traditional Chinese herbal medicines. After experimental optimizations, the best separation was achieved by using 75 mM phosphate buffer (pH 2.5) with 30% methanol (v/v). Compared with normal CZE injection, 51- and 38-fold improvement in concentration sensitivity was achieved for strychnine and brucine, respectively. The calibration curve was linear in the range of 0.1-5.0 µg mL(-1) for both strychnine and brucine, with the correlation coefficients of 0.9998 and 0.9997, respectively. The limits of detection (S/N=3) for both alkaloids were 0.01 µg mL(-1). The inter-day (n=8) and intra-day (n=5) reproducibilities expressed as the relative standard deviations for corrected peak area were less than 9.5%. The method was applied to determine strychnine and brucine in two Chinese herbal medicines, with recoveries ranging from 94.2% to 105.4%. The results indicated that the method is simple, rapid, reliable, and can be applied to determine strychnos alkaloids in traditional Chinese herbal medicines.

Carbon nanotubes reinforced hollow fiber solid phase microextraction for the determination of strychnine and brucine in urine.

A mixed matrix membrane (MMM), based on carbon nanotubes (CNTs) and hollow fiber (HF), was prepared and combined with solid phase microextraction (SPME) mode to determine strychnine and brucine in urine. This MMM was prepared by dispersing CNTs in water via surfactant assistance, and then immobilizing CNTs into the pores of HF by capillary forces and sonification. The prepared carbon nanotubes reinforced hollow fiber (CNTs-HF) was subsequently wetted by a few microliters of organic solvent (1-octanol), and then applied to extract the target analytes in direct immersion sampling mode. After extraction, analytes were desorbed via ultrasonic-assisted effect, and then detected via high-performance liquid chromatography (HPLC). To achieve the highest extraction efficiency, main extraction parameters such as the type and amount of surfactant, the diameter and doping level of CNTs, extraction time, desorption condition, pH value, stirring rate and volume of the donor phase were optimized. Under the optimum extraction conditions, the method showed good linearity ranges with correlation coefficients higher than 0.9990, good repeatability and batch-to-batch reproducibility with relative standard deviations (RSDs) less than 6% and 5% for strychnine and brucine, respectively, and low limits of detection (0.7 and 0.9 µg L(-1) for strychnine and brucine, respectively). The recoveries were in the range of 83.81-116.14% at three spiked levels. The developed method was successfully applied to real urine sample with mean relative recoveries of 94.28% and 91.30% for strychnine and brucine, respectively. The developed method shows comparable results against reference methods and is a simple, green, and cost-effective microextraction technique.

Brucine, an indole alkaloid from Strychnos nux-vomica attenuates VEGF-induced angiogenesis via inhibiting VEGFR2 signaling pathway in vitro and in vivo.

In this study, we investigated the mechanism of brucine in tumor angiogenesis. We found that brucine inhibits VEGF-induced cell proliferation, chemotactic motility, and the formation of capillary-like structures in HUVECs in a dose-dependent manner. Brucine suppresses VEGF- induced p-VEGFR2 kinase activity and inhibits neovascularization in vivo. Brucine inhibits the downstream protein kinases of VEGFR2, including Src, FAK, ERK, AKT and mTOR. And further downregulates levels of VEGF, NO, IL-6, IL-8, TNF-α and IFN-γ in HUVECs. Taken together, our study suggests that brucine potently suppresses angiogenesis by targeting VEGFR2 activation and may be a viable drug candidate in anti-angiogenesis and anti-cancer therapies.

Strynuxlines A and B, alkaloids with an unprecedented carbon skeleton from Strychnos nux-vomica.

The investigation of the seeds of Strychnos nux-vomica led to the isolation of two novel indole alkaloids, strynuxlines A (1) and B (2), with an unprecedented 6/5/9/6/7/6 hexacyclic ring system. Their structures and absolute configurations were elucidated on the basis of their MS, NMR, and ECD data. A plausible biosynthesis pathway of 1 and 2 is also proposed.

Effects of brucine combined with glycyrrhetinic acid or liquiritin on rat hepatic cytochrome P450 activities in vivo.

Abstract: The activities of four CYP450 enzymes (CYP3A, 1A2, 2El and 2C) and the mRNA expression levels of CYP1A2, 2El, 2Cll and 3A1 in rat liver were determined after Wistar rats were orally administered with brucine (BR) at three dosage levels (3, 15 and 60 mg.kg-1 per day) and the high dose of BR combined with glycyrrhetinic acid (GA, 25 mg.kg-1 per day) or liquiritin (LQ, 20 mg.kg-1 per day) for 7 consecutive days. Compared with the control, brucine caused 24.5% and 34.6% decrease of CYP3A-associated testosterone 6beta-hydroxylation (6betaTesto-OH) and CYP2C-associated tolbutamide hydroxylation (Tol-OH), respectively, and 146.1% increase of CYP2El-associated para-nitrophenol hydroxylation (PNP-OH) at the high dose level. On the other hand, (BR+GA) caused 51.4% and 33.5% decrease, respectively, of CYP2El-associated PNP-OH and CYP1A2-associated ethoxyresorufin-O-de-ethylation (EROD) as compared with the high dose of BR group. Meanwhile, (BR+LQ) caused 41.1% decrease of CYP2El-associated PNP-OH and 37.7% increase of CYP2C-associated Tol-OH. The results indicated that the co-administration of BR with GA or LQ had effect on mRNA expression and activities of the CYP450 enzymes mentioned above to some extent, and the in vivo antagonism of LQ on BR-induced CYPs adverse effects and the in vivo inhibitory action of GA on CYP2E1 and 1A2 might play an important role in the detoxification of Radix Glycyrrhizae against Strychnos nux-vomica L.

[In vitro absorption mechanism of strychnine and the transport interaction with liquiritin in Caco-2 cell monolayer model].

To study the effect of liquiritin (Liq) on the transport of strychnine (Str) in Caco-2 cell monolayer model, the transport parameters of Str, such as apparent permeability coefficient (P app (B-->A) and P app (A-->B)) and cumulative transport amount (TRcum), were determined and comparatively analyzed when Str was used solely and co-used with Liq. The effect of drug concentrations, conveying times, P-glycoprotein (P-gp) inhibitor verapamil and conveying liquor pH values on the transport of Str were also investigated. The results indicated that the absorption of Str in Caco-2 cell monolayer model was well and the passive transference was the main intestinal absorption mechanism of Str in the Caco-2 monolayer model, along with the excretion action mediated by P-gp. Liq enhanced the absorption of Str. Meanwhile, conveying liquor pH value had significant influence on the excretion transport of Str.

Use of Strychnos nux-vomica (Azraqi) seeds in Unani system of medicine: role of detoxification.

Some plants used in Unani system of medicine are toxic, even deadly poisonous. The drugs having such plants as their components are detoxified before they are dispensed to the patients. One such drug, capsule Hudar, has Strychnos nux-vomica L. (Azraqi) seeds as one of its components and is very effectively used to elevate blood pressure. Ancient manuscripts describe many methods of its detoxification. It has been found that the detoxification processes studied reduce the strychnine content, as determined either by using uv-vis spectrophotometer or HPLC, present in Strychnos nux vomica seeds which is responsible for Strychnos nux vomica toxicity. The decrease in strychnine amount was best when the seeds were immersed for detoxification in excess of water for 5 days, in milk for 2 days followed by their boiling in milk. Strychnine in small amounts has been reported to give subjective feeling of stimulation.

A three-phase microfluidic chip for rapid sample clean-up of alkaloids from plant extracts.

A three-phase microchip was developed for the rapid and efficient small-scale purification of alkaloids from plant extracts. As part of the development of such a three-phase microchip, first a two-phase microchip with two channels (3.2 cm and 9.3 cm) was used to study the extraction efficiency of strychnine nitrate and strychnine at various flow rates. Strychnine was extracted from a basic aqueous phase to a chloroform phase (extraction) or strychnine was extracted from a chloroform phase into an acidic aqueous phase (back extraction). Subsequently, the "simultaneous extraction and back extraction" of strychnine was carried out in a three-phase microchip. The experimental extraction rate and yield were compared with model data. At a residence time of 25 sec, 79.5% of strychnine was extracted into the acidic aqueous phase using the three-phase microchip. In general, a good correlation was found between experimental results and model data for both two- and three-phase extractions. Finally, the three-phase microchip was employed in the purification of alkaloids (strychnine and brucine) from Strychnos seed extracts.

Simple and rapid extraction, separation, and detection of alkaloids in beverages.

Implementation of an uncomplicated SPE process for the rapid extraction and preconcentration of the alkaloids, colchicine, strychnine, aconitine, and nicotine, from water, apple juice, and nonfat milk samples is presented. When coupled to analysis via micellar EKC (MEKC), the total analysis time per sample was less than 15 min for the water and juice samples and less than 20 min for the milk. The SPE process allowed for anywhere from a three to a fourteen-fold improvement in the LOD for each alkaloid when compared to detecting the alkaloids in a nontreated water sample matrix. Following SPE, the LODs for colchicine, strychnine, and nicotine were sufficient to meet levels from 150 to 5000 times more dilute than the LD(50) for a 50 kg individual drinking 12 oz of a contaminated beverage. Aconitine, on the other hand, was detected at approximately the LD(50) level. The percent recoveries for the SPE ranged from 37% to as high as 99%. Nicotine attained the highest recovery efficiencies, followed by colchicine, and finally, aconitine and strychnine, which were nearly identical. The greatest recovery efficiencies were achieved from apple juice and water, whereas nonfat milk yielded the lowest.

Spectral editing of organic mixtures into pure components using NMR spectroscopy and ultraviscous solvents.

A general technique is described that permits the extraction of a complete 1H NMR spectrum for components in organosoluble mixtures. The approach should find a wide range of applications considering that pure component spectra can be generated without the need for physical separation. This technique is especially significant for synthetic organic chemistry and the pharmaceutical industry due to the potential to isolate a product spectrum even in the presence of overlapping starting materials, byproducts, or degradation products. A viscous oil-based solvent system that can be temperature-manipulated from essentially a solid at one extreme to a freely flowing liquid at the other is employed. The system contains no protons and is miscible with common organic solvents. Through careful control of the temperature and thus solvent viscosity, the behavior of small molecules moves from the positive to the extreme of the negative NOE regime. Under such conditions, all protons in a molecule correlate with all other protons as propagation by spin diffusion becomes highly efficient, behavior normally only observed with rigid macromolecules in conventional solvents. Therefore, as long as one proton (or carbon signal in hybrid experiments) is resolved for a component in a mixture, the entire proton spectrum for that molecule can be cleanly extracted from a 2D NOESY spectrum (or from selective 1D NOE-based analogues). Preliminary results are highly encouraging, indicating that the approach may be feasible for a wide range of molecules and mixtures; however, in practice the exact types of structures, combinations of structures, and range of concentrations that can be cleanly extracted will become evident as the technique becomes better established.

Separation and determination of strychnine and brucine in Strychnos nux-vomica L. and its preparation by nonaqueous capillary electrophoresis.

An easy, rapid method for simultaneous determination of strychnine and brucine in Strychnos nux-vomica L. and its preparation was developed by nonaqueous capillary electrophoresis (NACE) without pretreatment for the first time. Optimum separation was achieved with a fused-silica capillary column (50 cmx75 microm i.d.) and a running buffer containing 30 mM ammonium acetate, 1.0% acetic acid and 15% acetonitrile (ACN) in methanol medium. The applied voltage was 30.0 kV. The analytes were detected by UV at 214 nm. The effects of concentration of ammonium acetate, acetic acid and organic modifier on electrophoretic behavior of the analytes were studied. The established method with sophoridine as internal standard was linear in the range of 5-1000 mg/mL for both strychnine and brucine. The extracts of Strychnos nux-vomica and its preparation could be directly injected for determination with recoveries ranging from 94.5 to 104%.

The anti-tumor effects of alkaloids from the seeds of Strychnos nux-vomica on HepG2 cells and its possible mechanism.

To screen the anti-tumor effects of the four alkaloids: brucine, strychnine, brucine N-oxide and isostrychnine from the seed of Strychnos nux-vomica, MTT assay was used to examine the growth inhibitory effects of these alkaloids on human hepatoma cell line (HepG2). Brucine, strychnine and isostrychnine revealed significant inhibitory effects against HepG2 cell proliferation, whereas brucine N-oxide didn't have such an effect. In addition, brucine caused HepG2 cell shrinkage, membrane blebbing, apoptotic body formation, all of which are typical characteristics of apoptotic programmed cell death. The results of flow cytometric analysis demonstrated that brucine caused dose-dependent apoptosis of HepG2 cells through cell cycle arrest at G0/G1 phase, thus preventing cells entering S or G2/M phase. Immunoblot results revealed that brucine significantly decreased the protein expression level of cyclooxygenase-2, whereas increased the expression caspase-3 as well as the caspase-3-like protease activity in HepG2 cells, suggesting the involvement of cyclooxygenase-2 and caspase-3 in the pro-apoptotic effects exerted by brucine. Therefore, this paper indicate that the major alkaloids present in the seed of Strychnos nux-vomica are effective against HepG2 cells proliferation, among which brucine proceed HepG2 cells death via apoptosis, probably through the participation of caspase-3 and cyclooxygenase-2.

The apoptotic effect of brucine from the seed of Strychnos nux-vomica on human hepatoma cells is mediated via Bcl-2 and Ca2+ involved mitochondrial pathway.

In an attempt to dissect the mechanism of Strychnos nux-vomica, 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-(4,5-dimethylthiazol-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 brucine-treated 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 [Ca2+], 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 [Ca2+]i caused by brucine was also suppressed by overexpression of Bcl-2 protein in HepG2 cells. From the facts given above, Ca2+ and Bcl-2 mediated mitochondrial pathway were found to be involved in brucine-induced HepG2 cell apoptosis.