Angela I. Calderón

Development of an Ultrafiltration-Liquid Chromatography/Mass Spectrometry (UF-LC/MS) Based Ligand-Binding Assay and an LC/MS Based Functional Assay for Mycobacterium tuberculosis Shikimate Kinase

Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets in the discovery of antimicrobial agents. In the current study, an ultrafiltration-liquid chromatography/mass spectrometry (UF-LC/MS) ligand based binding assay and an LC/MS based functional assay for Mycobacterium tuberculosis shikimate kinase (MtSK) were developed. Compounds 1, 2, 3, and 4 were tested for MtSK (1 microM) at a concentration of 1 microM. In order to evaluate the MtSK inhibitory activity, compounds 1-4 were tested at concentrations ranging from 0.05 to 1 microM, and the enzymatic activity was assessed by quantifying shikimate-3-phosphate (S3P) by LC/MS after 60 min incubation with 2 mM shikimic acid as a substrate. The EC(50) values of compounds 1, 2, 3, and 4 were 0.30, 0.24, 0.07, and 0.18 microM, respectively. The ligands and the S3P were analyzed using positive and negative electrospray LC/MS, respectively. The calibration curve for S3P was prepared with concentrations ranging from 4 to 125 microg/mL, and the lower detection limit (LOD) of S3P was identified as 1.95 microg/mL (9.75 ng on-column). This is the first application of UF-LC/MS and LC/MS in the development of ligand-binding and functional assays, respectively as a useful approach to screen MtSK inhibitors.

Forest plot as a tool to demonstrate the pharmaceutical potential of plants in a Tropical Forest of Panama

Based on literature analysis of 308 angiosperm species inventoried from a 50-hectare forest plot on Barro Colorado Island, Panama, 40 species were selected and 80 samples (two samples for every species; leaf + twig and stembark samples) were collected for investigation of their medicinal/pharmaceutical potential. Extracts of these 80 samples were tested in bioassay systems to assess cancer chemoprevention (eight assays), antiplasmodial, cytotoxicity, and anti-HIV activities. Details of bioassay techniques are described. Of the 40 species, 12 (30%) showed activity in one or more of the 11 bioassay systems used. These active species areBombacopsis (= Pachira) quinata, Calophyllum longifolium, Casearia commersoniana, Lozania pittieri, Maclura tinctoria, Mouriri myrtilloides, Olmedia aspera (= Trophis caucana), Pseudobombax septenatum, Spondias radlkoferi, Stylogyne standleyi, Turpinia occidentalis, andVochysia ferruginea. Because literature data on the chemistry ofBombacopsis (= Pachira) quinata, Lozan ia pittieri, Mouriri myrtilloides, Olmedia aspera (= Trophis caucana), Pseudobombax septenatum, andStylogyne standleyi, are lacking, and similar data on the other six species are deficient, further fractionation and isolation work on these active species potentially may yield novel, biologically active structures. This study demonstrates that a plot-based selection of plant species for evaluation of their potential medicinal/ pharmaceutical value has merit in achieving such a goal, and should be used in a program on plant drug discovery.ResumenA base de análisis de 308 especies de angiospermas inventariadas de una parcela de 50 hectáreas de un bosque en la Isla de Barro Colorado, Panama, 40 especies fueron seleccionadas y 80 muestras (hojas + ramitas y corteza de tallo, de coda especie) fueron coleccionadas para la investigaci00F3;n hacia su potencial medicinal/farmaceútico. Los extrados de estas 80 muestras fueron suministrados a bioensayos para detectar sus actividades en la prevención de cancer (8 tipos de ensayos), antipalúdica, citotoxicidad, y anti-HIV. Los detalles de los métodos de bioensayos se presentan. De las 40 especies, 12 (30%) demostraron actividad en uno o más de los 11 bioensayos empleados. Estas especies sonBombacopsis (= Pachira) quinata, Calophyllum longifolium, Casearia commersoniana, Lozania pittieri, Maclura tinctoria, Mouriri myrtilloides, Olmedia aspera (= Trophis caucana), Pseudobombax septenatum, Spondias radlkoferi, Stylogyne standleyi, Turpinia occidentalis, y Vochysia ferruginea. En vista de queBombacopsis (= Pachira) quinata, Lozania pittieri, Mouriri myrtilloides, Olmedia aspera (= Trophis caucana), Pseudobombax septenatum, y Stylogyne standleyi, carecen de datos químicos de la literatura, mientras que datos químicos sobre las otras seis especies son deficientes, un trabajo de fraccionamiento y aislamiento sobre estas especies potencialmente nos pueda dar compuestos novedosos, biologicamente activos. Este estudio demuestra que el método de selectión de especies de plantas a partir de especies inventariadas de una parcela de bosque, para someterlas a pruebas biológicas, tiene sus méritos para empleo más amplio.

Liquid chromatography/mass spectrometry based fingerprinting analysis and mass profiling of Euterpe oleracea (açaí) dietary supplement raw materials.

Chemical fingerprinting and mass profiling methods to identify biologically active compounds in botanical dietary supplements is gaining much attention in recent years. Euterpe oleracea (açaí) has been reported to be rich in health-beneficial chemical constituents. We have developed LC/MS based fingerprinting and mass profiling methods to identify fatty acids, anthocyanins and non-anthocyanin polyphenols in three processed raw materials; non-organic açaí powder (ADSR-1), raw-organic açaí powder (ADSR-2) and freeze-dried açaí powder (ADSR-3) that are used in the preparation of botanical dietary supplements. For LC/MS analysis of fatty acids and non-anthocyanin polyphenols, the açaí samples were extracted sequentially with dichloromethane followed by methanol. To study fingerprinting analysis of anthocyanins, açaí samples were extracted with acidic methanol-water. The LC separation of fatty acids, non-anthocyanin polyphenols and anthocyanins in açaí raw materials was achieved using a C18 column with a gradient mobile phase consisting of solvents A (0.1% formic acid in water), and B (0.1% formic acid in methanol). MS experiments were carried out with negative and positive mode electrospray ionization. LC/MS analysis of dichloromethane extracts of (ADSR-1), (ADSR-2) and (ADSR-3) açaí powders have shown to contain fatty acids, γ-linolenic acid, linoleic acid, palmitic acid, and oleic acid. Whereas, the fingerprinting analysis of methanol extracts of ADSR-1, ADSR-2 and ADSR-3 led to the identification of phenolic acids, anthocyanin and non-anthocyanin polyphenols. The results from our study may be useful for the authentication and quality assessment of açaí dietary supplement raw materials.

Development of binding assays to screen ligands for Plasmodium falciparum thioredoxin and glutathione reductases by ultrafiltration and liquid chromatography/mass spectrometry

To identify potential lead compounds for malaria drug discovery, ultrafiltration and liquid chromatography and mass spectrometry (UF and LC/MS) based binding assays were developed for the first time for Plasmodium falciparum thioredoxin (PfTrxR) and glutathione (PfGR) reductases. In the binding assays, curcuminoids (bis-demethoxycurcumin 1, demethoxycurcumin 2, and curcumin 3) were used to study the binding affinity for PfTrxR and PfGR enzymes. The optimum binding was observed when the curcumimoids mixture (1 microM) was incubated with 1 microM PfTrxR and 0.5 microM PfGR enzymes separately for 60 min at 25 degrees C. The peak areas of the ligands in the chromatogram corresponding to incubation with active PfTrxR and PfGR enzymes increased by 1.6- and 2.0-fold respectively compared to the chromatogram of test compounds incubated with denatured enzymes. Further, binding assay experiments were carried out for compound 2 under non-competitive and competitive incubation conditions with 1 microM PfTrxR and 0.5 microM PfGR enzymes, separately. The binding affinity of compound 2 was higher for both the enzymes under non-competitive incubation conditions. To validate the binding assay developed, we have tested bis-2,4-dinitrophenyl sulfide (4) which is reported as an inhibitor of PfTrxR and PfGR enzymes. Compound 4 showed greater binding affinity for both enzymes under competitive incubation conditions. The relative peak area of compound 4 increased by 3.2- and 6-fold when incubated with active PfTrxR (1 microM) and PfGR (0.5 microM) enzymes respectively compared to the peak areas of the compound in control experiments. The current method developed has a potential for automated high-throughput screening to rapidly determine the binding affinity of ligands for these enzymes.

Screening of Latin American plants for antiparasitic activities against malaria, Chagas disease, and leishmaniasis.

In order to explore rationally the medical potential of the plant biodiversity of the Central and South American region as a source of novel antiparasitic molecules, a multinational Organization of American States (OAS) project, which included the participation of multidisciplinary research centers from Argentina, Bolivia, Colombia, Costa Rica, Guatemala, Nicaragua and Panama, was carried out during the period 2001-2004. This project aimed at screening organic plant extracts for antitrypanosomal, antileishmanial and antimalarial activities and subsequently isolating and characterizing bioactive molecules. Plants for antiparasitic screening were selected from a database of ethnomedical uses of Latin American plants (PlanMedia) based on the amount of biological and chemical information available in the literature. We report here the evaluation of 452 extracts from 311 plant species in vitro screens against Plasmodium falciparum, Leishmania mexicana, and Trypanosoma cruzi. Out of 311 species tested, 17 plants (5.4%) showed antiparasitic activities at IC50 values ≤ 10 µg/mL. The most active plants were Acnistus arborescens (L.) Schltdl. (Solanaceae) (leaf, EtOH, IC50: 4 µg/mL) Monochaetum myrtoideum Naudin (Melastomataceae) (leaf, MeOH, IC50: 5 µg/mL) and Bourreria huanita (Lex.) Hemsl. (Boraginaceae) (branch, EtOH, IC50: 6 µg/mL). These were selectively active against P. falciparum, L. mexicana and T. cruzi, respectively.

Screening of natural compounds for ligands to PfTrxR by ultrafiltration and LC-MS based binding assay.

In our study, we have screened 133 structurally diverse natural compounds from the MEGx® collection of AnalytiCon Discovery and three synthetic hispolone analogs for binding affinity to Plasmodium falciparum thioredoxin reductase (PfTrxR) using an ultrafiltration (UF) and liquid chromatography (LC/MS) based ligand-binding assay newly developed in our laboratory. PfTrxR catalyzes the reduction of thioredoxin (PfTrx) protein. In reduced form, PfTrx is essentially involved in the antioxidative defense and redox regulation of P. falciparum. Nine compounds (yohimbine (1), catharanthine (2), vobasine (3), gnetifolin E (4), quinidine N-oxide (5), 11-hydroxycoronaridine (6), hispolone (7), hispolone methyl ether (8), and hernagine (9)) displayed binding affinity for PfTrxR at 1μM. The ranking order of compounds binding affinities for PfTrxR is 7>6>2>4>5>8>1>9>3. On the other hand, compounds 6, 7, 2 and 8 demonstrated specific binding to the active site of PfTrxR, when ligands were tested in an equimolar mixture of 1 μM.

Chemical Adulterants in Herbal Medicinal Products: A Review

Many herbal medicinal products have been found to contain synthetic prescription drugs as chemical adulterants. This has become evident by the number of toxicity cases and adverse reactions reported in which casualties were reported via analytical techniques that detected the presence of chemical adulterants in them, which could be responsible for their toxicity. The adulteration of herbal medicinal products with synthetic drugs continues to be a serious problem for regulatory agencies. This review provides up to date information on cases of toxicity, major chemical adulterants in herbal medicinal products, and current analytical techniques used for their detection.

Quantitative analysis of anthocyanins in Euterpe oleracea (açaí) dietary supplement raw materials and capsules by Q-TOF liquid chromatography/mass spectrometry

Context: Euterpe oleracea Mart. (Arecaceae) fruits and their dietary supplements are gaining much popularity internationally. Anthocyanins and their aglycons are responsible for the dense color of açaí fruit and are associated with a wide spectrum of health promoting effects. Objective: Quantitative analysis of anthocyanins in açaí dietary supplement raw materials; processed açaí powder (ADSR-1), organic açaí powder (ADSR-2), and nonorganic açaí powder (ADSR-3) by quadrupole-time-of-flight liquid chromatography/mass spectrometry (Q-TOF LC/MS) have been reported in this study. Materials and methods: The chromatographic separation for anthocyanins was achieved using a C-18 column with a gradient of 0.1% formic acid in water and 0.1% formic acid in methanol and acetonitrile (50:50, v/v). MS and MS/MS experiments were carried out on an electrospray ionization-Q-TOF LC/MS. Results: Except for ASDR-2, all the açaí samples were found to have cyanidin 3-glucoside (1), cyanidin 3-sambubioside (2), cyanidin 3-rutinoside (3), and peonidin 3-rutinoside (4). ASDR-2 contained anthocyanins 1 and 3. Among the açaí samples quantified, ADSR-3 showed higher concentration of anthocyanins compared to other raw materials and capsules tested in this study. Discussion and conclusion: The anthocyanins 1–4 present in ADSR-3 were 27.13 ± 0.37, 1.76 ± 0.04, 31.07 ± 0.49, and 3.46 ± 0.08 mg/100 g dry wt, respectively. The LOQ values for anthocyanins 1–4 were in the range of 2.44–9.76 ng/mL. Accuracy of the method was assessed by performing a recovery experiments. The intraday and interday variations (RSDs) were <10%. This is the first report on quantitation of anthocyanins in açaí dietary supplement raw materials and capsules.

Identification of oleamide in Guatteria recurvisepala by LC/MS-based Plasmodium falciparum thioredoxin reductase ligand binding method.

Our current research on applications of mass spectrometry to natural product drug discovery against malaria aims to screen plant extracts for new ligands to Plasmodium falciparum thioredoxin reductase (PfTrxR) followed by their identification and structure elucidation. PfTrxR is involved in the antioxidant defense and redox regulation of the parasite and is validated as a promising target for therapeutic intervention against malaria. In the present study, detannified methanol extracts from Guatteria recurvisepala, Licania kallunkiae, and Topobea watsonii were screened for ligands to PfTrxR using ultrafiltration and liquid chromatography/mass spectrometry-based binding experiments. The PfTrxR ligand identified in the extract of Guatteria recurvisepala displayed a relative binding affinity of 3.5-fold when incubated with 1 μM PfTrxR. The ligand corresponding to the protonated molecule m/z 282.2792 [M+ H]+ was eluted at a retention time of 17.95 min in a 20-min gradient of 95% B consisting of (A) 0.1%formic acid in 95% H₂O-5% ACN, and (B) 0.1% formic acid in 95% ACN-5% H₂O in an LC-QTOF-MS.Tandem MS of the protonated molecule m/z 282.2792 [M + H]+, C₁₈H₃₆NO (DBE: 2; error: 1.13 ppm) resulted in two daughter ions m/z 265.2516[M + H-NH₃]+ (DBE: 3; error: 0.35 ppm) and m/z 247.2405 [M + H-NH₃-H₂O] +, (DBE: 4; error:2.26 ppm). The PfTrxR ligand was identified as oleamide and confirmed by comparison of the retention time, molecular formula, accurate mass,and double bond equivalence with the standard oleamide. This is the first report on the identification of oleamide as a PfTrxR ligand from Guatteria recurvisepala R. E. Fr. and the corresponding in vitro activity against P. falciparum strain K1 (IC₅₀ 4.29 μg/mL).

Determination of antiplasmodial activity and binding affinity of curcumin and demethoxycurcumin towards PfTrxR

In our study, the inhibitory activity of curcuminoids towards Plasmodium falciparum thioredoxin reductase (PfTrxR) was determined using LC-MS-based functional assay and showed that only demethoxycurcumin (DMC) inhibited PfTrxR (IC50: 2 μM). In silico molecular modelling was used to ascertain and further confirm that the binding affinities of curcumin and DMC are towards the dimer interface of PfTrxR. The in vitro antiplasmodial activities of curcumin and DMC were evaluated and shown to be active against chloroquine (CQ)-sensitive (D6 clone) and moderately active against CQ-resistant (W2 clone) strains of Plasmodium falciparum while no cytotoxicity was observed against Vero cells.