Crude Alkaloid Extract Research Articles

Computer-Aided 13C NMR Chemical Profiling of Crude Natural Extracts without Fractionation.

A computer-aided, 13C NMR-based dereplication method is presented for the chemical profiling of natural extracts without any fractionation. An algorithm was developed in order to compare the 13C NMR chemical shifts obtained from a single routine spectrum with a set of predicted NMR data stored in a natural metabolite database. The algorithm evaluates the quality of the matching between experimental and predicted data by calculating a score function and returns the list of metabolites that are most likely to be present in the studied extract. The proof of principle of the method is demonstrated on a crude alkaloid extract obtained from the leaves of Peumus boldus, resulting in the identification of eight alkaloids, including isocorydine, rogersine, boldine, reticuline, coclaurine, laurotetanine, N-methylcoclaurine, and norisocorydine, as well as three monoterpenes, namely, p-cymene, eucalyptol, and α-terpinene. The results were compared to those obtained with other methods, either involving a fractionation step before the chemical profiling process or using mass spectrometry detection in the infusion mode or coupled to gas chromatography.

Protective effects of crude and alkaloidal extracts of Tamarindus indica against acute inflammation and nociception in rats

Objective: To investigate the anti-inflammatory and antinociceptive effects of total alkaloids extracted from the leaf of Tamarindus indica (T. indica) in rats. Methods: Acetic acid-induced pain and egg albumin-induced inflammation were used to inspect the anti-nociceptive and anti-inflammatory effects of the crude and alkaloidal extracts of T. indica at doses of 40 and 400 mg/kg, respectively. Sodium diclofenac was used as the control drug. Results: The percentage yields of crude methanol and alkaloidal extracts of T. indica were 2.85% and 0.98%, respectively. Screening of secondary metabolite of the crude extract revealed the presence of saponins, alkaloids, tannins, steroids, phenols and terpenes, while phlobatannins was not detected. The safe dose and LD50 were 400 and 750 mg/kg for crude methanol extract, respectively, while the safe dose and LD50 of alkaloidal extract were 40 and 57 mg/kg, respectively. The anti-inflammatory and antinociceptive effects of crude methanol extract and alkaloid extract of T. indica were significantly (P Conclusions: The results obtained indicated that the crude and alkaloidal extracts of the plant exhibited significant anti-inflammatory and antinociceptive activities, thus, supporting its folkloric use for the treatment of these conditions.

Preparative Separation of Lappaconitine from Aconitum leucostomum by HSCCC

This paper describes a system for the preparation of lappaconitine from Aconitum leucostmum Worosch., using HSCCC, and examines the effect of the two-immiscible solvent system (TISS) composed of chloroform- methanol-0.2 mol/L hydrochloric acid (4:1.5:2). Under the optimized condition, about 75 mg of lappaconitine, at a purity of 98% that was done by high performance liquid chromatography (HPLC), are obtained from 250 mg of the crude alkaloid extract. The compound was checked by HPLC-UV, electrospray ionization mass spectrometry (ESI-MS), 1H NMR and 13C NMR.

Isoquinoline alkaloid fractions of Fumaria officinalis: Characterization and evaluation of their antioxidant and antibacterial activities

Crude alkaloid extracts of Fumaria officinalis (Fumariaceae), used in Algerian traditional medicine for hepatobiliary diseases, were subjected to fractionation and GC–MS analysis. The total alkaloid (TA) content was 630mg/100g of dry weight. Fractionation led to three fractions: neutral (NF), acidic (AF) and basic (BF) with alkaloid levels of 300, 240 and 180mg/100g of dry weight, respectively. Eleven isoquinoline alkaloids were identified. The main component in all fractions was protopine, especially in the acidic fraction (AF) (51.75%). F. officinalis is usually classified by authors in the second chemotaxonomic group of Fumaria sp., while our results showing the presence of stylopine, especially in the basic fraction (2.08%), suggest it should be classified in the first chemotaxonomic group.The crude extract and fractions showed high antioxidant activity against the free radical DPPH (IC50 NF (15.74±0.04μg/mL)), the ABTS radical (IC50 BF (70.71±0.04μg/mL)), and the hypochlorous acid radical (IC50 NF (304.19±0.29μg/mL)), Total Antioxidant Capacity (TA (14.23±0.04mg eq ascorbic acid/g DM)), lipid peroxidation inhibition (IC50 TA (41.67±0.17μg/mL)) and ferric reducing power (FRP) (BF (3.49±0.11mg eq ascorbic acid/g DM)).Antibacterial activity was evaluated using liquid medium against Acinetobacter calcoaceticus, Propionibacterium acnes and Corynebacterium xerosis. Evaluation of minimum inhibitory concentration (MIC) showed low values, indicating an interesting antibacterial capacity. The MIC of 0.33mg/mL recorded against Propionibacterium acnes with alkaloid extracts showed the effectiveness of this plant as an antibacterial agent. The greater effectiveness of the acid fraction (AF) and the total alkaloid extract (TA) against the three strains tested was correlated to their richness in protopine with levels of 51.75% and 34.48%, respectively.

Harmal Extract Induces Apoptosis of HCT116 Human Colon Cancer Cells, Mediated by Inhibition of Nuclear Factor-κB and Activator Protein-1 Signaling Pathways and Induction of Cytoprotective Genes.

Colorectal cancer (CRC) is a major cause of morbidity and mortality, being the second most common type of cancer worldwide in both men and women. It accounts yearly for approximately 9% of all new cases of cancers. Furthermore, the current chemotherapeutic regimens seem unsatisfactory, so that exploration of novel therapeutic modalities is needed. The present study was undertaken to investigate the inhibitory effects of a crude alkaloid extract (CAERS) of a medicinal herb, Rhazya stricta, on proliferation of CRC HCT116 cells and to elucidate mechanisms of action. To achieve these aims, we utilized MTT, comet, DNA laddering and gene reporter assays, along with Western blot and RT-PCR analyses. We found that CAERS inhibited cell proliferation and induced apoptotic cell death in HCT116 cells. Hallmarks of morphological and biochemical signs of apoptosis were clearly evident. CAERS down-regulated DNA-binding and transcriptional activities of NF-κB and AP-1 proteins, while up-regulating expression of the Nrf-2 protein. It also down-regulated expression levels of the ERK MAPK, Bcl-2, cyclin D1, CDK-4, survivin and VEGF and up-regulated levels of Bax, caspase-3/7 and -9, p53, p21, Nrf-2. Markedly, it promoted mRNA expression levels of cytoprotective genes including the hemeoxygenase-1, NAD(P)H quinine oxidoreductase 1 and UDP-glucuronyltransferase. These findings indicate that CAERS exerts antiproliferative action on CRC cells through induction of apoptotic mechanisms, and suggest CAERS could be a promising agent for studying and developing novel chemotherapeutic agents aimed at novel molecular targets for the treatment of CRC.

A New Monoterpenoid Indole Alkaloid from Tabernaemontana corymbosa

Tabernaemontana corymbosa is used in folk medicine for the treatment of hypertension, sore throat, and abdominal pain by the Dais in Yunnan Province. The crude alkaloid extract of T. corymbosa were successively subjected to D-101 macroporous resin, MCI gel, and ODS column chromatography and purified via preparative HPLC to afford a new aspidospermar-type monoterpenoid indole alkaloid, named tabercarpamine K (1). The structure of 1 was elucidated using spectroscopic methods. Compared to the structure of vincamine, a hydroxyl group is substituted at C-19 and a proton, instead of a hydroxyl, is attached to C-16 in 1.

Studies on Optimization of Growth Parameters for Enhanced Production of Antibiotic Alkaloids by Isolated Marine actinomycetes

The aim of the present study is to optimize the growth conditions for improved production of alkaloids by promising marine actinomycetes isolated from marine sediments collected on Kakinada coast. The bioactive compounds were extracted from the isolated actinomycetes using organic solvents and screened for alkaloids using qualitative tests. The presence of alkaloids in the crude methanolic extract was confirmed by UV spectroscopic analysis and quantified by BCG method. The effect of pH, temperature, carbon and nitrogen sources on the growth and fermentative production of alkaloids was optimized. The strain was improved for enhanced production of alkaloids by physical and chemical mutagenesis. The antimicrobial activity of the crude alkaloid extract was determined by the well diffusion method. The isolated strain exhibited the highest growth and alkaloid production at pH 6 and temperature 30oC in 7 days. The alkaloid production was significantly increased 4.5-folds with UV treatment for 30 min and further 5.5-folds with ethidium bromide treatment (30µg/mL) for 1h. The resultant double mutant strain exhibited significantly high antimicrobial activity against S.aureus compared to other bacterial strains with MIC index less than 4. The isolated double mutant strain of actinomycetes can be a potential source of antibiotic alkaloids.

Effects of an alkaloid-rich extract from Mitragyna speciosa leaves and fluoxetine on sleep profiles, EEG spectral frequency and ethanol withdrawal symptoms in rats

BackgroundMany antidepressants are effective in alleviating ethanol withdrawal symptoms. However, most of them suppress rapid eye movement (REM) sleep. Thus, development of antidepressants without undesirable side effects would be preferable. Previously, crude alkaloid extract from Mitragyna speciosa (MS) Korth was found to produce antidepressant activities. It was hypothesized that the alkaloid extract from MS may attenuate ethanol withdrawal without REM sleep disturbance. MethodsAdult male Wistar rats implanted with electrodes over the frontal and parietal cortices were used for two separated studies. For an acute study, 10 mg/kg fluoxetine or 60 mg/kg alkaloid extract from MS were administered intragastrically. Electroencephalographic (EEG) signals were recorded for 3 h to examine sleep profiles and EEG fingerprints. Another set of animal was used for an ethanol withdrawal study. They were rendered dependent on ethanol via a modified liquid diet (MLD) containing ethanol ad libitum for 28 days. On day 29, fluoxetine (10 mg/kg) or alkaloid extract from MS (60 mg/kg) were administered 15 min before the ethanol-containing MLD was replaced with an isocaloric ethanol-free MLD to induced ethanol withdrawal symptoms. ResultsThe sleep analysis revealed that alkaloid extract from MS did not change any REM parameters which included average duration of each REM episode, total REM time, number of REM episode and REM latency whereas fluoxetine significantly suppressed all REM parameters and delayed REM latency. However, power spectral analysis revealed similar fingerprints for fluoxetine and alkaloid extract from MS characterized by decreasing powers in the slow frequency range in frontal and parietal cortical EEG. Neither treatment affected spontaneous motor activity. Finally, both alkaloid extract from MS and fluoxetine were found to significantly attenuate ethanol withdrawal-induced hyperexcitability (increases gamma activity) in both cortices and to reduce locomotor activity. ConclusionThe present study demonstrated that the alkaloid extract from MS alleviates ethanol withdrawal severity with no side effect on REM sleep. In addition, these data suggest that suppressive effects on slow frequency powers but not REM sleep may be hallmarks of effective antidepressants for ethanol withdrawal treatment.

Distribution of Alkaloides and Allelopathismof Selelected Weeds from a Post Harvest CropField

Though beneficial the weeds always pose an important biological constraint to crop productivity. The economic losses that are caused by these weeds globally costs billions of rupees every year. Allelopathic potentialities of plants are considered as an alternative natural way of weed management strategy for crop production as well as environmental benefits.Although the plants like the weeds are the great reservoirs of many new & potential phytochemicals,majority of them still remains unexplored either due to restricted habitat or because of being wild in nature.Hence the present survey was carried out taking ten (10) selected weeds of various stages of development from post harvest crop fields of Rice and Mustard in the OUAT Farm campus which have been left open for around two months. The Botanical authentication of the weeds were done by following the Texonomic literatures.They are Cardus nutans L, Cerastium fontanum Baumg., Cleome viscosa L., Euphorbia maculata L. ,Helianthus tuberosus L., Lactuca serriola L., Phyllanthus niruri L. ,Sinapsis arvensis L. ,Vernonia cinerea Scherb., Veronica arvensis L.Weeds were collected between January and February 2014 . Their crude alkaloidal contents were extracted and estimated from the dried powder of the whole plant following the standard methods (Harborne et al 1992). All plant samples indicate the presence of good amount of Alkaloid, highest being in Veronica persica Poiret.(17.35%) and lowest in Cardus nutans (3.07%). Variation of alkaloidal contents with variation in stage of development maximum reaching at the flowering stage, was indicated within the individuals of the same family. The quantity of the alkaloid was species specific.The allelopathic effect of the crude alkaloidal extract tested against the seed germination of Rice,Mung(Green gram ) and Mustard , indicates that the alkaloids are not so effective towards the growth of Rice but quite inhibitory towards the growth of Mung.This result is interpreted basing upon their chi-scquare values.

Effects of Mitragynine and a Crude Alkaloid Extract Derived from Mitragyna speciosa Korth. on Permethrin Elimination in Rats.

Detoxification and elimination of permethrin (PM) are mediated by hydrolysis via carboxylesterase (CES). Mitragyna speciosa (kratom) contains mitragynine (MG) and other bioactive alkaloids. Since PM and MG have the same catalytic site and M. speciosa is usually abused by adding other ingredients such as pyrethroid insecticides, the effects of MG and an alkaloid extract (AE) on the elimination of PM were investigated in rats. Rats were subjected to single and multiple pretreatment with MG and AE prior to receiving a single oral dose (460 mg/kg) of PM. Plasma concentrations of trans-PM and its metabolite phenoxybenzylalcohol (PBAlc) were measured. The elimination rate constant (kel) and the elimination half-life (t1/2 el) of PM were determined, as well as the metabolic ratio (PMR).A single and multiple oral pretreatment with MG and AE altered the plasma concentration-time courses of both trans-PM and PBAlc during 8–22 h, decreased the PMRs, delayed elimination of PM, but enhanced elimination of PBAlc. Results indicated that PM–MG or AE toxicokinetic interactions might have resulted from the MG and AE interfering with PM hydrolysis. The results obtained in rats suggest that in humans using kratom cocktails containing PM, there might be an increased risk of PM toxicity due to inhibition of PM metabolism and elimination.

In vitro antiplasmodial and HIV-1 neutralization activities of root and leaf extracts from Berberis holstii

The in vitro antiplasmodial activities and HIV neutralizing activities of six crude extracts from the leaves and roots of Berberis holstii were evaluated. The antiplasmodial activities were assessed against a chloroquine sensitive 3D7 strain of the malaria parasite, Plasmodium falciparum, and the HIV neutralizing activity were assessed against HBX2 and ZM53 pseudovirions. Parasite lactate dehydrogenase (pLDH) assay was used to measure parasite viability and Luciferase Reporter Gene assay was used to measure HIV neutralizing activity. The crude alkaloid extract of the roots showed the most potent anti-parasitic activity (IC50=0.17μg/mL) with a selectivity index (SI) of 701.2 while the DCM/MeOH extract also showed good activity (IC50=2.33μg/mL). The aqueous and total alkaloid extracts of the roots displayed significant anti-parasitic activity (IC50=3.64 and 3.85μg/mL, respectively). The aqueous extract of the leaves displayed good HIV neutralizing activity (IC50=5.84μg/mL) against the HXB2 pseudo virus. All the extracts tested displayed low cytotoxicity against TZM-bl cells.

Separation and Preparation of Five Cyclopamine Analogs from Rhizomes of Veratrum oxysepalum Turcz. by Two-Step High-Speed Counter-Current Chromatography

Five cyclopamine analogs including jervine, veratramine, pseudojervine, veratrosine, and verdine were isolated from the rhizomes of Veratrum oxysepalum Turcz. by an efficient two-step high-speed counter-current chromatography (HSCCC) method. In the filrst step, HSCCC, dichloromethane-methanol-water (4:3.5:2, v/v/v) was employed to obtain 24.5 mg of jervine, 18.2 mg of veratramine, 9.4 mg of pseudojervine, 20.8 mg of veratrosine, and 5.2 mg of verdine from 200.0 mg of crude alkaloid extracts, with the purities of 65.7, 97.5, 95.2, 98.3, and 98.1%, respectively. After the filrst HSCCC run, the jervine-fraction was subjected to the second step HSCCC using n-hexane-ethyl acetate-methanol-water (4:5:4:5, v/v/v/v) system for further purification and 12.7 mg of jervine with 95.8% purity was obtained. The structures of isolates were identified by liquid chromatography-quadruple time-of-flight tandem mass spectrometry (LC-QTOF MS/MS), 1H and 13C nuclear magnetic resonance (NMR) spectroscopy.

Staphylococcal Enterotoxin A Gene–Carrying Staphylococcus aureus Isolated from Foods and Its Control by Crude Alkaloid from Papaya Leaves

Staphylococcus aureus is a known pathogen causing intoxication by producing enterotoxins in food. Staphylococcal enterotoxin A is one of the enterotoxins commonly implicated in staphylococcal food poisoning. The ability of crude alkaloid extract from papaya leaves to inhibit the growth of S. aureus and staphylococcal enterotoxin A synthesis was investigated. Staphylococcal enterotoxin A gene-carrying S. aureus was isolated from raw milk and ready-to-eat foods. Crude alkaloid was extracted from ground, dried papaya leaves using ultrasonic-assisted extraction, and a MIC of the alkaloid was determined by the broth macrodilution method. Furthermore, S. aureus isolate was exposed to the crude alkaloid extract at one- and twofold MIC, and the expression of sea was subsequently analyzed using a quantitative reverse transcription real-time PCR. Ten isolates of S. aureus were obtained, and nine of those isolates were sea carriers. The yield of crude alkaloid extract was 0.48 to 1.82% per dry weight of papaya leaves. A MIC of crude alkaloid to S. aureus was 0.25 mg/ml. After exposure to the alkaloid at 0.25 and 0.5 mg/ml for 2 h, a significant increase in cycle threshold values of sea was observed. The sea was expressed 29 and 41 times less when S. aureus was exposed to crude alkaloid at one- and twofold MIC, respectively. This study revealed that crude alkaloid of papaya leaves could control staphylococcal enterotoxin A gene-carrying S. aureus by suppressing the expression of sea, in addition to the ability to inhibit the growth of S. aureus. The expression of sea was successfully quantified.

Distribution of reserpine in Rauvolfia species from India – HPTLC and LC–MS studies

Rauvolfia species are important medicinal plants due to the presence of bioactive indole alkaloids, especially reserpine. Reserpine content in the roots of six Rauvolfia species (R. hookeri, R. micrantha, R. serpentina, R. tetraphylla, R. verticillata, and R. vomitoria), were detected by high performance liquid chromatography coupled with electrospray ionization quadrapole time of flight tandem mass spectrometry (HPLC–ESI-QToF-MS/MS) and estimated by validated high performance thin layer chromatography (HPTLC) method. Reserpine has positively been identified in the crude alkaloid extracts through HPLC–ESI-QToF-MS/MS method by exact mass, isotopic peak pattern, tandem mass fragmentation pattern, and with authentic standard match. Excellent separation of reserpine was achieved in HPTLC using the solvent system hexane:acetone:methanol (6:3.5:0.5, v/v). The HPTLC estimation method was validated in terms of linearity, specificity, detection limit, quantitation limit, precision, accuracy, and repeatability. Among the six Rauvolfia species, reserpine content was highest in the exotic species R. vomitoria (689.5μg/g, dry wt.), while among the five Indian species the highest reserpine content was for R. tetraphylla (450.7μg/g, dry wt.). In the most common Indian Rauvolfia species, R. serpentina, the reserpine content was comparatively low (254.8μg/g, dry wt.). The endemic species R. micrantha possesses significant quantity of reserpine (422.1μg/g, dry wt.), making it a potential candidate for developing as a source of reserpine, replacing R. serpentina and R. tetraphylla that are endangered due to over exploitation.

Comparison of Antibacterial Activity of Crude Alkaloid and Saponin Extract from Phyllanthus niruri

Comparison of Antibacterial Activity of Crude Alkaloid and Saponin Extract from Phyllanthus niruri

Acetylcholinesterase inhibitory activity of Haplophyton cimicidum

In Oaxaca, México, Haplophyton cimicidum is used in combination with other plants as part of a folk strategy against arthropod pests of crops and cattle. Methanolic and crude alkaloid (acid–base extraction) extracts of H. cimicidum leaves were analysed by thin-layer chromatography (TLC) and assayed in vitro for acetylcholinesterase (AChE) inhibitory activity using Spodoptera frugiperda homogenates as source of enzyme. Derivatised chromatographic plates indicated the presence of indoles and alkaloids in the extracts. The crude alkaloid extract exhibited a higher number of compounds than the methanolic extract as judged by the number of spots on TLC plates. The crude alkaloid extract had a weak inhibition potential of AChE with a lower IC50 (93 μg mL− 1) than the methanolic extract (159 μg mL− 1). Indole alkaloids may be responsible for the activity, though a subsequent analysis of the extract components is necessary to determine the active alkaloid(s).

Effects of crude extracts from medicinal herbs Rhazya stricta and Zingiber officinale on growth and proliferation of human brain cancer cell line in vitro.

Hitherto, limited clinical impact has been achieved in the treatment of glioblastoma (GBMs). Although phytochemicals found in medicinal herbs can provide mankind with new therapeutic remedies, single agent intervention has failed to bring the expected outcome in clinical trials. Therefore, combinations of several agents at once are gaining increasing attractiveness. In the present study, we investigated the effects of crude alkaloid (CAERS) and flavonoid (CFEZO) extracts prepared from medicinal herbs, Rhazya stricta and Zingiber officinale, respectively, on the growth of human GBM cell line, U251. R. stricta and Z. officinale are traditionally used in folkloric medicine and have antioxidant, anticarcinogenic, and free radical scavenging properties. Combination of CAERS and CFEZO treatments synergistically suppressed proliferation and colony formation and effectively induced morphological and biochemical features of apoptosis in U251 cells. Apoptosis induction was mediated by release of mitochondrial cytochrome c, increased Bax : Bcl-2 ratio, enhanced activities of caspase-3 and -9, and PARP-1 cleavage. CAERS and CFEZO treatments decreased expression levels of nuclear NF-κBp65, survivin, XIAP, and cyclin D1 and increased expression level of p53, p21, and Noxa. These results suggest that combination of CAERS and CFEZO provides a useful foundation for studying and developing novel chemotherapeutic agents for the treatment of GBM.

Anti-plasmodial potential of crude alkaloidal extract of three plants used in traditional medicine in India

Malaria is one of the most important tropical diseases and the greatest cause of morbidity and mortality in India. The search for new antimalarial compounds has been necessitated by Plasmodium falciparum resistance to standard antimalarial drugs. Plants are important source of biologically active compounds and have potential for the development of novel antimalarial drugs. Since a number of alkaloids have been successfully used for the treatment of malaria since ancient time In this study the crude alkaloid extract of three young plants Carica papaya Linn. (Family: Caricaceae), Datura innoxia Mill. (Family: Solonaceae) and Ricinus communis Linn (Euphorbiaceae) were evaluated against Plasmodium falciparum 3D7. The mean inhibitory concentration (IC50), the mean cytotoxic concentration and the selectivity index were estimated. The cytotoxicity was estimated on Madin-Darby Canine Kidney (MDCK) cell line in maximum dose tested. The alkaloidal extract of C. papaya showed stronger antiplasmodial activity as compared to D. innoxia and R. communis, the IC50 values in the range of 28.35 to 93.17μg/mL. Results demonstrated alkaloids as the putative active compound showing promising antimalarial effect.

Influence of sample preparation methods on the quantitation of selected tropane alkaloids from herb ofDatura innoxiamill. by HPTLC

Summary The aim of our paper was the examination of various procedures of extraction and purification methods on the yield of atropine and scopolamine in Datura innoxia extracts determined by high-performance thin-layer chromatography (HPTLC). In our experiments, the following extraction methods were used: percolation, ultrasound-assisted extraction (USAE) at various temperatures, and microwave-assisted extraction (MASE). The highest extraction yields of atropine and scopolamine were obtained by ultrasound-assisted extraction method and aqueous acetic acid or aqueous tartaric acid used as extractants. Crude extracts of D. innoxia herb were purified by use of various methods: solid-phase extraction-strong cation exchanger (SPE-SCX), SPE-C18, and liquid-liquid extraction (LLE). The best results of purification with satisfactory %RSD values were obtained by liquid-liquid extraction by use of dichloromethane from aqueous crude alkaloid extract alkalized to pH 12. HPTLC was performed on pre-coated plates Kieselgel 60 F254 in horizontal DS chambers by use of mobile phase: methanol-acetone-aqueous ammonia (5:4.5:0.5). The alkaloids' content was determined in extracts by use of calibration curve method for atropine and scopolamine. Calibration curves were linear in range of 3.0–30 μg for scopolamine (R = 0.9980) and 2.6–26 μg for atropine (R = 0.9923). Limits of detection (LOD) and limits of quantification (LOQ) were 1.12 μg and 3.42 μg per spot for scopolamine and 0.89 μg and 2.82 μg per spot for atropine.

Preparative mass-spectrometry profiling of genotoxic pyrrolizidine alkaloids in Jacobaea vulgaris (syn. Senecio jacobaea) by spiral-coil countercurrent chromatography and ESI-MS/MS off-line continuous injection

Pyrrolizidine alkaloids (PAs) are genotoxic natural products. Almost 350 PAs are known with respective N-oxides. Legal regulations of PAs in food, and feed are discussed. There is a lack of available reference substances for targeted quantification [1]. PA-contamination was reported for honey, and bee products [2,3]. PA-contamination of retail ready-to-use lettuce was demonstrated. Morphological similarities of rocket salad (Diplotaxis tenuifolia, syn.: rucola), and the PA-plant Senecio vulgaris resulted in unintentional co-harvesting. In our study, PA-extract of J. vulgaris flower heads (Asteraceae) was fractionated by prep. spiral-coil countercurrent chromatography (spCCC, 5.7 L coil). The system n-hexane – EtOAc – MeOH – H2O (3:7:5:5) was used to separate 5 g of crude alkaloid extract. Obtained fractions were sequentially off-line injected to ESI-MS/MS. This target-guided isolation procedure [4] made use of selected ion traces of PAs. Elution of major (2, 5, 7), and minor (1, 3, 4, 6) PAs were monitored (altern. mode MS2, m/z 100 – 1500). The spCCC elution resulted in the fractionation of PAs, and one non-PA (8) in the extrusion-mode (8 not presented) [5]. Almost pure compounds were obtained for PAs 1, 2, 5, 7, and the non-PA 8 could be used as reference materials. Minor PAs were enriched and will be further purified with other separation techniques.