A thorough evaluation of matrix-free laser desorption ionization on structurally diverse alkaloids and their direct detection in plant extracts.

Alkaloids represent a major group of natural products (NPs), derived from highly diverse organisms. These structurally varied specialized metabolites are widely used for medicinal purposes and also known as toxic contaminants in agriculture and dietary supplements. While the detection of alkaloids is generally facilitated by GC- or LC-MS, these techniques do require considerable efforts in sample preparation and method optimization. Bypassing these limitations and also reducing experimental time, matrix-free laser desorption ionization (LDI) and related methods may provide an interesting alternative. As many alkaloids show close structural similarities to matrices used in matrix-assisted laser desorption ionization (MALDI), they should ionize upon simple laser irradiation without matrix support. With this in mind, the current work presents a systematic evaluation of LDI properties of a wide range of structurally diverse alkaloids. Facilitating a direct comparison between LDI and ESI-MS fragmentation, all tested compounds were further studied by electrospray ionization (ESI). Moreover, crude plant extracts of Atropa belladonna, Cinchona succirubra, and Colchicum autumnale were analyzed by LDI in order to evaluate direct alkaloid detection and dereplication from complex mixtures. Finally, dose-dependent evaluation of MALDI and LDI detection using an extract of Rosmarinus officinalis spiked with atropine, colchicine, or quinine was conducted. Overall, present results suggest that LDI provides a versatile analytical tool for analyzing structurally diverse alkaloids as single compounds and from complex mixtures. It may further serve various potential applications ranging from quality control to the screening for toxic compounds as well as the build up of MS databases. Graphical abstract.

Medicinal plants used in the treatment of Malaria: A key emphasis to Artemisia, Cinchona, Cryptolepis, and Tabebuia genera.

Malaria is one of the life-threatening parasitic diseases that is endemic in tropical areas. The increased prevalence of malaria due to drug resistance leads to a high incidence of mortality. Drug discovery based on natural products and secondary metabolites is considered as alternative approaches for antimalarial therapy. Herbal medicines have advantages over modern medicines, including fewer side effects, cost-effectiveness, and affordability encouraging the herbal-based drug discovery. Several naturally occurring, semisynthetic, and synthetic antimalarial medications are on the market. For example, chloroquine is a synthetic medication for antimalarial therapy derived from quinine. Moreover, artemisinin, and its derivative, artesunate with sesquiterpene lactone backbone, is an antimalarial agent originated from Artemisia annua L. A. annua traditionally has been used to detoxify blood and eliminate fever in China. Although the artemisinin-based combination therapy against malaria has shown exceptional responses, the limited medicinal options demand novel therapeutics. Furthermore, drug resistance is the cause in most cases, and new medications are proposed to overcome the resistance. In addition to conventional therapeutics, this review covers some important genera in this area, including Artemisia, Cinchona, Cryptolepis, and Tabebuia, whose antimalarial activities are finely verified.

Enantioselective resolution of biologically active dipeptide analogs by high-performance liquid chromatography applying Cinchona alkaloid-based ion-exchanger chiral stationary phases.

Sixteen pairs of enantiomeric dipeptides were separated on four chiral ion-exchanger-type stationary phases based on Cinchona alkaloids. Anion-exchangers (QN-AX, QD-AX) and zwitterionic phases [ZWIX(+)™ and ZWIX(-)™] were studied in a comparative manner. The effects of the nature and concentrations of the mobile phase solvent components and organic salt additives on analyte retention and enantioseparation were systematically studied in order to get a deeper insight into the enantiorecognition mechanism. Moreover, experiments were performed in the temperature range 10-50 °C to calculate thermodynamic parameters like changes in standard enthalpy, Δ(ΔH°), entropy, Δ(ΔS°), and free energy, Δ(ΔG°) on the basis of van't Hoff plots derived from the ln α vs. 1/T curves. Elution sequences of the dipeptides were determined in all cases and, with a few exceptions, they were found to be opposite on the pseudoenantiomeric stationary phases as of QN-AX/QD-AX and of ZWIX(+) and ZWIX(-). The stereoselective retention mechanism is based on electrostatically driven intermolecular interactions supported by additional interaction increments mainly determined by the absolute configuration of the chiral C8 and C9 atoms of the quinine and quinidine moieties.

Historical chemical annotations of Cinchona bark collections are comparable to results from current day high-pressure liquid chromatography technologies.

ETHNOPHARMACOLOGICAL RELEVANCE: Species of the genus Cinchona (Rubiaceae) have been used in traditional medicine, and as a source for quinine since its discovery as an effective medicine against malaria in the 17th century. Despite being the sole cure of malaria for almost 350 years, little is known about the chemical diversity between and within species of the antimalarial alkaloids found in the bark. Extensive historical Cinchona bark collections housed at the Royal Botanic Gardens, Kew, UK, and in other museums may shed new light on the alkaloid chemistry of the Cinchona genus and the history of the quest for the most effective Cinchona barks. AIM OF THE STUDY: We used High-Pressure Liquid Chromatography (HPLC) coupled with fluorescence detection (FLD) to reanalyze a set of Cinchona barks originally annotated for the four major quinine alkaloids by John Eliot Howard and others more than 150 years ago. MATERIALS AND METHODS: We performed an archival search on the Cinchona bark collections in the Economic Botany Collection housed in Kew, focusing on those with historical alkaloid content information. Then, we performed HPLC analysis of the bark samples to separate and quantify the four major quinine alkaloids and the total alkaloid content using fluorescence detection. Correlations between historic and current annotations were calculated using Spearman's rank correlation coefficient, before paired comparisons were performed using Wilcox rank sum tests. The effects of source were explored using generalized linear modelling (GLM), before the significance of each parameter in predicting alkaloid concentrations were assessed using chi-square tests as likelihood ratio testing (LRT) models. RESULTS: The total alkaloid content estimation obtained by our HPLC analysis was comparatively similar to the historical chemical annotations made by Howard. Additionally, the quantity of two of the major alkaloids, quinine and cinchonine, and the total content of the four alkaloids obtained were significantly similar between the historical and current day analysis using linear regression. CONCLUSIONS: This study demonstrates that the historical chemical analysis by Howard and current day HPLC alkaloid content estimations are comparable. Current day HPLC analysis thus provide a realistic estimate of the alkaloid contents in the historical bark samples at the time of sampling more than 150 years ago. Museum collections provide a powerful but underused source of material for understanding early use and collecting history as well as for comparative analyses with current day samples.

Analysis of free amino acids with unified chromatography-mass spectrometry-application to food supplements.

Amino acids are most often analyzed in reversed-phase liquid chromatography after a derivatization procedure to render them sufficiently hydrophobic and detectable with UV or fluorimetric detection. Simpler methods should be possible to avoid additional chemical reactions. We present an improved method to analyze free amino acids with unified chromatography, that is to say with a wide elution gradient starting with supercritical fluid chromatography (SFC) conditions (high percentage of carbon dioxide) and ending with high-performance liquid chromatography (HPLC) conditions (100% co-solvent). The mobile phase composition was carefully adjusted to permit the elution of 21 natural amino acids (among which 19 proteinogenic) with very good peak shapes from a zwitterionic cinchona-based stationary phase (Chiralpak ZWIX(+)). Chiral separation was not desired. The mobile phase finally selected comprised carbon dioxide and a co-solvent (methanol containing 2% water and 20 mM methanesulfonic acid), ranging from 10 to 100% in 7 min followed by 3 min re-equilibration at 25 °C. A reversed pressure gradient (15 to 11 MPa) and a reversed flow rate gradient (3 to 1 mL/min) were applied to avoid reaching the upper pressure limit of the pumping system (40 MPa) and to favor high chromatographic efficiency at every stage of the elution gradient. Detection was achieved with electrospray ionization-mass spectrometry (ESI(+)-MS). The method is then fast and straightforward as no derivatization step is necessary, and all isobaric species were chromatographically resolved. To demonstrate the applicability of the method, it was applied to the quantitation of amino acids in food supplements commonly consumed by sportsmen, containing taurine (a common natural amino acid) or branched-chain amino acids (BCAA), namely valine, and the isobaric leucine and isoleucine. A standard addition method was examined for sensitivity, linearity, repeatability and intermediate precision.

Automatised pharmacophoric deconvolution of plant extracts - application to Cinchona bark crude extract.

We present a development of the "Plasmodesma" dereplication method [Margueritte et al., Magn. Reson. Chem., 2018, 56, 469]. This method is based on the automatic acquisition of a standard set of NMR experiments from a medium sized set of samples differing by their bioactivity. From this raw data, an analysis pipeline is run and the data is analysed by leveraging machine learning approaches in order to extract the spectral fingerprints of the active compounds. The optimal conditions for the analysis are determined and tested on two different systems, a synthetic sample where a single active molecule is to be isolated and characterized, and a complex bioactive matrix with synergetic interactions between the components. The method allows the identification of the active compounds and performs a pharmacophoric deconvolution. The program is freely available on the Internet, with an interactive visualisation of the statistical analysis, at https://plasmodesma.igbmc.science.

Complementary enantioselectivity profiles of chiral cinchonan carbamate selectors with distinct carbamate residues and their implementation in enantioselective two-dimensional high-performance liquid chromatography of amino acids.

A cardinal requirement for effective 2D-HPLC separations is sufficient complementarity in the retention profiles of first and second dimension separations. It is shown that retention and enantioselectivity of chiral selectors derived from cinchona alkaloids can be conveniently modulated by structural variation of the carbamate residue of the quinine/quinidine carbamate ligand of such chiral stationary phases (CSP). A variety of aliphatic and aromatic residues have been tested in comparison to non-carbamoylated quinine CSP. Various measures of orthogonality have been utilized to derive the CSP that is most complementary to the tert-butylcarbamoylated quinine CSP (tBuCQN CSP), which is commercially available as Chiralpak QN-AX column. It turned out that O-9-(2,6-diisopropylphenylcarbamoyl)-modified quinine is most promising in this respect. Its implementation as a complementary CSP for the separation of amino acids derivatized with Sanger's reagent (2,4-dinitrophenylated amino acids) in the first dimension combined with a tBuCQN CSP in the second dimension revealed successful enantiomer separations in a comprehensive chiral×chiral 2D-HPLC setup. However, the degree of complementarity could be greatly enhanced when simultaneously the absolute configurations were exchanged from quinine to quinidine in the chiral selector of the first dimension separation resulting in opposite elution orders of the enantiomers in the two dimensions. The advantage of such a chiral×chiral over achiral×chiral 2D-HPLC setup, amongst others, is the perfect compatibility of the mobile phase because in both dimensions the identical eluent can be used.

Quantitative determination of major alkaloids in Cinchona bark by Supercritical Fluid Chromatography.

Chinoline alkaloids found in Cinchona bark still play an important role in medicine, for example as antimalarial and antiarrhythmic drugs. For the first time Supercritical Fluid Chromatography has been utilized for their separation. Six respective derivatives (dihydroquinidine, dihydroquinine, quinidine, quinine, cinchonine and cinchonidine) could be resolved in less than 7 min, and three of them quantified in crude plant extracts. The optimum stationary phase showed to be an Acquity UPC2 Torus DEA 1.7 µm column, the mobile phase comprised of CO2, acetonitrile, methanol and diethylamine. Method validation confirmed that the procedure is selective, accurate (recovery rates from 97.2% to 103.7%), precise (intra-day ≤2.2%, inter-day ≤3.0%) and linear (R2 ≥ 0.999); at 275 nm the observed detection limits were always below 2.5 µg/ml. In all of the samples analyzed cinchonine dominated (1.87%-2.30%), followed by quinine and cinchonidine. Their total content ranged from 4.75% to 5.20%. These values are in good agreement with published data, so that due to unmatched speed and environmental friendly character SFC is definitely an excellent alternative for the analysis of these important natural products.

Effects of quinine on gastric ulcer healing in Wistar rats.

Background Quinine (QT) is an important anti-malarial drug; however, there is little information about its effects on the gut. Therefore, this study aimed to investigate the effects of a therapeutic dose of QT on the healing of gastric ulcer in rats. Methods Male Wistar rats weighing 150-200 g were divided into three groups: control rats without ulcer (group 1), ulcerated rats treated with 1 mL/kg (p.o.) normal saline (NS) (group 2), and ulcerated rats treated with 10 mg/kg (p.o.) QT (group 3). Ulcers were induced by serosal application of 80 % acetic acid to the stomach of rats anaesthetized with 50 mg/kg thiopentone sodium and treatment was given three times daily. Healing was assessed on days 3, 7 and 10 after ulcer induction by macroscopic measurement of: ulcer area, histology, lipid peroxidation, superoxide dismutase activity and gastric mucus secretion. Results At day 3, there was no significant difference (p>0.05) in ulcer areas between NS- and QT-treated rats. By day 10, however, the percentage area healed in NS treated (59.6±2.35 %) was significantly higher (p<0.05) than in QT rats (49.0±2.20 %) and clearing of inflammatory cells and re-epithelization was greater in NS-treated group. By days 7 and 10, lipid peroxidation was significantly higher in QT animals, when compared with NS-treated rats and controls (p<0.05). Superoxide dismutase activity and mucus secretion were significantly (p<0.05) higher in NS-treated than QT-treated rats. Conclusions QT delayed ulcer healing by prolonging the inflammatory phase of healing, increasing oxidative stress, reducing antioxidant activity and gastric mucus secretion.

The Hidden History of a Famous Drug: Tracing the Medical and Public Acculturation of Peruvian Bark in Early Modern Western Europe (c. 1650-1720).

The history of the introduction of exotic therapeutic drugs in early modern Europe is usually rife with legend and obscurity and Peruvian bark is a case in point. The famous antimalarial drug entered the European medical market around 1640, yet it took decades before the bark was firmly established in pharmaceutical practice. This article argues that the history of Peruvian bark can only be understood as the interplay of its trajectories in science, commerce, and society. Modern research has mostly focused on the first of these, largely due to the abundance of medico-historical data. While appreciating these findings, this article proposes to integrate the medical trajectory in a richer narrative, by drawing particular attention to the acculturation of the bark in commerce and society. Although the evidence we have for these two trajectories is still sketchy and disproportionate, it can nevertheless help us to make sense of sources that have not yet been an obvious focus of research. Starting from an apparently isolated occurrence of the drug in a letter, this article focuses on Paris as the location where medical and public appreciation of the bark took shape, by exploring several contexts of knowledge circulation and medical practice there. These contexts provide a new window on the early circulation of knowledge of the bark, at a time when its eventual acceptance was by no means certain.

On the history of Cinchona bark in the treatment of Malaria.

How and when the medical value of Cinchona bark was discovered is obscure, but it is said that the powder was given to a European for malaria for the first time in the 1630s. The bark was brought to Europe by Spanish missionaries and it was recommended by the cardinal Juan de Lugo. In the 1660s, the use of Cinchona bark became known in England - and in Denmark by Thomas Bartholin. It was used for the treatment of malaria, but several debates on its value continued up to the 1730s. However, successful treatment of malaria was obtained by Thomas Sydenham, Robert Tabor and Francesco Torti. Sydenham emphasized a modern view that Cinchona bark was a unique specific drug for the treatment of malaria, and the treatment was fully accepted when Torti's Therapeutice specialis appeared. In the early 18th century, botanical expeditions were arranged in search of the most valuable Cinchona species for cultivation. The content of quinine was impor- tant, and determination of quinine was realized when Pierre Pelletier and Joseph Caventou isolated the alkaloid from the bark in 1820. Dutch plantations and quinine industry dominated the market, but the supply of quinine came to an end when the Japanese occupied Indonesia in 1942, cutting off the rest of the world from the main supplies of Cinchona. Synthetic antimalarials were developed and chloroquine became the drug of choice, but the intensive use of these drugs caused drug resistance. Chloroquine-resistant strains of P. falciparum are now treated with other drugs as artemisinin and artemether.

Composition of the endophytic filamentous fungi associated with Cinchona ledgeriana seeds and production of Cinchona alkaloids.

Four kinds of endophytic filamentous fungi (code names: CLS-1, CLS-2, CLS-3, and CLS-4) associated with the seeds of Cinchona ledgeriana (Rubiaceae) from West Java, Indonesia, were isolated. All of the isolates were classified into Diaporthe spp. based on phylogenetic analysis of the nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) including the 5.8S ribosomal DNA region. All four of these endophytic fungi produce Cinchona alkaloids, mainly quinine and quinidine, in synthetic liquid medium.

Comparison between 2 methods of solid-liquid extraction for the production of Cinchona calisaya elixir: an experimental kinetics and numerical modeling approach.

The purpose of this study is to compare the extraction process for the production of China elixir starting from the same vegetable mixture, as performed by conventional maceration or a cyclically pressurized extraction process (rapid solid-liquid dynamic extraction) using the Naviglio Extractor. Dry residue was used as a marker for the kinetics of the extraction process because it was proportional to the amount of active principles extracted and, therefore, to their total concentration in the solution. UV spectra of the hydroalcoholic extracts allowed for the identification of the predominant chemical species in the extracts, while the organoleptic tests carried out on the final product provided an indication of the acceptance of the beverage and highlighted features that were not detectable by instrumental analytical techniques. In addition, a numerical simulation of the process has been performed, obtaining useful information about the timing of the process (time history) as well as its mathematical description.

Science in the service of colonial agro-industrialism: the case of cinchona cultivation in the Dutch and British East Indies, 1852-1900.

The isolation of quinine from cinchona bark in 1820 opened new possibilities for the mass-production and consumption of a popular medicine that was suitable for the treatment of intermittent (malarial) fevers and other diseases. As the 19th century European empires expanded in Africa and Asia, control of tropical diseases such as malaria was seen as crucial. Consequently, quinine and cinchona became a pivotal tool of British, French, German and Dutch empire-builders. This comparative study shows how the interplay between science, industry and government resulted in different historical trajectories for cinchona and quinine in the Dutch and British Empires during the second half of the 19th century. We argue that in the Dutch case the vectors of assemblage that provided the institutional and physical framework for communication, exchange and control represent an early example of commodification of colonial science. Furthermore, both historical trajectories show how the employment of the laboratory as a new device materialised within the colonial context of agricultural and industrial production of raw materials (cinchona bark), semi-finished product (quinine sulphate) and plant-based medicines like quinine. Hence, illustrating the 19th century transition from 'colonial botany' and 'green imperialism' to what we conceptualise as 'colonial agro-industrialism'.

The use of an integrated molecular-, chemical- and biological-based approach for promoting the better use and conservation of medicinal species: a case study of Brazilian quinas.

ETHNOPHARMACOLOGICAL RELEVANCE: Quina is a popular name originally attributed to Cinchona pubescens Vahl (=Cinchona succirubra) and Cinchona. calisaya Wedd., species native from Peru that have the antimalarial alkaloid quinine. In Brazil, bitter barks substitutes for the Peruvian species began to be used centuries ago, and they still are sold in popular markets. To assess the authenticity and the conditions on which samples of quinas have been commercialized, using the DNA barcode, chemical and biological assays. MATERIALS AND METHODS: Starting with 28 samples of barks acquired on a popular market, 23 had their DNA extracted successfully. The regions matK and rbcL were amplified and sequenced for 15 and 23 samples, respectively. Phytochemical analyses were performed by chromatographic methods, and biological essays were done by antimalarial tests in vitro. RESULTS: The identified species belonged to six different families, many of them endangered or with no correlation with use in traditional medicine as a Brazilian quina. The absence of typical bitter chemical substances indicated that barks have been collected from other species or from very young trees. The results of biological essays confirm the lack of standardization of the sold materials. CONCLUSION: The integrated approaches proved to be efficient to evaluate medicinal plants sold in popular markets and can be useful for promoting their better use and conservation.

Chemical modifications of cinchona alkaloids lead to enhanced inhibition of human butyrylcholinesterase.

Butyrylcholinesterase (BChE) inhibitors were identified from a collection containing cinchonine, cinchonidine and synthetic derivatives, and further characterized using cytotoxicity and molecular docking studies. The most active ones were: (10 triple bond)-10,11-dibromo-10,11-dihydrocinchonidine (11), a competitive inhibitor with Ki = 3.45 +/- 0.39 microM, and IC50 BChE = 9.83 +/- 0.30 microM/human (h)BChE = 34.47 +/- 4.63 and O-(trimethylsilyl)cinchonine (15), a mixed inhibitor with Kiuc = 1.73 +/- 0.46 microM and Kic = 0.85 +/- 0.26 microM, and IC50 BChE = 0.56 +/- 0.14 microM/hBChE = 0.24 +/- 0.04. In cytotoxicity experiments, > or = 80% of the cells remained viable when exposed to concentrations of up to 80 microM of both inhibitors in four different cell lines, including neurons. Due to the bulkier trimethylsilyl side group of 15, it covered the active site of hBChE better than 11 with an OH-group while not being able to fit into the active site gorge of hAChE, thus explaining the selectivity of 15 towards hBChE.

Cinchona alkaloids from Cinchona succirubra and Cinchona ledgeriana.

Seven new cinchona alkaloids, cinchonanines A-G (1-7), and 29 known alkaloids were isolated from the barks of Cinchona surrirubra and C. ledgeriana collected from Yunnan Province in China. The new structures were elucidated by extensive spectroscopic analysis. All compounds were evaluated for their cytotoxicity against five human cancer cell lines. Compounds 2, 13, 14, and 15 showed moderate cytotoxicity.

An Empire's Extract: Chemical Manipulations of Cinchona Bark in the Eighteenth-Century Spanish Atlantic World.

In 1790, the Spanish Crown sent a "botanist-chemist" to South America to implement production of a chemical extract made from cinchona bark, a botanical medicament from the Andes used throughout the Atlantic World to treat malarial fevers. Even though the botanist-chemist's efforts to produce the extract failed, this episode offers important insight into the role of chemistry in the early modern Atlantic World. Well before the Spanish Crown tried to make it a tool of empire, chemistry provided a vital set of techniques that circulated among a variety of healers, who used such techniques to make botanical medicaments useful and intelligible in new ways.

Cuantificación de quinina en extractos de Cinchona pubescens y evaluación de la actividad antiplasmodial y citotóxica / Quantification of quinine in extracts from stems Cinchona pubescens and evaluation of antiplasmodial and cytotoxic activity

Plants belonging to the genus Cinchona L. (Rubiaceae), whose active ingredient is quinine, was used for centuries to treat malaria. Plants of this genus are a potential source of new structural templates in the search for new antimalarial candidates. This study aimed to the identification, quantification of quinine and other metabolites present in extracts of different polarity of the stems of Cinchona pubescens Vahl. , oxoquinovic acid isolation, antiplasmodial activity, and measuring its cytotoxic effect. The results show a high activity to antiplasmodial alkaloids extract (IC50 = 2.20 +/- 0.0325 ug/mL), cytotoxicity (CC50 = 80.2 +/- 12.2 ug/mL), and a quinine content of 21.3+/-0.0247 ppm. The compound known as acid antiplasmodial activity oxoquinovic presented in IC50 = 11.3 +/- 0.741 ug/mL, and cytotoxicity CC50 = 72.4 +/- 3.85 ug/mL. These results motivate phytochemical studies in the search for active structural analogues quinine and quinolinic core as a source of new antimalarial agents.

Las plantas pertenecientes al género Cinchona L. (familia Rubiaceae), cuyo principio activo es la quinina, fueron utilizadas durante siglos para tratar la malaria. Este género es una fuente potencial de nuevas plantillas estructurales en la búsqueda de nuevos candidatos antimaláricos. El presente trabajo tuvo como objetivo la identificación y, cuantificación de la quinina y de otros metabolitos presentes en los extractos de diferente polaridad, de los tallos de Cinchona pubescens Vahl. , el aislamiento del ácido oxoquinóvico, la actividad antiplasmodial y, además, la medición de su efecto citotóxico. Los resultados muestran una alta actividad antiplasmodial para el extracto de los alcaloides (IC50 = 2,20 +/- 0,0325 ug/mL), una baja citotoxicidad (CC50 = 80,2 +/- 12,2 ug/mL), y un alto contenido de quinina el cual fue 21,3+/-0,0247 ppm. El compuesto ácido oxoquinóvico presentó una actividad antiplasmodial de IC50 = 11,3 +/- 0.741 ug/mL, y una citotoxicidad de CC50 = 72,4 +/- 3,85 ug/mL. Estos resultados motivan los estudios fitoquímicos en la búsqueda de principios activos y análogos estructurales en diferentes especies de Cinchonas como una fuente de nuevos agentes antimaláricos.