Alkaloids Analysis of Habranthus cardenasianus (Amaryllidaceae), Anti-Cholinesterase Activity and Biomass Production by Propagation Strategies.

Plants in the Amaryllidaceae family synthesize a diversity of bioactive alkaloids. Some of these plant species are not abundant and have a low natural multiplication rate. The aims of this work were the alkaloids analysis of a Habranthus cardenasianus bulbs extract, the evaluation of its inhibitory activity against cholinesterases, and to test several propagation strategies for biomass production. Eleven compounds were characterized by GC-MS in the alkaloid extract, which showed a relatively high proportion of tazettine. The known alkaloids tazettine, haemanthamine, and the epimer mixture haemanthidine/6-epi-haemanthidine were isolated and identified by spectroscopic methods. Inhibitory cholinesterases activity was not detected. Three forms of propagation were performed: bulb propagation from seed, cut-induced bulb division, and micropropagated bulbs. Finally, different imbibition and post-collection times were evaluated in seed germination assays. The best propagation method was cut-induced bulb division with longitudinal cuts into quarters (T1) while the best conditions for seed germination were 0-day of post-collection and two days of imbibition. The alkaloids analyses of the H. cardenasianus bulbs showed that they are a source of anti-tumoral alkaloids, especially pretazettine (tazettine) and T1 is a sustainable strategy for its propagation and domestication to produce bioactive alkaloids.

Pseudolycorine N-oxide, a new N-oxide from Narcissus tazetta.

A new N-oxide, Pseudolycorine N-oxide (1) was characterised along with eleven known alkaloids homolycorine (2), O-methylmaritidine (3), 8-O-demethylhomolycorine (4), homolycorine N-oxide (5), lycorine (6), narciclasine (7), pseudolycorine (8), ungeremine (9), 8-O-demethylmaritidine (10), zefbetaine (11) and lycorine N-oxide (12), from Narcissus tazetta. Their structures were established on the basis of spectroscopic data analysis. The extract, fractions and isolated compounds were screened for in vitro cytotoxicity against two human cancer cell lines, human cervical cancer (SiHa) and human epidermoid carcinoma (KB) cells. The study demonstrated the cytotoxic potential of extract and its chloroform and n-butanol fractions. Further, the results revealed the bioactive potential of narciclasine, pseudolycorine and homolycorine alkaloids. However, new N-oxide (1) was not active against these cell lines.

The individual components of commercial isometamidium do not possess stronger trypanocidal activity than the mixture, nor bypass isometamidium resistance.

The four components present in the trypanocidal treatment Samorin, the commercially available formulation of isometamidium, were separated and purified by column chromatography. These compounds as well as the Samorin mixture and the other phenanthridine trypanocide, homidium, were tested on Trypanosoma congolense and wild type, diamidine- and isometamidium-resistant Trypanosoma brucei brucei strains using an Alamar blue drug sensitivity assay. EC50 values obtained suggest that M&B4180A (2) was the most active of the components, followed by M&B38897 (1) in all the strains tested, whereas M&B4596 (4) was inactive. Samorin was found to be significantly more active than any of the individual components alone, against T. congolense and all three T. b, brucei strains. Samorin and all its active constituents displayed reduced activity against the previously characterised isometamidium-resistant strain ISMR1.

Drug quality analysis of isometamidium chloride hydrochloride and diminazene diaceturate used for the treatment of African animal trypanosomosis in West Africa.

BACKGROUND: Diminazene diaceturate (DA) and isometamidium chloride hydrochloride (ISM) are with homidium bromide, the main molecules used to treat African Animal Trypanosomosis (AAT). These drugs can be purchased from official suppliers but also from unofficial sources like local food markets or street vendors. The sub-standard quality of some of these trypanocides is jeopardizing the efficacy of treatment of sick livestock, leading thus to economic losses for the low-resource farmers and is contributing to the emergence and spread of drug resistance. The objective of this study was to assess the quality of trypanocidal drugs sold in French speaking countries of West Africa. In total, 308 drug samples including 282 of DA and 26 of ISM were purchased from official and unofficial sources in Benin, Burkina Faso, Côte d'Ivoire, Mali, Niger and Togo. All samples were analysed at LACOMEV (Dakar, Senegal), a reference laboratory of the World Organisation for Animal Health, by galenic inspection and high performance liquid chromatography. RESULTS: The results showed that 51.90% of the samples were non-compliant compared to the standards and were containing lower quantity of the active ingredient compared to the indications on the packaging. The non-compliances ranged from 63.27% in Togo to 32.65% in Burkina Faso (61.82% in Benin, 53.84% in Mali, 50% in Côte d'Ivoire, 47.36% in Niger). The rates of non-compliance were not statistically different (P = 0.572) from official or unofficial suppliers and ranged from 30 to 75% and from 0 to 65% respectively. However, the non-compliance was significantly higher for ISM compared to DA (P = 0.028). CONCLUSIONS: The high non-compliance revealed in this study compromises the efficacy of therapeutic strategies against AAT, and is likely to exacerbate chemoresistance in West Africa. Corrective actions against sub-standard trypanocides urgently need to be taken by policy makers and control authorities.

Green techniques in comparison to conventional ones in the extraction of Amaryllidaceae alkaloids: Best solvents selection and parameters optimization.

An undisputed trend in sample preparation at present is to meet the requirements of green chemistry especially in the field of natural products. Green technology continuously pursues new solvents to replace common organic solvents that possess inherent toxicity. Over the past two decades, non-ionic surfactants have gained enormous attention from the scientific community. The micelle-mediated extraction and cloud-point preconcentration (CPE) methods offer a convenient alternative to the conventional extraction systems. Recently, natural deep eutectic solvents (NDESs) have emerged as green and sustainable solvents for efficient extraction of bioactive compounds or drugs. They are generally composed of neutral, acidic or basic compounds that form liquids of high viscosity when mixed in certain molar ratio. The presented work aimed to comprehensively compare and evaluate the potential and effectiveness of NDES as well as non-ionic surfactants (Genapol X-080, Triton X-100 and Triton X-114) for extraction of Amaryllidaceae alkaloids from Crinum powellii bulbs as representative example of plant material, in comparison to the conventional solvents (methanol, ethanol and water).A new validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous quantitation of three alkaloids markers, lycorine, crinine and crinamine, in the bulbs of C. powellii. Extraction efficiency of the targeted alkaloids from the bulb matrix with organic and ecofriendly (green) solvents were studied. Results revealed that NDES and surfactants were significantly more efficient in alkaloid extraction than previous methods requiring the consumption of organic solvents and water. Genapol X-80 demonstrated 138%, 149% and 145%, while choline chloride: fructose (5:2): H2O (35%) NDES mixture demonstrated 243%, 225% and 238% of the total alkaloidal extraction capacity of ethanol, methanol and water, respectively at 50 °C for extraction time 1 h using ultrasonication for all experiments. Furthermore, Box-Behnken response surface design combined with the overall desirability value were successfully employed to optimize and study the individual and interactive effect of process variables such as extraction temperature, time and surfactant %, for Genapol X-80, and sonication extraction temperature, time and water concentration, for choline chloride: fructose: H2O NDES mixture, on the alkaloidal yield from C. powellii. It was evident that parameters interacting together can act in synergism if adjusted properly according to the optimized conditions to obtain maximum alkaloids extractability. It is for the first time that the efficiency of micelle-mediated extraction has been compared to that of natural deep eutectic solvents for the extraction of alkaloids and the results thoroughly discussed.

[Simultaneous determination of diminazene aceturate and isometamidium chloride residues in cattle tissues by high performance liquid chromatography with solid-phase extraction].

A method for the simultaneous determination of trypanocidal diminazene aceturate (DIM) and isometamidium chloride (ISM) that containing benzamidine groups in cattle tissues was developed by high performance liquid chromatography (HPLC) with solid-phase extraction (SPE). The tissue samples were extracted with different proportions of water-acetonitrile, then were cleaned up by Oasis WCX cartridges. DIM and ISM were separated by HPLC with a Spherisorb CN column (250 mm×4.6 mm, 5 µm). Acetonitrile-0.05 mol/L ammonium formate solution (pH 2.4) was used as mobile phases with gradient elution. The detection wavelength of UV was set at 380 nm. The limits of detection (LODs) and the limits of quantification (LOQs) of DIM and ISM in cattle tissues were 0.01 mg/kg and 0.025 mg/kg, respectively. The correlation coefficients (r) of DIM and ISM in cattle tissues were not less than 0.9993. The average recoveries of DIM and ISM at three spiked levels were 82.2%-97.6% with the intra-day relative standard derivations (RSDs) of 0.3%-5.2% (n=5) and inter-day RSDs of 1.3%-5.2% (n=15). The method was successfully applied to the analysis of DIM and ISM in cattle tissues. The method is rapid, sensitive and repeatable for the determination of diminazene aceturate and isometamidium chloride in cattle tissues.

[Application of Coagulant in the Analysis of Lycorine and Galanthamine in Processed Foods].

Analytical method by HPLC and LC-MS/MS for determining lycorine and galanthamine in processed food was newly developed. In this method, coagulant which has never been used in food analysis was applied on cleanup process. With coagulant approach, removal of interfering substances on determination for analytes was easily achieved. The method using HPLC showed recovery of 95.4-102.9% on both analytes with repeatability of less than 2.9% and reproducibility of less than 2.9%. The method using LC-MS/MS showed recovery of 97.4-107.6% with repeatability of less than 5.7% and reproducibility of less than 5.7%. On HPLC method, limit of quantification for lycorine was 0.004 g/kg and that of galanthamine was 0.006 g/kg. On LC-MS/MS method, limit of quantification for lycorine was 0.0008 g/kg and that of galanthamine was 0.0005 g/kg.

Chromatographic and spectroscopic analysis of the components present in the phenanthridinium trypanocidal agent isometamidium.

The chromatographic isolation and characterisation of the four compounds present in the quaternary phenanthridine veterinary trypanocidal agent, isometamidium chloride hydrochloride (ISM), is reported. The isolated compounds were unambiguously characterised using spectroscopic (NMR, UV, IR and MS) methods as 3-amino-8-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium (1a) and related isomers, 8-amino-3-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium, 3,-8-diamino-7-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium and 3,-8-bis[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium. During the course of this study, it was realised that the nature of the solvent used in the NMR study was critical as in DMSO-d6 the quaternary group in the compounds was reduced to dihydro forms (e.g. 2a).

Use of potentiometric fluorophores in the measurement of mitochondrial reactive oxygen species.

Mitochondrial reactive oxygen species (ROS) are implicated in signal transduction, inflammation, neurodegenerative disorders, and normal aging. Net ROS release by isolated brain mitochondria derived from a mixture of neurons and glia is readily quantified using fluorescent dyes. Measuring intracellular ROS in intact neurons or glia and assigning the origin to mitochondria are far more difficult. In recent years, the proton-motive force crucial to mitochondrial function has been exploited to target a variety of compounds to the highly negative mitochondrial matrix using the lipophilic triphenylphosphonium cation (TPP(+)) as a "delivery" conjugate. Among these, MitoSOX Red, also called mito-hydroethidine or mito-dihydroethidium, is prevalently used for mitochondrial ROS estimation. Although the TPP(+) moiety of MitoSOX enables the manyfold accumulation of ROS-sensitive hydroethidine in the mitochondrial matrix, the membrane potential sensitivity conferred by TPP(+) creates a daunting set of challenges not often considered in the application of this dye. This chapter provides recommendations and cautionary notes on the use of potentiometric fluorescent indicators for the approximation of mitochondrial ROS in live neurons, with principles that can be extrapolated to nonneuronal cell types. It is concluded that mitochondrial membrane potential changes render accurate estimation of mitochondrial ROS using MitoSOX difficult to impossible. Consequently, knowledge of mitochondrial membrane potential is essential to the application of potentiometric fluorophores for the measurement of intramitochondrial ROS.

Quantititative determination of lycorine and galanthamine in Galanthus trojanus and G. cilicicus by HPLC-DAD.

Lycorine and galanthamine have various biological activities. A reliable HPLC method coupled with DAD detection was developed and validated for the determination of galanthamine and lycorine in Galanthus trojanus and G. cilicicus. A simple method for the extraction of the alkaloids in low-mass plant samples was employed utilizing columns pre-packed with diatomaceous earth (Extrelut). This method was applied to the aerial parts and bulbs of G. trojanus and G. cilicicus (Amaryllidaceae) collected during the flowering season. The chromatographic separation was performed using an isocratic system with a mobile phase of trifluoroacetic acid-water-acetonitrile (0.01:92.5:7.5) applied at a flow rate of 1 mL min(-1) and using a diode array detector. Validation procedures showed that the method was specific, accurate and precise. The highest amount of lycorine (0.012%) was detected in the bulbs of G. trojanus collected from Can (Canakkale), whereas the aerial parts of this species collected from Bayramiç (Canakkale) was not found to contain this alkaloid. In G. cilicicus samples, lycorine was only determined in the bulbs, giving yields of 0.004%; galanthamine yields were between 0.015-0.016%, but none of the G. trojanus samples contained this latter alkaloid.

Simultaneous determination of galanthamine and lycorine in Lycoris radiata by a capillary electrophoresis with an electrochemiluminescence method.

A novel capillary electrophoresis with electrochemiluminescence determination method was developed for the determination of two alkaloids based on the electrochemiluminescence signal enhancement effect of the tertiary amine group on tris(2,2'-bipyridyl)ruthenium(II). A linear relationship between the electrochemiluminescence peak area and concentrations of galanthamine and lycorine in the range of 0.07 ∼ 17 µg/mL and 0.07 ∼ 18 µg/mL was obtained and the detection limit was 0.008 and 0.002 µg/mL, respectively. The method is selective, simple, and convenient. It had been successfully applied to the analysis of galanthamine and lycorine in Lycoris radiata samples purchased from a local market.

Comparative analysis of lycorine in wild plant and callus culture samples of Hymenocallis littoralis by HPLC-UV method.

The Hymenocallis littoralis, an ornamental and medicinal plant, had been traditionally used for wound healing. In the present study, an analytical method using HPLC with ultraviolet detection was developed for the quantification of lycorine in the extracts of different parts of wild plant and tissue culture samples of H. littoralis. The separation was achieved using a reversed-phase column. The method was found to be accurate, repeatable, and sensitive for the quantification of minute amount of lycorine present in the samples. The highest lycorine content was found in the bulb extract (2.54 ± 0.02 µg/mg) whereas the least was in the root extract (0.71 ± 0.02 µg/mg) of the wild plants. Few callus culture samples had high content of lycorine, comparable to that of wild plants. The results showed that plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) alone at 4.5 µM (2.58 ± 0.38 µg/mg) or a combination of 2,4-D at 9.00 µM with 4.5 µM of 6-benzylaminopurine (BAP), were the optimum concentrations for the production of high lycorine (2.45 ± 0.15 µg/mg) content in callus culture. The present analytical method could be of value for routine quantification of lycorine in the tissue culture production and standardization of the raw material or extracts of H. littoralis.

Chromatographic retention behaviour, modelling and separation optimisation of the quaternary ammonium salt isometamidium chloride and related compounds on a range of reversed-phase liquid chromatographic stationary phases.

This paper describes the reversed-phase liquid chromatographic behaviour of the trypanocidal quaternary ammonium salt isometamidium chloride and its related compounds on a range of liquid chromatographic phases possessing alkyl and phenyl ligands on the same inert silica. In a parallel study with various extended polar selectivity phases which possessed different hydrophobic/silanophilic (hydrogen bonding) activity ratios, the chromatographic retention/selectivities of the quaternary ammonium salts was shown to be due to a co-operative mechanism between hydrophobic and silanophilic interactions. The highly aromatic and planar isometamidium compounds were found to be substantially retained on stationary phases containing aromatic functionality via strong π-π interactions. The chemometric approach of principal component analysis was used to characterise the chromatographic behaviour of the isometamidium compounds on the differing phases and to help identify the dominant retention mechanism(s). Two-dimensional (temperature/gradient) retention modelling was employed to develop and optimise a rapid liquid chromatography method for the separation of the six quaternary ammonium salts within 2.5 min which would be suitable for bioanalysis using liquid chromatography-mass spectrometry. This is the first reported systematic study of the relationship between stationary phase chemistries and retention/selectivity for a group of quaternary ammonium salts.

Overexpression of SOD in retina: need for increase in H2O2-detoxifying enzyme in same cellular compartment.

In retinitis pigmentosa (RP), various mutations cause rod photoreceptor cell death leading to increased oxygen levels in the outer retina, progressive oxidative damage to cones, and gradual loss of cone cell function. We have been exploring the potential of overexpressing components of the endogenous antioxidant defense system to preserve cone cell function in rd10(+/+) mice, a model of RP. rd10(+/+) mice deficient in superoxide dismutase 1 (SOD1) showed increased levels of superoxide radicals and carbonyl adducts (a marker of oxidative damage) in the retina and more rapid loss of cone function than rd10(+/+) mice with normal levels of SOD1. This suggests that SOD1 is an important component of the antioxidant defense system of cones, but increased expression of SOD1 in rd10(+/+) mice increased oxidative damage and accelerated the loss of cone function. Coexpression of SOD1 with glutathione peroxidase 4 (Gpx4), which like SOD1 is localized in the cytoplasm, but not with catalase targeted to the mitochondria, reduced oxidative damage in the retina and significantly slowed the loss of cone cell function in rd10(+/+) mice. Gene transfer resulting in increased expression of SOD2, but not coexpression of SOD2 and mitochondrial Gpx4, resulted in high levels of H(2)O(2) in the retina. These data suggest that to provide benefit in RP, overexpression of an SOD must be combined with expression of a peroxide-detoxifying enzyme in the same cellular compartment.

Determination of pseudolycorine in the bulb of lycoris radiata by capillary electrophoresis combined with online electrochemiluminescence using ultrasonic-assisted extraction.

A novel method for determination of pseudolycorine in the bulb of lycoris radiata by capillary electrophoresis coupled with online electrochemiluminescence detection with ultrasonic-assisted extraction has been developed. The effects of several factors such as detection potential, concentration and pH of phosphate buffer, separation voltage, injection time, ultrasonic power and extraction time were investigated. Under optimal conditions, the linear concentration range for pseudolycorine was 0.002-2 µg/mL with a correlation coefficient of 0.9991. Relative standard deviations of migration time and peak areas were 1.4 and 3.2%, respectively. The limit of detection (S/N=3) was 0.46 ng/mL. The proposed method can be successfully applied to the determination of pseudolycorine in the bulb of lycoris radiata.

Spectrofluorometry assays for oxidative stress and apoptosis, with cell viability on the same microplates: a multiparametric analysis and quality control.

Our objective was to compare neutral red (NR) tests performed separately and on the same microplates as hydroethidine, H2DCF-DA and YO-PRO®-1 assays, and to compare toxicity ratios calculated with the two methods. Human trabecular meshwork cells (HTM-3) were exposed to different concentrations of benzalkonium chloride (BAC), to PBS and to diluted solutions of latanoprost and bimatoprost. Microplate fluorescence spectrophotometry assays were performed: NR uptake for cell viability evaluation, hydroethidine and H2DCF-DA for reactive oxygen species (ROS), and YO-PRO®-1 for apoptosis. The four NR assays presented identical toxicological profiles and correlation coefficients between them were close to one. There was no difference between toxicity ratios for ROS assays calculated by the two methods, with high correlation coefficients. However, in the apoptosis assay, ratios calculated with the double staining technique were more accurate. Thus, it is possible and recommended to perform NR assays on the same microplates as the other assays. This double staining procedure could constitute a quality control procedure and would allow multiparametric analysis.

Determination of isometamidium residues in animal-derived foods by liquid chromatography-tandem mass spectrometry.

In this paper, a new determination method for isometamidium residues in animal-derived foods was developed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Isometamidium residues in bovine tissues and milk were extracted with the mixed solution of acetonitrile and 0.25 mol/L of ammonium formate-methanol (v/v, 1:1), concentrated and degreased, and determined by LC-MS/MS with quantification by external standard method. The results showed that the peak area of chromatogram was linearly related to the concentration of isometamidium in the range of 1-100 µg/L, and the limits of detection (LOD) and quantification (LOQ) were 0.05 µg/kg and 5 µg/kg, respectively. The average recoveries of spiked samples were in the range of 73.8-93.9% with relative standard deviations ranged from 2.3% to 7.5%. This method is simple, accurate and suitable for the identification and quantification for isometamidium in animal-derived foods.

Anti-inflammatory activity of Crinum asiaticum Linne var. japonicum extract and its application as a cosmeceutical ingredient.

Crinum asiaticum Linne var. japonicum has long been used as a rheumatic remedy, as an anti-pyretic and as an anti-ulcer treatment, and for the alleviation of local pain and fever in Korea and Malaysia. In order to investigate the possibility of Crinum asiaticum Linne var. japonicum extract as a cosmetic ingredient, we measured its anti-inflammatory effect by its inhibition of iNOS (inducible nitric oxide synthase) and the release of PGE2, IL-6, and IL-8. We also measured its anti-allergic effect by its inhibition of beta-hexosamidase release. An HPLC experiment after extraction with 95% EtOH at pH 3.5 showed that Crinum asiaticum Linne var. japonicum was mainly composed of lycorine (up to 1%), a well-known immunosuppressor. The content of lycorine varied, depending on the type of plant tissue analyzed and the extraction method. In an anti-inflammatory assay for inhibition of nitric oxide formation on lipopolysaccharide (LPS)-activated mouse macrophage RAW 264.7 cells, the ethanol extract of Crinum asiaticum showed an inhibitory activity of NO production in a dose-dependent manner (IC50 = 58.5 microg/ml). Additional study by RT-PCR demonstrated that the extract of Crinum asiaticum significantly suppressed the expression of the iNOS gene. Moreover, the extract of Crinum asiaticum did not show any cytotoxicity, but did show a cell proliferation effect against LPS (a 10 approximately 60% increase in cell viability). In an assay to determine inhibition of the H2O2-activated release of PGE2, IL-6, and IL-8 in human normal fibroblast cell lines, the release of PGE2 and IL-6 was almost completely inhibited above concentrations of 0.05% and 1%, respectively. Moreover, the release of IL-8 was completely inhibited over the entire range of concentration (>0.0025%). In order to investigate the skin-sensitizing potentials of the extract of Crinum asiaticum, a human clinical test was performed after repeated epicutaneous 48-h applications under an occlusive patch (RIPT). The repeated and single cutaneous applications of Crinum asiaticum Linne var. japonicum extract under the occlusive patch did not provoke any cumulative irritation and sensitization reactions. The result showed that the extract of Crinum asiaticum Linne var. japonicum has a sufficient anti-inflammatory effect. Therefore, Crinum asiaticum Linne var. japonicum extract may be useful for development as an ingredient in cosmetic products.

How to deal with lipophilic and volatile organic substances in microtiter plate assays.

Microtiter plate-based assays are a promising technique for toxicity assessment of substances. Chemicals with physicochemical properties such as high volatility and/or high lipophilicity, however, can be lost from the exposure solution during an experiment, so that exposure concentrations are not consistent. The aim of the present study was to determine and reduce the proportion of the reference compounds phenanthrene and phenanthridine lost during exposure in the zebra fish (Danio rerio) embryo test regime. It could be shown that under the standard exposure regime (48 h), the concentration of phenanthrene decreased strongly, by more than 99%, whereas that of phenanthridine decreased by 17% during a 48-h experiment. After modifications to the microtiter plate exposure regime, the phenanthrene concentration showed a decrease of only 40%, while the phenanthridine concentration remained unchanged. The major processes of substance loss could be assigned to accumulations of these substances into the glue of commercially available adhesive foils and the polystyrene walls of the microtiter plates. Furthermore, by investigating the sorption capacity of different plastics, it was found that the phenanthrene concentration decreased less when using a plexiglass specimen (28%) compared with the same-sized polystyrene specimen (94%). Moreover, it was found, for a constant exposure regime, that concentration profiles of different phenanthrene concentrations in the microtiter plate assay during an experiment were similar. A mathematical method is proposed to predict concentration profiles in an exposure solution by scaling a determined profile.