Investigation of Tropane Alkaloids in Genetically Transformed <Emphasis Type="Italic">Atropa belladonna</Emphasis> L. Cultures

The purpose of the present study was to determine the tropane alkaloid content of genetically transformed hairy root cultures of Atropa belladonna L. Determination of alkaloids was performed by HPLC method. Samples were extracted with chloroform – methanol - cc. ammonia 15:5:1 (v/v/v). Crude extracts were purified on Extrelut columns. HPLC separation was performed on Luna C8 reversed phase column. An isocratic mixture of acetonitrile – 30 mM phosphate buffer - methanol 12.2:79.7:8.1(v/v/v) was used as eluent. Peaks were identified by addition of standards and diode-array detection. Hyoscyamine, scopolamine and apoatropine were determined by external standard method at 210 nm. We measured the alkaloid content of genetically transformed in vitro cultures (hairy roots and reorganised plants) cultivated on Gamborg B5 basic media. The highest hyoscyamine and scopolamine content was found in hairy root clone #K₅ (0,223 m/m%) and in hairy root clone #K₄ (0,018 m/m%) respectively. Alkaloid contents were higher in the hairy roots than in the reorganised plants.     

LC-DAD and LC–MS–MS Analysis of Piperidine Alkaloids of Lobelia inflata L. (In Vitro and In Vivo)

An LC-DAD method was developed for determination of lobeline from in vitro and in vivo cultures of Lobelia inflata. Samples were extracted with 0.1 N HCl–acetonitrile (1:1, v/v), and purified by solid-phase extraction. Optimized conditions resulted in high recovery. LC separations were performed on an Eurosphere C8 reversed-phase column using 30:70 (v/v) acetonitrile–0.1% trifluoroacetic acid as a mobile phase. Quantitative determination of lobeline was performed by external standard method at 250 nm, in the range of 2.4–80 μg mL⁻¹. Validation studies proved that the repeatability of the method was good and the recovery was satisfactory. In vitro organized cultures contained considerable amount of lobeline (herb: 175 μg g⁻¹, root: 100 μg g⁻¹). When these cultures were transplanted into the open field, the lobeline content increased significantly (herb: 323 μg g⁻¹, root: 833 μg g⁻¹). Plants obtained from seed propagation contained 382 μg g⁻¹ lobeline in the herb. For direct characterization of di-substituted piperidine alkaloids in extracts of L. inflata, tandem mass spectrometric method was developed using electrospray ionization. Analysis was performed in the positive ion mode on a triple quadropole LC–MS system. LC separations were achieved on Eurosphere C8 column with a modified mobile phase (acetonitrile–30 mM ammonium formate, pH 2.80) to ensure proper molecular ionization. The identification and structural elucidation of the alkaloids were performed by comparing their changes in molecular mass (ΔM), full-scan MS–MS spectra with those of lobeline, norlobelanine and lobelanidine. These alkaloids and ten other derivatives were identified in the plant extracts. Three piperidine alkaloids were reported in L. inflata for the first time.

     

Alkaloid Composition of Chelidonium majus L. Studied by Different Chromatographic Techniques

The greater celandine (Chelidonium majus L.) is a well known source of isoquinoline alkaloids with therapeutic value. The aim of our work was to develop a simple and fast method to determine the alkaloid composition in Chelidonium plant organs. TLC-densitometry seemed to be the most convenient analytical technique for routine, fast investigations and its precision was controlled by using HPLC. A densitometric method using Silica gel 60 F₂₅₄ with chloroform-methanol 60:30 (v/v) and methylene chloride-methanol 97:3 (v/v) as mobile phases has been developed to quantify the main alkaloids (chelidonine, chelerythrine, sanguinarine, coptisine and berberine). The application of TLC permitted utilizing the fluorescence of alkaloids without purification and made the detection extremely sensitive. The samples were further investigated by our reliable HPLC method. Analysis was performed on a C₁₈ column with acetonitrile-methanol-30 mM ammonium formate (pH = 2.8) 14.7:18.0:67.3 (v/v/v) as mobile phase and alkaloids were determined at 280 nm by using external standards. Our TLC-densitometric method elaborated is a simple technique for accurate and reproducible determination of Chelidonium alkaloids. The results of the two chromatographic methods showed good agreement.     

Determination of Visnagin inAmmi visnagaHairy Root Cultures Using Solid-Phase Extraction and High-Performance Liquid Chromatography

A high-performance liquid chromatographic method was developed for separation of the furochromone fraction and for determination of visnagin inAmmi visnagahairy root cultures. Lyophilized samples were extracted with chloroform:methanol (1:1, v/v) and purified on solid-phase extraction cartridges. HPLC analyses were performed on a Eurospher 100-C₈Knauer column and the mobile phase was 29:28:526:417 (v/v/v/v) acetonitrile:tetrahydrofuran:30 mM citric acid (pH 3.0):methanol. Quercetin was used as internal standard. Peaks were identified by addition of authentic standards and/or by diode-array detection.     

Analysis of Non-volatile Constituents in Dracocephalum Species by HPLC and GC-MS

In this paper we identify/determine the composition of the extracts of Dracocephalum moldavica L. and D. ruyschiana L. with special emphasis on their flavonoids, aliphatic, aromatic carboxylic acids and sugars. The plant materials were extracted using methanol-acetone 1:1 (v/v) acidified with HCl (0.05%). Composition of extract's most red fractions was identified by HPLC, while the constituents of the entire extract were identified and quantified as their trimethylsilyl-(oxime) ether/ester derivatives by GC-MS. On the basis of HPLC analyses - (performed on Phenomenex Luna 5 µm C18 column, 250× 4.6 mm I.D.; eluent: acetonitrile - trifluoroacetic acid (0.1 %); isocratic/gradient conditions) - delphinidin, cyanidin and pelargonidin (in traces) were identified by spectral analysis and on the basis of authentic standard's addition. GC-MS analyses were carried out immediately with extracts, as well as subsequently to extract's hydrolysis (trifluoroacetic acid: 2M, 2h, 4h). Constituents were identified and quantified as their trimethylsilyl-(oxime) ether/ester derivatives, from a single run, on the basis of their total (TIC) and selective fragment ion (SIM) responses. Calculations were related to the dry matter content of extracts. As main constituents monosaccharides, sugar alcohols di- and trisaccharides, aliphatic (phosphoric, succinic, levulinic, malic, tartaric, fatty acids) and aromatic (quinic, chlorogenic, caffeic, ferulic, 3,4-dihydroxyphenyllactic, rosmarinic) carboxylic acids and their corresponding esters; apigenin, luteolin flavon aglycons and tocopherol, in total 33 constituents were quantitated partly by their TIC, partly by their SIM responses. Identification/quantification proved to be in total of 35–69% (expressed in the dry matter content of extracts).     

HPLC Analysis of Alizarin and Purpurin Produced by Rubia tinctorum L. Hairy Root Cultures

We have determined the quantities of the anthraquinones alizarin and purpurin, with especial regard to their effective antigenotoxic activity, in genetically transformed hairy root cultures of Rubia tinctorum L. Hairy roots were cultured on solid and in liquid Gamborg B5 and 1/2 NMS media in a shaking cabinet and in a bioreactor. Methanolic extracts of lyophilized hairy roots were hydrolysed, and then purified by solid phase extraction with good recovery. A new HPLC method was developed for the determination of alizarin and purpurin. The analysis was performed on a Luna C8 RP column using a 45:55 (v/v) mixture of acetonitrile:20 mM ammonium formate-formic acid buffer (pH 3.00) as eluent. Peaks were identified by addition of standards and by diode-array detection. External standardization allowed the determination of alizarin and purpurin with good sensitivity and reliability. The maximum purpurin content was observed in cultures cultivated on solid B5 medium (5.94 mg g⁻¹). The highest alizarin content was measured in cultures cultivated on solid 1/2 NMS medium (2.14 mg g⁻¹).

     

LC-MS Analysis of Antioxidant Plant Phenoloids

Extracts of selected medicinal plants with promising integral antioxidative capacity were examined by high-performance liquid chromatography (HPLC) coupled with diode-array detection (DAD) and on-line mass spectrometry (ESI-MS or APCI-MS). These techniques allowed determination of the main components of each extract, which may serve us thereby providing a typical “finger-print” in the identification of the plants. More specifically various flavonol aglycones, flavon- and flavonol-glycosides, flavonol-diglycosides were detected in herbs of Solidago canadensis chemovarieties, in leaves of Filipendula ulmaria and in the herb of Viola tricolor species.     

HPLC‐ESI‐MS/MS of brain neurotransmitter modulator lobeline and related piperidine alkaloids in Lobelia inflata L

There is a renewed interest in lobelia alkaloids because of their activity on the central nervous system. Lobeline, the most active of them, a nicotinic receptor ligand and neurotransmitter transporter inhibitor, is a candidate pharmacotherapy for metamphetamine abuse. In the present work, high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry in positive ion mode was used for investigating the alkaloid profile in Lobelia inflata L. Chromatographic separations were achieved on a Gemini C6‐phenyl reversed‐phase column providing good peak shape and improved selectivity. Being mostly 2,6‐disubstituted piperidines, lobelia alkaloids presented abundant [M + H]⁺ ions with typical fragmentation. Identification was possible from a few specific ions, especially those resulting from excision of one of the substituents. Based on fragmentation pattern of lobeline as reference compound, 52 alkaloids were identified in the aqueous methanolic extract of L. inflata in contrast to the previously known some 20. Structural variability of these alkaloids identified arises basically from their substituents which can be phenyl‐2‐ketoethyl‐ or phenyl‐2‐hydroxyethyl units as well as their methyl‐, ethyl‐ or propyl‐ homologues attached in different combinations. Several propyl homologue lobelia alkaloids and five hydroxypiperidine derivatives were found in the plant at the first time. In addition to 8‐O‐esters of 2‐monosubstituted piperidine alkaloids previously reported by us in L. inflata, a 3‐hydroxy‐3‐phenylpropanoic acid ester of hydroxyallosedamine ring‐substituted was also identified as a new natural product. High‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry can be successfully applied to Lobeliacae plant samples in the routine screening for new and known bioactive constituents, quality control of the crude drug, lobelia herba, alkaloid production studies, breeding and chemotaxonomy. Copyright © 2015 John Wiley & Sons, Ltd.     

HPLC Determination of Flavonoids in Hairy-Root Cultures of <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi

A simple and sensitive liquid chromatographic method has been established for determination of the main flavonoid components of Scutellaria baicalensis Georgi (Lamiaceae) roots and hairy-root cultures. Wogon, the dried root of the plant, is used in traditional Chinese medicine for treatment of bronchitis, hepatitis, tumors, and inflammatory diseases. Lyophilized hairy roots were extracted with methanol. The crude extracts were purified by SPE on Supelco LC-8 cartridges. HPLC separations were performed on a Eurospher 100-C₈ reversed-phase column. The mobile phase was a gradient prepared from mixtures of acetonitrile and 0.1% trifluoroacetic acid. Peaks were identified by addition of standards and/or by diode-array detection. Baicalein 7-O-glucuronide (baicalin), wogonin 7-O-glucuronide (wogonoside), baicalein, wogonin, and acteoside were determined by the external standard method at 280 nm. We found that the aglycon (baicalein and wogonin) content of the transformed roots was consistently higher than that of the intact root from Siberia.