Influence of different elicitors on BIA production in Macleaya cordata.

Sanguinarine (SAN) and chelerythrine (CHE) have been widely used as substitutes for antibiotics for decades. For a long time, SAN and CHE have been extracted from mainly Macleaya cordata, a plant species that is a traditional herb in China and belongs to the Papaveraceae family. However, with the sharp increase in demand for SAN and CHE, it is necessary to develop a new method to enhance the supply of raw materials. Here, we used methyl jasmonate (MJ), salicylic acid (SA) and wounding alone and in combination to stimulate aseptic seedlings of M. cordata at 0 h, 24 h, 72 h and 120 h and then compared the differences in metabolic profiles and gene expression. Ultimately, we found that the effect of using MJ alone was the best treatment, with the contents of SAN and CHE increasing by 10- and 14-fold, respectively. However, the increased SAN and CHE contents in response to combined wounding and MJ were less than those for induced by the treatment with MJ alone. Additionally, after MJ treatment, SAN and CHE biosynthetic pathway genes, such as those encoding the protopine 6-hydroxylase and dihydrobenzophenanthridine oxidase enzymes, were highly expressed, which is consistent with the accumulation of SAN and CHE. At the same time, we have also studied the changes in the content of synthetic intermediates of SAN and CHE after elicitor induction. This study is the first systematic research report about using elicitors to increase the SAN and CHE in Macleaya cordata.

Intracellular Accumulation as an Indicator of Cytotoxicity to Screen Hepatotoxic Components of Chelidonium majus L. by LC-MS/MS.

A novel strategy was developed to identify hepatotoxic compounds in traditional Chinese medicines (TCMs). It is based on the exposure of HL-7702 cells to a TCM extract, followed by the identification and further determination of potential hepatotoxic compounds accumulated in the cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a case study, potential hepatotoxic components in Chelidonium majus L. were screened out. Five alkaloids (sanguinarine, coptisine, chelerythrine, protopine, and chelidonine) were identified by LC-MS/MS within 10 min, and their intracellular concentrations were first simultaneously measured by LC-MS/MS with a run time of 4 min. A cell viability assay was performed to assess the cytotoxicity of each alkaloid. With their higher intracellular concentrations, sanguinarine, coptisine, and chelerythrine were identified as the main hepatotoxic constituents in Ch. majus. The study provides a powerful tool for the fast prediction of cytotoxic components in complex natural mixtures on a high-throughput basis.

Rapid identification of "mad honey" from Tripterygium wilfordii Hook. f. and Macleaya cordata (Willd) R. Br using UHPLC/Q-TOF-MS.

Cases of honey poisoning have been reported widely, meaning there is a need for methods that detect "mad honey" or honey contaminated with plant-derived toxins to protect human health. In this study, we compared whole flower extracts and honey from Tripterygium wilfordii Hook. f. (TwHf) and Macleaya cordata (Willd) R. Br (McRB) using QuEChERS (quick, easy, cheap, effective, rugged, and safe) and ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS). The results revealed several compounds common to whole flowers and honey samples. Triptolide and protopine were selected as potential markers for identifying "mad honeys" from these plants. The developed method can easily detect different honey varieties that were spiked with 5% TwHf and McRB honey samples. Additionally, 90 commercial honey samples were analyzed and determined as free from contamination. The method described in this report could be useful for studies on honey from other poisonous nectar and pollen plants.

The comparative pharmacokinetic study of Yuanhu Zhitong prescription based on five quality-markers.

BACKGROUND: According to the compatibility theory, therapeutic effects of Chinese medicine prescription are generally attributed to the synergism of multi-herbs. Quality-markers, as the crucial effective components, play a key role in the interaction of compatibility. Pharmacokinetic studies could illustrate the interaction between multiple components in dynamics perspective. PURPOSE: This study aims to establish a rapid, reliable and sensitive ultra performance liquid chromatography coupled with tandem mass spectrometry method for simultaneous determination of corydaline, tetrahydropalmatine, protopine, imperatorin and isoimperatorin (quality-markers of Yuanhu Zhitong prescription) in rat plasma, and then applied to the comparative pharmacokinetic study for clearing the interaction of compatibility in Yuanhu Zhitong prescription. METHODS: Five quality-markers were separated on a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) by gradient elution with 0.1% formic acid-water and 0.1% formic acid-acetonitrile. Detection was performed in the positive ionization and multiple reaction monitoring mode. The analytical method was validated and successfully applied to the comparative pharmacokinetic study after oral administration of Yuanhu Zhitong prescription and single-herb extracts. RESULTS: Calibration curves showed good linearity over the concentration ranges of 0.25-500 ng/ml for corydaline, tetrahydropalmatine and isoimperatorin, 0.1-200 ng/ml for protopine, and 0.5-500 ng/ml for imperatorin, respectively. Compared with Rhizoma corydalis group, AUC0-t and AUC0-∞ significantly increased (p < 0.01 for corydaline and tetrahydropalmatine, and p < 0.05 for protopine) after oral administration of Yuanhu Zhitong prescription extract. Meanwhile, Cmax of corydaline and tetrahydropalmatine increased remarkably, from 93.00 µg/l to 196.35 µg/l for corydaline (p < 0.05) and 181.62 µg/l to 311.22 µg/l for tetrahydropalmatine (p < 0.01). In addition, MRT0-t and MRT0-∞ of corydaline, as well as Tmax of protopine in Yuanhu Zhitong prescription group were obviously delayed compared to Rhizoma corydalis group (p < 0.01). CONCLUSION: These achieved results indicated that the compatibility of Rhizoma corydalis and Radix Angelicae dahuricae lead to greater absorption of corydaline, tetrahydropalmatine and protopine, which would be help to better understand the compatibility mechanism of Yuanhu Zhitong prescription.

Identification of quality markers of Yuanhu Zhitong tablets based on integrative pharmacology and data mining.

BACKGROUND: The quality evaluation of traditional Chinese medicine (TCM) formulations is needed to guarantee the safety and efficacy. In our laboratory, we established interaction rules between chemical quality control and biological activity evaluations to study Yuanhu Zhitong tablets (YZTs). Moreover, a quality marker (Q-marker) has recently been proposed as a new concept in the quality control of TCM. However, no appropriate methods are available for the identification of Q-markers from the complex TCM systems. PURPOSE: We aimed to use an integrative pharmacological (IP) approach to further identify Q-markers from YZTs through the integration of multidisciplinary knowledge. In addition, data mining was used to determine the correlation between multiple constituents of this TCM and its bioactivity to improve quality control. METHODS: The IP approach was used to identify the active constituents of YZTs and elucidate the molecular mechanisms by integrating chemical and biosynthetic analyses, drug metabolism, and network pharmacology. Data mining methods including grey relational analysis (GRA) and least squares support vector machine (LS-SVM) regression techniques, were used to establish the correlations among the constituents and efficacy, and dose efficacy in multiple dimensions. RESULTS: Seven constituents (tetrahydropalmatine, α-allocryptopine, protopine, corydaline, imperatorin, isoimperatorin, and byakangelicin) were identified as Q-markers of YZT using IP based on their high abundance, specific presence in the individual herbal constituents and the product, appropriate drug-like properties, and critical contribution to the bioactivity of the mixture of YZT constituents. Moreover, three Q-markers (protopine, α-allocryptopine, and corydaline) were highly correlated with the multiple bioactivities of the YZTs, as found using data mining. Finally, three constituents (tetrahydropalmatine, corydaline, and imperatorin) were chosen as minimum combinations that both distinguished the authentic components from false products and indicated the intensity of bioactivity to improve the quality control of YZTs. CONCLUSIONS: Tetrahydropalmatine, imperatorin, and corydaline could be used as minimum combinations to effectively control the quality of YZTs.

Enrichment of chelidonine from Chelidonium majus L. using macroporous resin and its antifungal activity.

Chelidonium majus L. (greater celandine) has been used as an herbal medicine for several centuries. This study investigated an efficient method to purify chelidonine from the extract of C. majus L. using macroporous adsorption resins and evaluated the antifungal activity of chelidonine against Botryosphaeria dothidea as a model strain. Static adsorption and desorption tests revealed that D101 was the optimal resin for chelidonine purification. Pseudo-second-order kinetics model and Freundlich equation model were the most suitable for evaluating the endothermic and spontaneous adsorption processes of chelidonine on D101. Dynamic adsorption and desorption tests on D101 columns showed that the concentration of chelidonine increased 14.16-fold, from 2.67% to 37.81%, with the recovery yield of 80.77%. The antifungal activity of enriched chelidonine products was studied with B. dothidea. The results showed that the EC50 of crude extracts, enriched chelidonine products, and chelidonine standard against B. dothidea were 3.24mg/mL, 0.43mg/mL, and 0.77mg/mL, respectively. The result of antifungal activity test showed that chelidonine had the potential to be a useful antifungal agent. Moreover, the enrichment method of chelidonine was highly efficient, low cost, and harmless to the environment for industrial applications.

Identification of allocryptopine and protopine metabolites in rat liver S9 by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

RATIONALE: Allocryptopine (AL) and protopine (PR) have been extensively studied because of their anti-parasitic, anti-arrhythmic, anti-thrombotic, anti-inflammatory and anti-bacterial activity. However, limited information on the pharmacokinetics and metabolism of AL and PR has been reported. Therefore, the purpose of the present study was to investigate the in vitro metabolism of AL and PR in rat liver S9 using a rapid and accurate high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC/QqTOFMS) method. METHODS: The incubation mixture was processed with 15% trichloroacetic acid (TCA). Multiple scans of AL and PR metabolites and accurate mass measurements were automatically performed simultaneously through data-dependent acquisition in only a 30-min analysis. The structural elucidations of these metabolites were performed by comparing their changes in accurate molecular masses and product ions with those of the precursor ion or metabolite. RESULTS: Eight and five metabolites of AL and PR were identified in rat liver S9, respectively. Among these metabolites, seven and two metabolites of AL and PR were identified in the first time, respectively. The demethylenation of the 2,3-methylenedioxy, the demethylation of the 9,10-vicinal methoxyl group and the 2,3-methylenedioxy group were the main metabolic pathways of AL and PR in liver S9, respectively. In addition, the cleavage of the methylenedioxy group of the drugs and subsequent methylation or O-demethylation were also the common metabolic pathways of drugs in liver S9. In addition, the hydroxylation reaction was also the metabolic pathway of AL. CONCLUSIONS: This was the first investigation of in vitro metabolism of AL and PR in rat liver S9. The detailed structural elucidations of AL and PR metabolites were performed using a rapid and accurate HPLC/QqTOFMS method. The metabolic pathways of AL and PR in rat were tentatively proposed based on these characterized metabolites and early reports. Copyright © 2016 John Wiley & Sons, Ltd.

Introduction of macarpine as a novel cell-permeant DNA dye for live cell imaging and flow cytometry sorting.

BACKGROUND INFORMATION: Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti-inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time-lapse microscopy and flow-cytometric cell sorting. RESULTS: Living A-375 and MEF cells stained with MA were monitored by time-lapse microscopy for 24 h. Mitoses were observed at MA concentrations up to 0.5 µg/ml during the first 2-3 h. After this period of time, cells treated with MA at concentrations of 0.75 and 0.5 µg/ml underwent apoptosis. Cells cultivated with MA at concentration of 0.25 µg/ml or lower survived throughout the 24 h period. Toxicity of MA was dependent on light wavelength and frequency of image capturing. The intensity of MA fluorescence decreased during the incubation. MA concentration of 0.1 µg/ml was identified as the most suitable for live cell imaging with respect to fluorescence intensity and toxicity. MA at the concentration 10 µg/ml was used for sorting of enhanced green fluorescent protein (EGFP)-labelled neurons and fibroblasts yielding profiles similar to those obtained with DRAQ5. Contrary to DRAQ5, MA-stained cells survived in culture, and the sorted cells lost the MA signal suggesting reversible binding of the dye to the DNA. CONCLUSION: The results proved that MA may readily be used for chromosomes depicting and mitosis monitoring by time-lapse microscopy. In addition, MA has shown to be a suitable probe for sorting of EGFP-labelled cells, including neurons, that survived the labelling process. SIGNIFICANCE: In consideration of the results, we highly anticipate an onward use of MA in a broad range of applications based on live cell sorting and imaging, for example, cell synchronisation and monitoring of proliferation as an important experimental and/or diagnostic utility.

Microchip electrophoretic separation and fluorescence detection of chelerythrine and sanguinarine in medicinal plants.

A new method has been developed for separation of chelerythrine and sanguinarine in medicinal plants used in traditional Chinese medicine (TCM). The separation is achieved by microchip electrophoresis (CE) using laser-induced fluorescence detection. The CE separation is achieved by using a hydro-organic medium as the electrolyte buffer. The experimental results are consistent with the prediction by theory in terms of resolution and migration speed because of the low Joule heat generated in microchip CE. In addition, formamide was found to have a potential for separation of molecules with similar chemical structures. Based on these findings, a run buffer containing 50% formamide was used to separate chelerythrine (CHE) and sanguinarine (SAN). The influencing factors, such as solvent of run buffer, pH of buffer, separation distance, and separation voltage, were optimized. Baseline separation of chelerythrine and sanguinarine was achieved within 120 s under an electrical voltage of 1.8 kV. Good linearity was observed in the concentration range of 0.15-550 µg mL(-1) (r=0.9993) for CHE and in the range of 0.3-600 µg mL(-1) (r=0.9998) for SAN. A low limit of detection (LOD) was achieved because of the high sensitivity achieved by laser-induced fluorescence detection (i.e. 5.0 ng mL(-1) and 2.0 ng mL(-1) for CHE and SAN, respectively). The contents of CHE are found to be 641.8±7.5 and 134.0±2.3 mg/kg in extracts of Macleaya cordata and Chelidonium majus, respectively, with good recovery of above 99%. The corresponding values for SAN found in these Chinese herbal extracts are 681.8±7.9 mg/kg and 890.5±8.9 mg/kg, respectively.

Simultaneous quantitative determination of sanguinarine, chelerythrine, dihydrosanguinarine and dihydrochelerythrine in chicken by HPLC-MS/MS method and its applications to drug residue and pharmacokinetic study.

A specific and reliable HPLC-MS/MS method was developed and validated for simultaneously determination of sanguinarine, chelerythrine and their metabolites (dihydrosanguinarine and dihydrochelerythrine) in chicken tissue for the first time. This is important because these compounds are related to the use of a naturally occurring and novel feed additive with many benefits, but the levels of these compounds must be strictly controlled. The compounds were extracted by acetonitrile and 1% HCl-methanol solution successively and then separated on a C18 column. A triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source was used for detection. Quantification was performed using multiple reaction monitoring with positive mode. The method was validated in terms of specificity, linearity, precision, accuracy and stability. The calibration curves were linear over the concentration range of 0.5-100.0ng/g for sanguinarine, 0.5-100.0ng/g for chelerythrine, 0.2-100.0ng/g for dihydrosanguinarine and 0.1-100ng/g for dihydrochelerythrine, respectively. All of the recovery rates of the four analytes were over 85%. The RSD of intra-day and inter-day precision was less than 5.0%, and the relative error was all within 12.0%. This validated method has been successfully applied to assess the drug residue and metabolite residue characteristics of sanguinarine and chelerythrine in chicken tissue after oral administration of the extracts of Macleaya cordata (Willd.) R. Br, and to investigate the pharmacokinetic parameters of sanguinarine and dihydrosanguinarine in chicken plasma.

In vitro and in vivo antiplasmodial activity of three Rwandan medicinal plants and identification of their active compounds.

In our previous study, we reported the interesting in vitro antiplasmodial activity of some Rwandan plant extracts. This gave rise to the need for these extracts to also be evaluated in vivo and to identify the compounds responsible for their antiplasmodial activity. The aim of our study was, on the one hand, to evaluate the antiplasmodial activity in vivo and the safety of the selected Rwandan medicinal plants used in the treatment of malaria, with the objective of promoting the development of improved traditional medicines and, on the other hand, to identify the active ingredients in the plants. Plant extracts were selected according to their selectivity index. The in vivo antiplasmodial activity of aqueous, methanolic, and dichloromethane extracts was then evaluated using the classical 4-day suppressive test on Plasmodium berghei infected mice. The activity of the plant extracts was estimated by measuring the percentage of parasitemia reduction, and the survival of the experimental animals was recorded. A bioguided fractionation was performed for the most promising plants, in terms of antiplasmodial activity, in order to isolate active compounds identified by means of spectroscopic and spectrometric methods. The highest level of antiplasmodial activity was observed with the methanolic extract of Fuerstia africana (> 70 %) on days 4 and 7 post-treatment after intraperitoneal injection and on day 7 using oral administration. After oral administration, the level of parasitemia reduction observed on day 4 post-infection was 44 % and 37 % with the aqueous extract of Terminalia mollis and Zanthoxylum chalybeum, respectively. However, the Z. chalybeum extract presented a high level of toxicity after intraperitoneal injection, with no animals surviving on day 1 post-treatment. F. africana, on the other hand, was safer with 40 % mouse survival on day 20 post-treatment. Ferruginol is already known as the active ingredient in F. Africana, and ellagic acid (IC50 = 175 ng/mL) and nitidine (IC50 = 77.5 ng/mL) were identified as the main active constituents of T. mollis and Z. chalybeum, respectively. F. africana presented very promising antiplasmodial activity in vivo. Although most of the plants tested showed some level of antiplasmodial activity, some of these plants may be toxic. This study revealed for the first time the role of ellagic acid and nitidine as the main antimalarial compounds in T. mollis and Z. chalybeum, respectively.

Fluorescence quenching investigation on the interaction of glutathione-CdTe/CdS quantum dots with sanguinarine and its analytical application.

Water-soluble glutathione (GSH)-capped core/shell CdTe/CdS quantum dots (QDs) were synthesized. In pH5.4 sodium phosphate buffer medium, the interaction between GSH-CdTe/CdS QDs and sanguinarine (SA) was investigated by spectroscopic methods, including fluorescence spectroscopy and ultraviolet-visible absorption spectroscopy. Addition of SA to GSH-CdTe/CdS QDs results in fluorescence quenching of GSH-CdTe/CdS QDs. Quenching intensity was in proportion to the concentration of SA in a certain range. Investigation of the quenching mechanism, proved that the fluorescence quenching of GSH-CdTe/CdS QDs by SA is a result of electron transfer. Based on the quenching of the fluorescence of GSH-CdTe/CdS QDs by SA, a novel, simple, rapid and specific method for SA determination was proposed. The detection limit for SA was 3.4 ng/mL and the quantitative determination range was 0.2-40.0 µg/mL with a correlation coefficient of 0.9988. The method has been applied to the determination of SA in synthetic samples and fresh urine samples of healthy human with satisfactory results.

Optimization of the separation and determination of nitidine and chelerythrine in Zanthoxylum nitidum by high-performance liquid chromatography with fluorescence detection.

A new ion-pair high-performance liquid chromatography method with fluorescence detection was developed for the determination of nitidine and chelerythrine in Zanthoxylum nitidum. To optimize the separation of the two compounds in reversed-phase liquid chromatography, a response surface method (Box-Behnken designs) was used. Three important factors: concentration of ion pair agent, mobile phase composition and buffer pH, were studied for their contribution to the analytes' response, leading to a total of 17 experiments performed on a Kromasil C18 column. The experimental responses were fitted into a second-order polynomial to predict the best conditions. The optimal mobile phase conditions were predicted to be acetonitrile-sodium dodecyl sulphate (17.8 mM, 20 mM citric acid, pH 2.98, 57:43, v/v). The proposed method was validated according to International Conference on Harmonization guidelines and it is suggested to be appropriate for the routine quality control analysis of nitidine and chelerythrine in Zanthoxylum nitidum.

Optimization of the extraction conditions and quantification by RP-LC analysis of three alkaloids in Zanthoxylum nitidum roots.

CONTEXT: A classic traditional Chinese medicine, Zanthoxylum nitidum (Roxb.) DC. widely used in China, exhibits anticancer, anti-inflammatory and antianalgesic activities. Alkaloids are one of the main bioactive components. It is urgent to develop a simple and reliable method to determine the main alkaloids in Z. nitidum roots. OBJECTIVE: To determine the three alkaloids in Z. nitidum roots, a reversed-phase liquid chromatographic (RP-LC) method combined with an optimum extraction condition was established. MATERIALS AND METHODS: A method involving four-factor-three-level orthogonal array design including the extracting solvent and the RP-LC condition was assayed. Twenty batches were collected from different areas of the Guangxi Province at different harvesting times. The determined alkaloids were nitidine chloride (NC, 1), ethoxychelerythrine (2) and liriodenine (3). The stable mobile phase was a C18 packing, and the mobile phase was acetonitril-aqueous phosphoric acid-triethylamine-buffer solution. RESULTS: The optimum extraction and detection conditions have been determined in the process of quantification of Z. nitidum root alkaloids. The three alkaloids were detected simultaneously in the 20 batches of samples. The results clearly showed that alkaloid concentrations differed significantly among Z. nitidum collected from various collection areas. DISCUSSION AND CONCLUSION: We have established an optimum extraction and detection conditions in the process of quantification the three alkaloids in Z. nitidum roots. From this research, the most influenced factor on Z. nitidum roots was the collecting location, and the next factor was the harvesting time. The collecting location and the harvesting time should be considered as the high-quality medicinal herbs factors.

Identification and quantification of the main active anticancer alkaloids from the root of Glaucium flavum.

Glaucium flavum is used in Algerian folk medicine to remove warts (benign tumors). Its local appellations are Cheqiq el-asfar and Qarn el-djedyane. We have recently reported the anti-tumoral activity of Glaucium flavum root alkaloid extract against human cancer cells, in vitro and in vivo. The principal identified alkaloid in the extract was protopine. This study aims to determine which component(s) of Glaucium flavum root extract might possess potent antitumor activity on human cancer cells. Quantitative estimation of Glaucium flavum alkaloids was realized by HPLC-DAD. Glaucium flavum effect on human normal and cancer cell viability was determined using WST-1 assay. Quantification of alkaloids in Glaucium flavum revealed that the dried root part contained 0.84% of protopine and 0.07% of bocconoline (w/w), while the dried aerial part contained only 0.08% of protopine, glaucine as the main alkaloid, and no bocconoline. In vitro evaluation of the growth inhibitory activity on breast cancer and normal cells demonstrated that purified protopine did not reproduce the full cytotoxic activity of the alkaloid root extract on cancer cell lines. On the other hand, bocconoline inhibited strongly the viability of cancer cells with an IC50 of 7.8 µM and only a low cytotoxic effect was observed against normal human cells. Our results showed for the first time that protopine is the major root alkaloid of Glaucium flavum. Finally, we are the first to demonstrate a specific anticancer effect of Glaucium flavum root extract against breast cancer cells, which can be attributed, at least in part, to bocconoline.

Association of mustard oil as cooking media with carcinoma of the gallbladder.

PURPOSE: Carcinoma of the gallbladder (CaGB) is a common health problem in Northern India. Exact causative factors are still obscure. Dietary habits are also known to be a major factor in the gallbladder carcinogenesis. Mustard oil is mostly used as cooking media, which is adulterated by sanguinarine, diethylnitrosamine and repeated frying. We tried to find out the association of mustard oil as cooking media with CaGB. METHODS: Twenty patients each of CaGB (group I) and cholelithiasis (group II) were included in the study. Sanguinarine and diethylnitrosamine (DEN) were extracted from the tissue and blood samples from both groups. Mean and standard error of mean of the concentration of the sanguinarine and DEN were calculated. Mann-Whitney U test was applied to test the level of significance between the two groups. RESULTS: The mean concentration of tissue sanguinarine in both groups (I and II) was 195.18 ng/mg and 24.05 ng/mg, respectively, and the difference was statistically highly significant (p < 0.001). The estimated concentration of blood sanguinarine was 230.96 ng/mL and 14.0 ng/mL in group I and II, respectively, and the difference was statistically highly significant (p < 0.001). The concentration of DEN in the tissue sample was 38.08 ng/mg in CaGB and 2.51 ng/mg in cholelithiasis patient, and these values were statistically highly significant (p < 0.001). Similarly, blood DEN concentration was 119.05 ng/mL and 4.22 ng/mL in group I and II, respectively, and the difference was statistically highly significant (p < 0.001). CONCLUSION: There is an increase in concentration of sanguinarine and diethylnitrosamine in CaGB blood and tissue in comparison to the cholelithiasis group suggesting an association with carcinoma of the gallbladder.

Establishment of a sanguinarine-producing cell suspension culture of Argemone mexicana L (Papaveraceae): induction of alkaloid accumulation.

A protocol for the induction of a cell suspension culture of Argemone mexicana is described. This suspension has been kept for over 3 years producing sanguinarine, a benzophenanthridine-type alkaloid. Sanguinarine levels can be increased by exposing these cultures to yeast or fungal elicitation.

[Simultaneous determination of four alkaloids in Corydalis decumbens (Thunb.) Pers. by high performance liquid chromatography-tandem mass spectrometry].

A method for the analysis of 4 alkaloids in Corydalis decumbens (Thunb.) Pers. was developed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-MS/MS). The sample was extracted in methanol by ultrasonic, filtered and diluted with methanol for further analysis. The analysis was performed on a C18 column (150 mm x 2.1 mm, 3.5 microm) using a gradient elution program with the mobile phase of 0.2% acetic acid solution and acetonitrile. The analyte was determined by an electrospray ionization tandem mass spectrometry in multiple reactions monitoring (MRM) mode. The qualitative and quantitative analyses were based on the retention times and characteristic ion pairs consisting of one parent ion and two fragment ions of the analyte. The limits of detection (LODs) for 4 alkaloids were in the range of 0.02 - 0.2 microg/L, and the limits of quantification (LOQs) were in the range of 0.07 - 0.66 microg/L. The average recoveries were in the range of 93.6% - 103.5% for 4 alkaloids with the relative standard deviations below 3.8%. This method is reliable, sensitive and reproducible, and it can be used for the quality control of Corydalis decumbens (Thunb.) Pers. sample.

Identification of benzo[c]phenanthridine metabolites in human hepatocytes by liquid chromatography with electrospray ion-trap and quadrupole time-of-flight mass spectrometry.

The metabolism of the benzo[c]phenanthridine alkaloids was studied using human hepatocytes which are an excellent model system for biotransformation studies. For analysis of the alkaloids and their metabolites, an electrospray quadrupole ion-trap mass spectrometry (ESI ion-trap MS) connected to a reversed phase chromatographic system based on cyanopropyl modified silica was used. The optimized experimental protocol allowed simultaneous analysis of the alkaloids and their metabolites and enabled study of their uptake into and interconversion in human hepatocytes. The results show that formation of the dihydro metabolite which may be followed by specific O-demethylenation/O-demethylation processes, is probably the main route of biotransformation (detoxification) of the benzo[c]phenanthridines in human hepatocytes. The structure of the main O-demethyl metabolite (2-methoxy-12-methyl-12,13-dihydro-[1,3]dioxolo[4',5':4,5]benzo[1,2-c]phenanthridin-1-ol; 336.1 m/z,) was proposed by the multi-stage MS and quadrupole time-of-flight MS methods using chemically synthesized standard.