Qualitative analysis of licorice and strychnine decoction before and after combination using UPLC-QE-Orbitrap-MS.

BACKGROUND AND OBJECTIVE: Glycyrrhiza glabra L. (GG) and Strychnos nux-vomica L. (NV) are traditional Chinese medicines (TCMs). Changes in the chemical composition may occur before and after the GG-NV compatibility. Ultra-performance liquid chromatography Q-exactive Orbitrap mass spectrometry (UPLC-QE-Orbitrap-MS) was applied here to study the difference in the components of the GG and NV decoctions before and after they were combined. The changes in the chemical composition of GG and NV before and after the combination were determined. METHODS: The precise molecular weight, retention time, and fragment ion peak of the different components of the decoctions before and after compatibility were obtained through UPLC-QE-Orbitrap-MS. Differential analysis methods, such as principal component analysis, were used for comparison. RESULTS: In the positive ion mode, 200 new components were added, whereas six components were lost. In the negative ion mode, 144 new compounds were identified, whereas three components were missing. CONCLUSIONS: The compatibility difference between GG and NV was studied through UPLC-QE-Orbitrap-MS. The chemical composition of GG and NV changed before and after compatibility, and a class of compounds different from GG and NV was identified in the co-decoction. This study provides an experimental basis for subsequent research into detoxification mechanisms of the GG-NV combination and offers a new analytical method for investigating the compatibility of various other TCM pairs.

Cross-Linked Poly-4-Acrylomorpholine: A Flexible and Reversibly Compressible Aligning Gel for Anisotropic NMR Analysis of Peptides and Small Molecules in Water.

Determination of the solution conformation of both small organic molecules and peptides in water remains a substantial hurdle in using NMR solution conformations to guide drug design due to the lack of easy to use alignment media. Herein we report the design of a flexible compressible chemically cross-linked poly-4-acrylomorpholine gel that can be used for the alignment of both small molecules and cyclic peptides in water. To test the new gel, residual dipolar couplings (RDCs) and J-coupling constants were used in the configurational analysis of strychnine hydrochloride, a molecule that has been studied extensively in organic solvents as well as a small cyclic peptide that is known to form an α-helix in water. The conformational ensembles for each molecule with the best fit to the data are reported. Identification of minor conformers in water that cannot easily be determined by conventional NOE measurements will facilitate the use of RDC experiments in structure-based drug design.

Determination of the conformational states of strychnine in solution using NMR residual dipolar couplings in a tensor-free approach.

Small molecules with rotatable bonds can occupy different conformational states in solution as a consequence of their thermal fluctuations. The accurate determination of the structures of such states, as well as of their statistical weights, has been challenging because of the technical difficulties in extracting information from experimental measurements, which are normally averaged over the conformational space available. Here, to achieve this objective, we present an approach based on a recently proposed tensor-free method for incorporating NMR residual dipolar couplings as structural restraints in replica-averaged molecular dynamics simulations. This approach enables the information provided by the experimental data to be used in the spirit of the maximum entropy principle to determine the structural ensembles of small molecules. Furthermore, in order to enhance the sampling of the conformational space we incorporated the metadynamics method in the simulations. We illustrate the method in the case of strychnine, determining the three major conformational states of this small molecule and their associated occupation probabilities.

HPLC and GC-MS: Identification and Quantification of Strychnine and Brucine in Strychnos nux-vomica leaves.

In this study, we have aimed to analyze the phytochemical composition of this plant and the concentration of strychnine and brucine. The identification of bioactive compounds was done by GC-MS with NIST Library. Strychnine and Brucine were quantified using HPLC. Twenty one medicinal bio active compounds were identified from the Strychnos nux-vomica leaf ethanolic extract. Strychnine is showing 28.43% purity and brucine was not detected in GCMS analysis. Quantified the concentration of strychnine (0.6 mg in 500mg of extract) and brucine (1.6 mg in 500mg of extract) was done by HPLC against Strychnine and Brucine standard. These compounds are having natural properties of Anti-inflammatory, Hypocholesterole, Cancer preventive, Hepatoprotective, Antimicrobial, Antioxidant, Cardio protective, Antiaging, Antialzheimeran, Antidermatitic, Immunostimulant, Anthepatotoxic, biosynthesis of steroid hormones, Nematicide, Antiandrogenic, 5-alpha reductase inhibitor, antipsychotic, analgesic, apoptotic effect, antidepressant, antidote for snake poisoning and diabetic activity.

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.

Variability of Stimulant Levels in Nine Sports Supplements Over a 9-Month Period.

Many studies have found that some dietary supplement product labels do not accurately reflect the actual ingredients. However, studies have not been performed to determine if ingredients in the same dietary supplement product vary over time. The objective of this study was to assess the consistency of stimulant ingredients in popular sports supplements sold in the United States over a 9-month period. Three samples of nine popular sports supplements were purchased over the 9-month period. The 27 samples were analyzed for caffeine and several other stimulants (including adulterants). The identity and quantity of stimulants were compared with stimulants listed on the label and stimulants found at earlier time points to determine the variability in individual products over the 9-month period. The primary outcome measure was the variability of stimulant amounts in the products examined. Many supplements did not contain the same number and quantity of stimulants at all time points over the 9-month period. Caffeine content varied widely in five of the six caffeinated supplements compared with the initial measurement (-7% to +266%). In addition, the stimulants-synephrine, octopamine, cathine, ephedrine, pseudoephedrine, strychnine, and methylephedrine-occurred in variable amounts in eight of the nine products. The significance of these findings is uncertain: the sample size was insufficient to support statistical analysis. In our sample of nine popular sports supplements, the presence and quantity of stimulants varied over a 9-month period. However, future studies are warranted to determine if the variability found is significant and generalizable to other supplements.

A Drosophila Gustatory Receptor Required for Strychnine Sensation.

Strychnine is a potent, naturally occurring neurotoxin that effectively protects plants from animal pests by deterring feeding behavior. In insects, such as the fruit fly, Drosophila melanogaster, bitter-tasting aversive compounds are detected primarily through a family of gustatory receptors (GRs), which are expressed in gustatory receptor neurons. We previously described multiple GRs that eliminate the behavioral avoidance to all bitter compounds tested, with the exception of strychnine. Here, we report the identity of a strychnine receptor, referred to as GR47a. We generated a mutation in Gr47a and found that it eliminated strychnine repulsion and strychnine-induced action potentials. GR47a was narrowly tuned, as the responses to other avoidance compounds were unaffected in the mutant animals. This analysis supports an emerging model that Drosophila GRs fall broadly into two specificity classes-one class is comprised of core receptors that are broadly required, whereas the other class, which includes GR47a, consists of narrowly tuned receptors that define chemical specificity.

A new method for the simultaneous analysis of strychnine and brucine in Strychnos nux-vomica unprocessed and processed seeds using a carbon-paste electrode modified with multi-walled carbon nanotubes.

INTRODUCTION: Strychnos nux-vomica L. (Loganiaceae), widely used in folk medicine, is grown extensively in southern Asian countries. Its major bioactive constituents are strychnine and brucine, which are frequently used in traditional herbal medicines for treatment of nervous diseases, vomiting and traumatic pain. OBJECTIVE: A new method using a carbon-paste electrode modified with multi-walled carbon nanotubes (CNT/CPE) was developed and validated for single or simultaneous determination of strychnine and brucine in Strychnos nux-vomica seeds. Additionally, an environmentally friendly method was successfully applied to reduce the levels of strychnine and brucine in seeds. MATERIALS AND METHODS: Cyclic voltammetry, chronocoulometry and differential pulse voltammetry were used with multi-walled carbon nanotube modified carbon-paste electrodes. RESULTS: The peak currents increase linearly with the strychnine and brucine concentrations in the ranges of 50-1000 and 5-355 µ m, and the detection limits for strychnine and brucine were 0.43 and 0.28 µ m, respectively. Of the processing methods used, the greatest reduction in the strychnine and brucine levels was observed in samples processed using milk and saltwater. CONCLUSION: A new, sensitive and selective method was developed for the measurement of strychnine and brucine. This method was successfully applied to the determination of strychnine and brucine in unprocessed and processed Strychnos nux-vomica seed.

[Determination of brucine and strychnine in rat after cutaneous administration of semen strychni niosome gel by LC-MS/MS].

OBJECTIVE: A sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the determination of brucine and strychnine in rat plasma. METHOD: Samples were extracted by ethyl acetate-n-butanol (7: 3). Chromatographic separation was operated on ZORBAX XDB-C18 column with gradient elution of acetonitrile-methanol-water (0.05% acetic acid and 10 nmol x L(-1) ammonium formate contained), followed by LC-MS/MS in positive electrospray ionization. Quantification was carried out on multiple reaction monitoring (MRM) of the transition m/z 395.2/324.2, m/z 335.2/184.2 and m/z 199.1/171.1 for brucine, strychnine and tacrine (internal standard), respectively. RESULT: The method was linear in the range of 0.195-100 and 0.07840 microg x L(-1) for brucine and strychnine, with coefficient correlation 0.994 and 0.996 respectively. The recoveries of extraction were 78.9% - 102.4% for brucine and 95.2% - 106.1% for strychnine. Precision, accuracy, stability and matrix effect of the analytes met the requirement. The method was applied to a pharmacokinetic study of brucine and strychnine after cutaneous administration of Semen Strychni niosome gel. The C(max) were (26.20 +/- 5.81) and (12.50 +/- 3.00) microg x L(-1) while the AUC(0-infinity), were (193.75 +/- 39.43) and (98.25 +/- 28.54) microg x h x L(-1) of the two components. CONCLUSION: We conclude that the niosomes may reduce the systemic exposures and prolong the local release of brucine and strychnine.

[LC-MS/MS analysis of determination of strychnine and brucine in formaldehyde fixed tissue].

OBJECTIVE: To establish a method for determination of strychnine and brucine in formaldehyde fixed tissue by LC-MS/MS analysis. METHODS: The samples were pretreated with solid phase extraction using SCX cartridges and separated on SB-C18 column with mobile phase 0.1% formic acid : 0.1% formic acid-acetonitrile (75:25). Electrospray ionization (ESI) source was utilized and operated in positive ion mode. Multiple reactions monitoring (MRM) mode was applied. External standard method was applied for quantitation. RESULTS: The chromatographic separation of strychnine and brucine in formaldehyde fixed nephritic and hepatic tissues resulted successfully. The standard curve was linear in the range of 0.002-2.0 microg/g for strychnine and brucine in formaldehyde fixed tissues, and the correlation coefficient was more than 0.996. The limits of detection (LOD) of strychnine and brucine in nephritic tissues were 0.06ng/g and 0.03 ng/g, respectively. The LOD of both chemicals were 0.3 ng/g in hepatic tissues. The extraction recovery rate was more than 74.5%. The precision of intra-day and inter-day were both less than 8.2%. CONCLUSION: Strychnine and brucine can be sensitive to be determined in formaldehyde fixed tissue by LC-MS/MS analysis. It can be applied in the forensic toxicological analysis.

Interproton distance determinations by NOE--surprising accuracy and precision in a rigid organic molecule.

The accuracy inherent in the measurement of interproton distances in small molecules by nuclear Overhauser enhancement (NOE) and rotational Overhauser enhancement (ROE) methods is investigated with the rigid model compound strychnine. The results suggest that interproton distances can be established with a remarkable level of accuracy, within a few percent of their true values, using a straight-forward data analysis method if experiments are conducted under conditions that support the initial rate approximation. Dealing with deviations from these conditions and other practical issues regarding these measurements are discussed.

HSQC-ADEQUATE correlation: a new paradigm for establishing a molecular skeleton.

Various experimental methods have been developed to unequivocally identify vicinal neighbor carbon atoms. Variants of the HMBC experiment intended for this purpose have included 2J3J-HMBC and H2BC. The 1,1-ADEQUATE experiment, in contrast, was developed to accomplish the same goal but relies on the (1) J(CC) coupling between a proton-carbon resonant pair and the adjacent neighbor carbon. Hence, 1,1-ADEQUATE can identify non-protonated adjacent neighbor carbons, whereas the 2J3J-HMBC and H2BC experiments require both neighbor carbons to be protonated to operate. Since 1,1-ADEQUATE data are normally interpreted with close reference to an HSQC spectrum of the molecule in question, we were interested in exploring the unsymmetrical indirect covariance processing of multiplicity-edited GHSQC and 1,1-ADEQUATE spectra to afford an HSQC-ADEQUATE correlation spectrum that facilitates the extraction of carbon-carbon connectivity information. The HSQC-ADEQUATE spectrum of strychnine is shown and the means by which the carbon skeleton can be conveniently traced is discussed.

Ammonium sulfate gradient loading of brucine into liposomes: effect of phospholipid composition on entrapment efficiency and physicochemical properties in vitro.

BACKGROUND: Brucine, the major active alkaloid constituent extracted from traditional Chinese herbal medicine Nux vomica, had been found to possess remarkable antitumor, analgesic, and anti-inflammatory activities. In this study, we attempted to encapsulate brucine into liposomes to improve its therapeutic effects. The entrapment efficiency (EE) and the stability of liposomes are two key factors associated with the therapeutic effects of liposomal drugs. We developed a novel liposome-based brucine formulation that was composed of soybean phosphatidylcholine (SPC) and hydrogenated soybean phosphatidylcholine (HSPC). METHOD: The liposomes with different phospholipid composition were characterized for their EE, vesicle size, drug release profile, and leakage in vitro. RESULTS: The molar ratio of HSPC/SPC = 1:9 was determined as the optimum ratio. Compared with conventional liposomes composed of only SPC or HSPC, EE of the brucine-loaded novel liposomes was increased markedly, especially at high drug/lipid molar ratios. The results of drug release showed that the novel liposomes were more stable than the conventional SPC liposomes in the presence of fetal calf serum. In addition, the results of the leakage experiments revealed that the novel liposomes also had better stability in phosphate buffer solution (PBS) with respect to drug retention. Although the conventional HSPC liposomes is more stable than the novel liposomes, the novel liposomes composed of 10% HSPC and 90% SPC may still have promising application potential because HSPC is much more expensive than SPC. CONCLUSION: Taken together, efficient encapsulation of brucine into the novel liposomes, their improved stability, and the price of phospholipids indicate that the novel liposomes may act as promising carriers for active alkaloids such as brucine.

A simple, cost-effective and rapid method for simultaneous determination of Strychnos nux-vomica alkaloids in blood and Ayurvedic medicines based on ultrasound-assisted dispersive liquid-liquid microextraction-thin-layer chromatography-image analysis.

A simple, rapid, cost-effective and green analytical method is developed based on ultrasound-assisted dispersive liquid-liquid microextraction (US-DLLME) coupled to thin-layer chromatography (TLC)-image analysis for the simultaneous determination of two major alkaloids of Strychnos nux-vomica L i.e., strychnine and brucine. The method is composed of three steps, namely (i) US-DLLME by injecting a mixture of 100-µL chloroform (extraction solvent) and 1-mL methanol (disperser solvent) in 5 mL of aqueous sample, followed by ultrasonication and centrifugation, (ii) TLC of 20 µL of sedimented phase with methanol: ammonia (100:1.5, v/v) as the mobile phase and visualization under ultraviolet radiation (254 nm) and (iii) photography of TLC plate and quantification of spots by image analysis using freely available imageJ software (National Institute of Health, Bethesda, MD, USA). The limit of detection and limit of quantification for both alkaloids were found to be in the range of 0.12-0.15 and 0.36-0.48 µg/spot, respectively. The method was found to be linear in the range of 0.5-5 µg/spot with correlation coefficient (R2) of 0.995 and 0.997 for strychnine and brucine, respectively. The developed method was successfully applied for the determination of strychnine and brucine in Ayurvedic formulations and blood samples. The method does not require any sophisticated instrument and handling skills and can be adopted for rapid analysis of strychnine and brucine in forensic toxicological laboratories.

[A comparative study on HPLC fingerprint of Semen Strychni and the processed].

OBJECTIVE: To establish the fingerprint of Semen Strychni and the processed by HPLC. METHODS: The HPLC Method was used, the chromatgraphic conditions were as follows: Chromatographic column: Lichrospher C18 (250 mm x 4.6 mm, 5 microm), mobile phase: solvent A consisted of water-ethanoic acid-triethylamine (100: 0.2: 0.2), solvent B contained acetonitril (gradient elution), column temperature: 30 degrees C, flow rate: 1.0 mL/min, detection wavelength: 254 nm. RESULTS: The similar degree of 10 batch samples buiding sharing mode of crude drug and processed product was above 0.9. 18 common peaks in chromatograms were separated from 10 batches of Semen Strychni samples and 21 common peaks in chromatograms were separated from 10 batches of the processed. CONCLUSION: This study establishes the HPLC fingerprint commonmode, and researches the difference between Semen Strychni and the processed in HPLC fingerprint.

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.

Microdialysis combined with RRLC-MS/MS for the pharmacokinetics of two major alkaloids of Bi qi capsule and the potential roles of P-gp and BCRP on their penetration.

Bi qi capsule (BQC) is a traditional Chinese medicine prescription that is clinically used for the treatment of rheumatoid arthritis. Strychnine and brucine, as two typical kinds of alkaloids, are the primary active and neurotoxic constituents of BQC. In this study, a sensitive and reliable rapid resolution liquid chromatography-tandem mass spectrometry (RRLC-MS/MS) quantitative method was used to determine the concentrations of brucine and strychnine in rat brain and blood dialysates. The blood-brain barrier (BBB) penetration of free brucine and strychnine and their pharmacokinetic characteristics were investigated by the validated RRLC-MS/MS method coupled with in vivo microdialysis for the first time. The dialysate brain-blood AUC ratios of brucine were 0.098, 0.44 and 0.40 respectively at 0.4, 0.8 and 1.6 g kg-1 doses of BQC, and the dialysate brain-blood AUC ratios of strychnine were 0.20, 1.25 and 2.06 respectively at 0.4, 0.8 and 1.6 g kg-1 doses of BQC. The high brain-blood AUC ratios of brucine and strychnine were observed in medium and high dose groups of BQC. In addition, the effects of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) on brucine and strychnine across BBB were also studied using the above method as well as molecular docking. The results prompted that brucine was the substrate of P-gp, and strychnine might be the inhibitor of P-gp. Brucine and strychnine showed high brain penetration, so it is very important to well control the clinic dosage of BQC and manufactory quality for avoiding the side effects and obtaining good therapeutic efficacy. Our study could be further used in investigating BBB penetration for other drugs caused neurotoxicity.

Development and validation of a TLC-densitometric method for the simultaneous quantitation of strychnine and brucine from Strychnos spp. and its formulations.

A simple, sensitive, and specific thin-layer chromatography densitometric method has been developed for the simultaneous quantitation of strychnine and brucine. These two marker compounds are quantitated in the seeds of Strychnos nux-vomica, Strychnos ignatii, and its formulations. The method involves densitometric evaluation of strychnine and brucine after resolving it by high-performance TLC on silica gel plate with toluene-ethyl acetate-diethyl amine-methanol (7:2:1:0.3 v/v) as the mobile phase. The method is validated for precision (interday and intraday), repeatability, and accuracy. The relationship between the concentration of standard solutions and the peak response is linear within the concentration range of 160 to 480 ng/spot for strychnine and 80 to 480 ng/spot for brucine. Instrumental precision is found to be 0.54 and 0.78 (% CV), and repeatability of the method is 1.01 and 1.06 (% CV) for strychnine and brucine, respectively. Accuracy of the method is checked by recovery study conducted at three different levels and the average percentage recovery is found to be 99.13% for strychnine and 100.16% for brucine. The proposed HPTLC method for the simultaneous quantitation of strychnine and brucine is found to be simple, precise, specific, sensitive, and accurate, and it can be used for routine quality control of raw material of Strychnos spp. It also can be applied in quantitating any of these marker compounds in other formulations.

HPLC-UV method for nicotine, strychnine, and aconitine in dairy products.

The toxic nitrogen alkaloids nicotine, strychnine, and aconitine were quantitated in whole milk, skim milk, and cream using solid-phase extraction cleanup and HPLC-UV with dual wavelength detection. Samples were extracted in McIlvaine's buffer with EDTA and then partitioned with aqueous acetonitrile and hexane. The aqueous phase was concentrated and passed through an OASIS HLB column. The column was eluted with methylene chloride/ammonium hydroxide, 1 mL/1 microL, v/v. The eluent was acidified with hydrochloric acid and evaporated. The sample was diluted for HPLC with acetonitrile/phosphate buffer pH 7.4. Chromatography was performed on an Xterra RP-18 column using a gradient of acetonitrile and ammonium bicarbonate buffer at pH 9.8. Nicotine and strychnine were monitored at 260 nm; aconitine was monitored at 232 nm. Calibration curves were generated from external standards in the range 0.2-10 microg/mL using 1/x weighting. Mean recoveries in whole milk spiked between 0.1 and 10 ppm were the following: nicotine 89.2%, strychnine 75.7%, and aconitine 85.1%. Mean recoveries in skim milk spiked between 0.1 and 10 ppm were the following: nicotine 72.1%, strychnine 78.2%, and aconitine 82.9%. Mean recoveries in cream spiked between 0.2 and 20 ppm were the following: nicotine 87.9%, strychnine 76.9%, and aconitine 82.0%. Relative standard deviations of recovery were less than 20% in each case.

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%.