Molecular Identification of Endophytic Bacteria in Leucojum aestivum In Vitro Culture, NMR-Based Metabolomics Study and LC-MS Analysis Leading to Potential Amaryllidaceae Alkaloid Production.

In this study, endophytic bacteria belonging to the Bacillus genus were isolated from in vitro bulblets of Leucojum aestivum and their ability to produce Amaryllidaceae alkaloids was studied. Proton Nuclear Magnetic Resonance (1H NMR)-based metabolomics combined with multivariate data analysis was chosen to compare the metabolism of this plant (in vivo bulbs, in vitro bulblets) with those of the endophytic bacteria community. Primary metabolites were quantified by quantitative 1H NMR (qNMR) method. The results showed that tyrosine, one precursor of the Amaryllidaceae alkaloid biosynthesis pathway, was higher in endophytic extract compared to plant extract. In total, 22 compounds were identified including five molecules common to plant and endophyte extracts (tyrosine, isoleucine, valine, fatty acids and tyramine). In addition, endophytic extracts were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) for the identification of compounds in very low concentrations. Five Amaryllidaceae alkaloids were detected in the extracts of endophytic bacteria. Lycorine, previously detected by 1H NMR, was confirmed with LC-MS analysis. Tazettine, pseudolycorine, acetylpseudolycorine, 1,2-dihydro-chlidanthine were also identified by LC-MS using the positive ionization mode or by GC-MS. In addition, 11 primary metabolites were identified in the endophytic extracts such as tyramine, which was obtained by decarboxylation of tyrosine. Thus, Bacillus sp. isolated from L. aestivum bulblets synthesized some primary and specialized metabolites in common with the L.aestivum plant. These endophytic bacteria are an interesting new approach for producing the Amaryllidaceae alkaloid such as lycorine.

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.

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.

Kinetic study of the rearrangement of deuterium-labeled 4'-O-methylnorbelladine in Leucojum aestivum shoot cultures by mass spectrometry. Influence of precursor feeding on amaryllidaceae alkaloid accumulation.

Alkaloids from plants of the family Amaryllidaceae have important pharmacological properties and can be regarded as derivatives of the common precursor 4'-O-methylnorbelladine (6) via intramolecular oxidative phenol coupling. Their biosynthetic pathway, particularly in Leucojum aestivum, has not yet been totally elucidated. Therefore, shoot cultures of this plant were subcultured in medium containing the labeled precursor 4'-O-methyl-d(3)-norbelladine (3) at various concentrations (0.05, 0.10, and 0.20 g/L) and were incubated for various periods of time (15, 30, and 40 days). The aim of this work was to study the influence of this precursor on both labeled and native alkaloid accumulation. Biotransformation into galanthamine (1) and lycorine (2) in shoot cultures was demonstrated using HPLC coupled to mass spectrometry. A maximal amount of 0.16% of 1 referred to the dry weight was obtained at day 15 in shoots fed with 0.10 g/L of precursor. In addition, a 20.5% dry weight of 2 was reached after 40 days of feeding with 0.20 g/L of precursor.

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.

LCMS and GCMS for the screening of alkaloids in natural and in vitro extracts of Leucojum aestivum.

HPLC coupled to a mass spectrometer (MS) was used for the analysis of galanthamine and lycorine in natural extracts of Leucojum aestivum and in their in vitro cultures grown with a precursor (ACC), inhibitors (AgNO(3), STS), or an absorber (KMnO(4)) of ethylene. The maximum galanthamine (0.002%) and lycorine (0.02%) concentrations in tissue cultures were obtained in the presence of KMnO(4). GCMS was used to investigate underivatized alkaloid mixtures from L. aestivum. Seven alkaloids were identified in in vivo bulbs. KMnO(4) led to the highest diversity of alkaloids in tissue culture extracts.

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.

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 electrochemiluminescence determination of galanthamine, homolycorine, lycorenine, and tazettine in Lycoris radiata by capillary electrophoresis with ultrasonic-assisted extraction.

After ultrasonic-assisted extraction, four lycoris radiata alkaloids: galanthamine, homolycorine, lycorenine, and tazettine were determined by capillary electrophoresis electrochemiluminescence. Polyvinylpyrrolidone was added to the running buffer (RB) to obtain better resolution. Experimental conditions influencing the determination were examined, including the additives, detection potential, separation voltage, injection voltage and time, and RB pH and concentration. Under optimal experimental conditions, the baseline separation of the four alkaloids occurred within 16min. The proposed method displayed the following linear ranges (in ng/mL): galanthamine [60-5000], homolycorine [40-5000], lycorenine [5.0-1500], and tazettine [8.0-2500]. The detection limits in ng/mL, (S/N=3), were galanthamine [14], homolycorine [11], lycorenine [1.8], and tazettine [3.1]. Intra-day and inter-day RSDs for the four alkaloids of the six replicates were less than 2.7% and 3.1%, respectively. The recoveries in% were: tazettine [102.5], lycorenine [98.20], galanthamine [97.30], and homolycorine [98.33].

Analysis of Amaryllidaceae alkaloids from Narcissus by GC-MS and capillary electrophoresis.

Amaryllidaceae are known as ornamental plants, furthermore some species of this family contain galanthamine, an acetylcholinesterase inhibitor approved for the treatment of Alzheimer's disease, and other alkaloids with interesting pharmacological activity. In the present work, the quali- and quantitative analysis of Amaryllidaceae-type alkaloids in the bulbs of Narcissus species is presented using different analytical approaches. Extracts of Narcissus pseudonarcissus cv. Carlton and Narcissus jonquilla Quail, were first examined by GC-MS using a Rtx-5 MS (programmed temperature) and the major alkaloids were identified. Together with galanthamine, high contents of haemanthamine, were found. Galanthamine was reliably quantified by GC-MS, whereas haemanthamine partly decomposed under the GC conditions, thus alternative analytical methods were investigated. Firstly, reversed-phase HPLC-ESI-MS was applied to identify and isolate at semipreparative levels haemanthamine. The compound was fully characterized by MS/MS and (1)H NMR and then used as a reference substance. The quantitation of both galanthamine and haemanthamine was then accomplished by capillary electrophoresis with spectrophotometric detection. A non-aqueous (NACE) approach was selected in order to use a running buffer fully compatible with samples in organic solvent. In particular, a mixture methanol-acetonitrile (75:25, v/v) containing ammonium acetate (90 mM) was used as a background electrolyte. The same analytical sample was subjected to GC-MS and NACE analysis; the different selectivity displayed by these techniques allowed different separation profiles that can be useful in phytochemical characterization of the extracts. The GC-MS and NACE methods were validated and applied to the quantitation of galanthamine (GC-MS and NACE) and haemanthamine (NACE) in bulbs of N. jonquilla.

Chemical evaluation and anticholinesterase activity of Hippeastrum puniceum (Lam. ) Kuntz bulbs (Amaryllidaceae)

Hippeastrum puniceum is a species that belongs to the Amaryllidaceae family. A particular characteristic of this family is the consistent and very specific presence of isoquinoline alkaloids, which have demonstrated a wide range of biological activities such as antioxidant, antiviral, antifungal, antiparasitic, and acetylcholinesterase inhibitory activity, among others. In the present work, fifteen alkaloids were identified from the bulbs of Hippeastrum puniceum (Lam.) Kuntz using a GC-MS approach. The alkaloids 9-O-demethyllycoramine, 9-demethyl-2α-hydroxyhomolycorine, lycorine and tazettine were isolated through chromatographic techniques. The typical Amaryllidaceae alkaloids lycorine and tazettine, along with the crude and ethyl acetate extract from bulbs of the species were evaluated for their inhibitory potential on α-amylase, α-glucosidase, tyrosinase and acetylcholinesterase activity. Although no significant inhibition activity was observed against α-amylase, α-glucosidase and tyrosinase from the tested samples, the crude and ethyl acetate extracts showed remarkable acetylcholinesterase inhibitory activity. The biological activity results that correlated to the alkaloid chemical profile by GC-MS are discussed herein. Therefore, this study contributed to the knowledge of the chemical and biological properties of Hippeastrum puniceum (Lam.) and can subsidize future studies of this species

[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.

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.

6-O-methylkrigeine, a new amaryllidaceae alkaloid from Nerine huttoniae Schönland.

A novel Amaryllidaceae alkaloid, 6-O-methylkrigeine, has been isolated from Nerine huttoniae and the configuration was elucidated by 1-D and 2-D (1)H-NMR, (13)C-NMR, HRMS and optical rotation.

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.

Analysis of Amaryllidaceae alkaloids from Zephyranthes robusta by GC-MS and their cholinesterase activity.

From the bulbs of Zephyranthes robusta Baker (Amaryllidaceae), seven known compounds, belonging to four structural types of Amaryllidaceae alkaloids, were identified and quantified by GC-MS. The alkaloid extract from the bulbs showed promising acetylcholinesterase and butyrylcholinesterase inhibitory activities against HuAChE (IC50 = 35.9 +/- 3.5 microg/mL) and HuBuChE (IC50 = 190.9 +/- 8.2 microg/mL).

New method for the study of Amaryllidaceae alkaloid biosynthesis using biotransformation of deuterium-labeled precursor in tissue cultures.

Biotransformation of deuterated-4'-O-methylnorbelladine into alkaloids galanthamine and lycorine in tissue cultures of Leucojum aestivum was demonstrated using HPLC coupled to mass spectrometry. GC-MS screening was also carried to investigate other native and deuterated alkaloids. A total of six labeled alkaloids were identified indicating that 4'-O-methyl-d(3)-norbelladine is incorporated into three different groups of Amaryllidaceae alkaloids that are biosynthesized by three modes of intramolecular oxidative phenol coupling.

Highly efficient, selective and sensitive molecular screening of acetylcholinesterase inhibitors of natural origin by solid-phase extraction-liquid chromatography/electrospray ionisation-octopole-orthogonal acceleration time-of-flight-mass spectrometry and novel thin-layer chromatography-based bioautography.

Highly efficient, selective and sensitive molecular screening of natural acetylcholinesterase (AChE) inhibitors was developed and comprised optimized pressurized liquid extraction (PLE) of plant materials followed by highly selective solid-phase extraction (SPE) using Oasis HLB cartridges. Pure alkaloidal fractions were analyzed by a newly developed high-performance liquid-chromatography (HPLC) on a 3 microm Atlantis HILIC silica stationary phase combined with recently introduced electrospray ionisation (ESI) octopole-orthogonal acceleration time-of-flight (oa TOF)-mass spectrometry (MS) with high mass accuracy (about 2 ppm) and high sensitivity (absolute limit of detection (LOD) for galanthamine was about 43 fg at signal-to-noise 13:1). Moreover, a newly developed and validated TLC-bioautography permit galanthamine sensitivities at pg levels. In this way, more potent than galanthamine AChE inhibitor namely 1,2-dihydrogalanthamine in Narcissus jonquilla 'Pipit' extract could be found (with IC(50) value 0.19 microM lower of about 42% than that of galanthamine).