Liquid chromatography mass spectrometry simultaneous determination of vindoline and catharanthine in rat plasma and its application to a pharmacokinetic study

Vinblastine and vincristine, both of which are bisindole alkaloids derived from vindoline and catharanthine, have been used for cancer chemotherapy; their monomeric precursor molecules are vindoline and catharanthine. A simple and selective liquid chromatography mass spectrometry method for simultaneous determination of vindoline and catharanthine in rat plasma was developed. Chromatographic separation was achieved on a C₁₈ (2.1 × 50 mm, 3.5 µm) column with acetonitrile–0.1% formic acid in water as mobile phase with gradient elution. The flow rate was set at 0.4 mL/min. An electrospray ionization source was applied and operated in positive ion mode; selective ion monitoring mode was used for quantification. Mean recoveries were in the range of 87.3–92.6% for vindoline in rat plasma and 88.5–96.5% for catharanthine. Matrix effects for vindoline and catharanthine were measured to be between 95.3 and 104.7%. Coefficients of variation of intra‐day and inter‐day precision were both <15%. The accuracy of the method ranged from 93.8 to 108.1%. The method was successfully applied in a pharmacokinetic study of vindoline and catharanthine in rats. The bioavailability of vindoline and catharanthine were 5.4 and 4.7%, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis and study of a molecularly imprinted polymer for the specific extraction of indole alkaloids from Catharanthus roseus extracts

Two molecularly imprinted polymers (MIP) for catharanthine and vindoline have been synthesized in order to specifically extract these natural indole alkaloids from Catharanthus roseus by solid-phase extraction (SPE). Each MIP was prepared by thermal polymerisation using catharanthine (or vindoline) as template, methacrylic acid (or itaconic acid) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross-linking agent and acetonitrile (or acetone) as porogenic solvent.For catharanthine-MIP, a SPE protocol (ACN–AcOH 99/1 washing and MeOH–AcOH 90/10 elution) allows a good MIP/NIP selectivity (imprinting factor 12.6). The specificity of catharanthine-MIP versus related bisindole alkaloids was assessed by cross-reactivity study. The catharanthine-MIP specifically retained catharanthine and its N-oxide analogue but displayed a weak cross-reactivity for other Vinca alkaloids (vinorelbine, vincristine, vinblastine, vindoline, vinflunine). It appears that the catharanthine-like unit of these molecules are hardly trapped in catharanthine cavities located in the MIP, probably due to the sterical hindrance of the vindoline moiety. Finally, the MIP-SPE applied to C. roseus extract enabled quantitative recovery of catharanthine (101%) and the total removal of vindoline. Its capacity was determined and was equal to 2.43 μmol g⁻¹.Vindoline is a weaker base than catharanthine, so the vindoline-MIP was achieved with a strong acidic monomer (itaconic acid) to increase vindoline–monomer interactions and a modified washing solvent (ACN–HCOOH 99/1) to reduce non-specific interactions. The influence of the amount of HCOOH (protic modifier) percolated during the washing step upon the elution yield and the imprinting factor for vindoline was investigated. This preliminary optimisation of the washing step, and in particular the number of moles of acid percolated, seems useful to emphasize the use of MIP in conditions of high selectivity or high yield. A compromise was obtained with an imprinting factor equal to 7.6 and an elution recovery of 33%. However MIP-vindoline failed to achieve a specific extraction of vindoline since catharanthine was also extracted probably because of strong non-specific interactions occurring between catharanthine and the sorbent.     

Isolation of indole alkaloids from Catharanthus roseus by centrifugal partition chromatography in the pH-zone refining mode.

Centrifugal partition chromatography (CPC) in the pH-zone refining mode allowed a preparative and efficient isolation of vindoline, vindolinine, catharanthine and vincaleukoblastine from a crude mixture of Catharanthus roseus alkaloids. The separation protocol was tested with a synthetic mixture of vindoline, catharanthine and vincaleukoblastine. The fraction content was analyzed and the results compared with theoretical chromatograms obtained by numerical simulation. The increase in injected sample mass results in an improvement of the purity of the isolated compounds. This observation, confirmed by theory, is of prime importance for the development of preparative pH-zone refining CPC as a preparative separation method.     

Total synthesis of indole and dihydroindole alkaloids. IX. Studies on the synthesis of bisindole alkaloids in the vinblastine-vincristine series. The biogenetic approach.

A detailed study of the reaction of catharanthine N-oxide and vindoline has been carried out employing various conditions. Under optimum conditions, which involve low temperatures and trifluoroacetic anhydride as reagent, 3′, 4′-dehydrovinblastine (XIII, R = COOCH₃), in reasonable yields is essentially the exclusive product. However two additional products, 18′ (epi)- 3′, 4′-dehydrovinblastine (XIV, R = COOCH₃) and 1′-hydroxy- 3′, 4′-dehydrovinblastine (XVI, R = COOCH₃) are also often isolated. The reaction, which follows the course of a Polonovski-type fragmentation process, has been extended to the N-oxide derivatives of dihydrocatharanthine and decarbomethoxycatharanthine to provide again a series of bisindole alkaloid derivatives, also vinblastines. A mechanistic rationale is provided to explain the various results obtained.     

New Insights into the Mechanism and an Expanded Scope of the Fe(III)-Mediated Vinblastine Coupling Reaction

A definition of the scope of aromatic substrates that participate with catharanthine in an Fe(III)-mediated coupling reaction, an examination of the key structural features of catharanthine required for participation in the reaction, and the development of a generalized indole functionalization reaction that bears little structural relationship to catharanthine itself are detailed. In addition to providing insights into the mechanism of the Fe(III)-mediated coupling reaction of catharanthine with vindoline suggesting the reaction conducted in acidic aqueous buffer may be radical mediated, the studies provide new opportunities for the preparation of previously inaccessible vinblastine analogs and define powerful new methodology for the synthesis of indole-containing natural and unnatural products.     

高效液相色谱法同时测定长春花中的文多灵、长春质碱和脱水长春碱

运用高效液相色谱（HPLC）梯度洗脱同时测定了长春花中的文多灵、长春质碱和脱水长春碱等3种生物碱。色谱柱为Waters 5C18-MS-Ⅱ(4.6 mm×250 mm),流动相为甲醇-1%二乙胺(磷酸调pH至7.3)系统,梯度洗脱,检测波长220 nm,柱温25 ℃。实验结果表明,文多灵、长春质碱和脱水长春碱等3种生物碱分别在0.03～1 mg/mL,0.03～1 mg/mL和0.01～0.5 mg/mL时线性关系良好,平均加样回收率分别为96.8%,97.0%和96.4%,相对标准偏差(RSD)分别为1.53%,1.37%和1.96%。用该法同时检测了不同干旱条件处理下长春花样品中的文多灵、长春质碱和脱水长春碱,方法准确、快速、简便。     

A simplified procedure for indole alkaloid extraction from Catharanthus roseus combined with a semi-synthetic production process for vinblastine

Dried leaves of Catharanthus roseus were extracted with aqueous acidic 0.1 M solution of HCl. Alkaloid-embonate complexes were obtained as precipitates by treating the extract with an alkaline (NaOH) solution of embonic acid (4,4-methylene-bis-3-hydroxynaphtalenecarboxylic acid). The precipitate mainly consisted of catharanthine and vindoline embonates and it was directly used as the starting material for a semi-synthesis of the anti-cancer bisindole alkaloid vinblastine. The coupling reaction involved oxidation of catharanthine in aqueous acidic medium by singlet oxygen ((1)O2), continuously produced in situ by the reaction between H2O2 with NaClO. An excess of NaBH4 was used for the reduction step. Analysis of the reaction mixture indicated a maximum yield of 20% for vinblastine at pH 8.3, based on the initial amount of catharanthine concentration. Direct-injection electrospray ionization mass spectrometry in positive ion mode was used for the identification of vinblastine. The mass spectra of vinblastine were dominated by the corresponding protonated molecular ion [M+H]+ at m/z 811 and the characteristic fragment ions matched with those of the standard compound.     

Alkaloid Production in Catharanthus roseus Cell Cultures. VII.. Effect of parameter changes and catabolism studies on cell line PRL No. 953

Catharanthus roseus cells suspended in production medium showed the presence of four Aspidosperma-type alkaloids, however, no vindoline. Cells grown in media with the pH adjusted to 7.0 produced 3.3 mg total alkaloids/g dry weight. At pH 5.0, 1.7 mg of alkaloid/g dry weight was produced. (S)-Adenosyl-methionine did not stimulate the production of vindoline. When added to cell suspensions for 21 days, vindoline and catharanthine were degraded to non-alkaloidal substances, not dimerized to bisindole alkaloids.     

Voltammetric oxidation of vinblastine and related compounds

The indole alkaloids, vinblastine, catharanthine, and vindoline, gave oxidation waves in mixed aqueous/organic media by cyclic, normal-pulse, and differential-pulse voltammetry (d.p.v). Of the systems investigated, d.p.v. at carbon paste anodes in pH 5.6 acetate buffer in ethanol/water (1:1) is recommended for general analytical work; calibration graphs were linear up to about 1 mM, with limits of detection of 4, 20, and 1.5 μM for vinblastine, catharanthine, and vindoline, respectively. Results obtained by cyclic voltammetry as well as by pulse techniques suggest that electron transfer is preceded by deprotonation and followed by additional chemical reactions. Products of the electrode processes form mechanically unstable films on the electrode surface.     

Simultaneous determination of vinblastine and its monomeric precursors vindoline and catharanthine in Catharanthus roseus by capillary electrophoresis-mass spectrometry

Catharanthus roseus is an important dicotyledonous medicinal plant that contains various anticancer components, such as vinblastine (VLB) and its monomeric precursors (vindoline and catharanthine). A capillary electrophoresis-mass spectrometry (CE-MS) approach for the simultaneous determination of three components was developed in this work. Baseline separation for three components was achieved by using a running buffer consisting of 20 mM ammonium acetate and 1.5% acetic acid in <20 min. Quantification of three components was assigned in positive-ion mode at a protonated molecular ion [M+H](+). The CE-MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the above components. The detection limits of VLB, catharanthine and vindoline are 0.8, 0.1 and 0.1 μg/mL, respectively. The precision was not more than 4.54% and the mean recovery of the analytes was 95.04-97.04%. The CE-MS method was successfully applied to determine VLB and its monomeric precursors in real sample C. roseus.     

Stereoselective conversion of anhydrovinblastine into vinblastine utilizing an anti-vinblastine monoclonal antibody as a chiral mould

Dimeric indole alkaloid, anhydrovinblastine, which can be obtained from catharanthine and vindoline in a high yield, was converted stereoselectively into vinblastine through alternating oxidation-reduction with oxygen and NaBH(3)CN in the presence of anti-vinblastine monoclonal antibody.     

Production of 3', 4'-anhydrovinblastine: a unique chemical synthesis

Preliminary Investigations have led to the discovery that ferric ion can couple catharanthine and vindoline, in aqueous acidic media, to produce 3',4'-anhydrovinblastine as the major product. A conversion of 77% could be realized under optimized conditions.     

Optimization of Tabersonine Methoxylation to Increase Vindoline Precursor Synthesis in Yeast Cell Factories

Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.     

PHOTOCHEMICAL ONE-POT SYNTHESIS OF VINBLASTINE and VINCRISTINE

The naturally occurring cytostatic dimer alkaloids vinblastine 1 and vincristine 2 were photochemically synthesized in slightly acidic aqueous solution from the monomer alkaloids catharanthine 3 and vindoline 4. Multi-centre reactions should obviously be involved and some of the principal photochemistry-associated phenomena here discussed are quite likely to be characteristic even to the biosynthetic reactions yielding vinblastine 1 and vincristine 2 in the cells of Catharanthus roseus.     

Addition of Vindoline to p-Benzoquinone: Regiochemistry,Stereochemistry and Symmetry Considerations

Vindoline and catharanthine are the major alkaloids of Catharanthus roseus and are extracted in large quantities to prepare the pharmaceutically important Vinca type alkaloids vincaleukoblastine, vincristine and navelbine. The higher yield of vindoline relative to catharanthine makes it an attractive substrate for developing new chemistry and adding value to the plant. In this context, we have reacted vindoline with a selection of electrophiles among which benzoquinone. Conditions were developed to optimize the synthesis of a mono-adduct, of five bis-adducts, and of tri-adducts and tetra-adducts, several of these adducts being mixtures of conformational isomers. Copper(II) was added to the reactions to promote reoxidation of the intermediate hydroquinones and simplify the reaction products. The structures were solved by spectroscopic means and by symmetry considerations. Among the bis-isomers, the 2,3-diadduct consists of three unseparable species, two major ones with an axis of symmetry, thus giving a single set of signals and existing as two different species with indistinguishable NMR spectra. The third and minor isomer has no symmetry and therefore exhibits nonequivalence in the signals of the two vindoline moieties. These isomers are designated as syn (minor) and anti (major) and there exists a high energy barrier between them making their interconversion difficult. DFT calculations on simplified model compounds demonstrate that the syn-anti interconversion is not possible at room temperature on the NMR chemical shift time scale. These molecules are not rigid and calculations showed a back-and-forth conrotatory motion of the two vindolines. This “windshield wiper” effect is responsible for the observation of exchange correlations in the NOESY spectra. The same phenomenon is observed with the higher molecular weight adducts, which are also mixtures of rotational isomers. The same lack of rotations between syn and anti isomers is responsible for the formation of four tri-adducts and of seven tetra-adducts. On a biological standpoint, the mono adduct displayed anti-inflammatory properties at the 5 μM level while the di-adducts and tri-adducts showed moderate cytotoxicity against Au565, and HeLa cancer cell lines.     

Isolation of Catharanthus alkaloids by solid-phase extraction and semipreparative HPLC

A combination of moderate-pressure chromatography on C18 sorbent and preparative HPLC is developed for rapid isolation of alkaloids from Catharanthus roseus. The procedure is optimized for vindoline and catharanthine with respective yields of 3 and 2 mg per 1 g of dried leaves of the plant. The methodology is also applied for identification of the above and other alkaloids from cultured plant cells.     

Biosynthesis of the Indole Alkaloids. Cell-free Systems from Catharanthus roseus Plants

Cell-free systems from Catharanthus roseus plants are utilized for various studies relating to the biosynthesis of indole alkaloids. Tryptamine ( 5 ) and secologanin ( 6 ), two fundamental building units, are shown to be incorporated into the alkaloid vindoline ( 7 ). In another study, catharanthine ( 18 ) and vindoline ( 7 ) are utilized by this enzyme system and coupled to the important bisindole biointermediate 3′,4′-anhydrovinblastine

1 The previously [20] used name for 17 , 3′, 4′-dehydrovinblastine, is incorrect.

( 17 ). The latter substance is, in turn, incorporated and converted to the natural alkaloids leurosine ( 8 ), Catharine ( 9 ) and vinblastine ( 10 ), thereby providing information about the biosynthesis of these complex molecules. High pressure liquid chromatography assay of the enzymic mixture sheds light on the enzymes involved in the coupling of 18 and 7 .     

Screening of Catharanthus roseus secondary metabolites by high-performance liquid chromatography

Two direct HPLC analytical methods for the screening of the major indole alkaloids of Catharanthus roseus hairy roots and their iridoid precursors have been developed. Photodiode array and fluorescence detection were performed. The separation was achieved on a reversed-phase C18 column. The first method allowed the separation of catharanthine, serpentine, tabersonine, vindoline, vinblastine, and vincristine in 20 min. Ajmalicine, tryptophan, tryptamine and secologanine were separated using the second method in 13 min. The identification of the compounds was based on the retention time and the comparison of UV spectra with those of authentic standards. A simplified alkaloid extraction method was developed in order to accelerate sample preparation. The assays were successfully used to quantify major compounds of the secondary metabolism of hairy root cultures of C. roseus, thus providing a reliable tool for rapid screening of C. roseus secondary metabolite samples. In these cultures, ajmalicine, serpentine, catharanthine, tabersonine, and tryptamine were detected, but tryptophan, vindoline, vinblastine and vincristine were not.     

Disproof of the Overall Enzymatic Biosynthesis of Vindoline from Tryptamine and Secologanin by Cell-Free Extracts from the Leaves of Catharanthus roseus (L.) G. DON

A cell-free extract, which was isolated from the leaves of mature Catharanthus roseus plants by a previously published procedure, does not convert a mixture of secologanin and radiolabelled tryptamine to vindoline, as was recently claimed. The radioactivity in the purified alkaloid extract determined by earlier workers is certainly due to ‘impurities’ in the presumed ‘vindoline’. This was shown by extensive purification of the alkaloid extract (which contained added unlabelled vindoline as a carrier) followed by chemical conversion of vindoline to two derivatives and subsequent purification, ultimately giving unlabelled deacetylvindoline.