Phenolic variation among Chamaecrista nictitans subspecies and varieties revealed through UPLC-ESI(-)-MS/MS chemical fingerprinting.

INTRODUCTION: Comparative analysis of metabolic features of plants has a high potential for determination of quality control of active ingredients, ecological or chemotaxonomic purposes. Specifically, the development of efficient and rapid analytical tools that allow the differentiation among species, subspecies and varieties of plants is a relevant issue. Here we describe a multivariate model based on LC-MS/MS fingerprinting capable of discriminating between subspecies and varieties of the medicinal plant Chamaecrista nictitans, a rare distributed species in Costa Rica. METHODS: Determination of the chemical fingerprint was carried out on a LC-MS (ESI-QTOF) in negative ionization mode, main detected and putatively identified compounds included proanthocyanidin oligomers, several flavonoid C- and O-glycosides, and flavonoid acetates. Principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA) and cluster analysis of chemical profiles were performed. RESULTS: Our method showed a clear discrimination between the subspecies and varieties of Chamaecrista nictitans, separating the samples into four fair differentiated groups: M1 = C. nictitans ssp. patellaria; M2 = C. nictitans ssp. disadena; M3 = C. nictitans ssp. nictitans var. jaliscensis and M4 = C. nictitans ssp. disadena var. pilosa. LC-MS/MS fingerprint data was validated using both morphological characters and DNA barcoding with ITS2 region. The comparison of the morphological characters against the chemical profiles and DNA barcoding shows a 63% coincidence, evidencing the morphological similarity in C. nictitans. On the other hand, genetic data and chemical profiles grouped all samples in a similar pattern, validating the functionality of our metabolomic approach. CONCLUSION: The metabolomic method described in this study allows a reliably differentiation between subspecies and varieties of C. nictitans using a straightforward protocol that lacks extensive purification steps.

New Botanical Anxiolytics for Use in Companion Animals and Humans.

As part of our ongoing research into botanical therapies for anxiety disorders, the neotropical vine Souroubea sympetala was chosen for study as a phytochemical discovery strategy focusing on rare Central American plant families. When orally administered to male Sprague-Dawley rats, the crude plant extract, its ethyl acetate fraction, supercritical carbon dioxide fraction, or its isolated triterpenes reduced anxiety and/or fear-related behavior in standardized behavioral models. Pharmacological studies showed that the extracts acted at the benzodiazepine GABAA receptor and reduced corticosterone levels. A preparation containing Souroubea fortified with a second triterpene containing plant, Platanus occidentalis, was shown to be safe in a 28-day feeding trial with beagles at 5 times the intended dose. Subsequent trials with beagles in a thunderstorm model of noise aversion showed that the material reduced anxiety behaviors and cortisol levels in dogs. The formulation has been released for the companion animal market in Canada and the USA under the Trademark "Zentrol." Ongoing research is exploring the use of the material in treatment of anxiety and post-traumatic stress in humans.

Antifungal Saponins from the Maya Medicinal Plant Cestrum schlechtendahlii G. Don (Solanaceae).

Bioassay-guided fractionation of the crude extract (80% EtOH) of the leaves of Cestrum schlechtendahlii, a plant used by Q'eqchi' Maya healers for treatment of athlete's foot, resulted in the isolation and identification of two spirostanol saponins (1 and 2). Structure elucidation by MS, 1D-NMR, and 2D-NMR spectroscopic methods identified them to be the known saponin (25R)-1ß,2α-dihydroxy-5α-spirostan-3-ß-yl-O-α-L-rhamnopyranosyl-(1 → 2)-ß-D-galactopyranoside (1) and new saponin (25R)-1ß,2α-dihydroxy-5α-spirostan-3-ß-yl-O-ß-D-galactopyranoside (2). While 2 showed little or no antifungal activity at the highest concentration tested, 1 inhibited growth of Saccharomyces cerevisiae (minimum inhibitory concentration (MIC) of 15-25 µM), Candida albicans, Cryptococcus neoformans, and Fusarium graminearum (MIC of 132-198 µM).

Inhibition of bacterial quorum sensing and biofilm formation by extracts of neotropical rainforest plants.

Bacterial biofilms are responsible for many persistent infections by many clinically relevant pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. Biofilms are much more resistant to conventional antibiotics than their planktonic counterparts. Quorum sensing, an intercellular communication system, controls pathogenesis and biofilm formation in most bacterial species. Quorum sensing provides an important pharmacological target since its inhibition does not provide a selective pressure for resistance. In this study, we investigated the quorum sensing and biofilm inhibitory activities of 126 plant extracts from 71 species collected from neotropical rainforests in Costa Rica. Quorum sensing and biofilm interference were assessed using a modified disc diffusion bioassay with Chromobacterium violaceum ATCC 12,472 and a spectrophotometric bioassay with Pseudomonas aeruginosa PA14, respectively. Species with significant anti-quorum sensing and/or anti-biofilm activities belonged to the Meliaceae, Melastomataceae, Lepidobotryaceae, Sapindaceae, and Simaroubaceae families. IC50 values ranged from 45 to 266 µg/mL. Extracts of these active species could lead to future development of botanical treatments for biofilm-associated infections.

Hypoglycemic and antihyperglycemic effect of Witheringia solanacea in normal and alloxan-induced hyperglycemic rats.

AIM OF THE STUDY: Witheringia solanacea is a small shrub that belongs to the Solanaceae family. The plant is used as an antidiabetic in Costa Rican herbal medicine. The aim of this study was to evaluate the hypoglycemic and antihyperglycemic activity of the aqueous extract of W. solanacea leaves in rodent models. MATERIALS AND METHODS: A crude extract of W. solanacea leaves was prepared in boiling water and the aqueous filtrate was lyophilized. A single oral dose of 250, 500 and 1000 mg/kg of the extract was evaluated for hypoglycemic activity in a glucose tolerance test in normal rats and for antihyperglycemic activity in alloxan-induced (140 mg/kg) diabetic rats. The blood glucose level was determined at different times by the glucose oxidase method. RESULTS: Dosage of 500 and 1000 mg/kg of the extract significantly decreased (p<0.05) blood glucose levels in the glucose tolerance test in normal rats after 1 h, there was no significant difference observed at 250 mg/kg. Dose of 500 mg/kg of the extract significantly reduced (p<0.05) blood glucose levels in alloxan induced hyperglycemic rats at 4 and 5 h. CONCLUSIONS: In the present study, the hypoglycemic and antihyperglycemic potential of the W. solanacea was demonstrated in rats. These results give support to the traditional use of W. solanacea as antidiabetic herbal medicine.

Anxiolytic activity of a supercritical carbon dioxide extract of Souroubea sympetala (Marcgraviaceae).

The purpose of this work was to develop an extraction technique to yield a betulinic acid-(BA) enriched extract of the traditional anti-anxiety plant Souroubea sympetala Gilg (Marcgraviaceae). Five extraction techniques were compared: supercritical carbon dioxide extraction (SCE), conventional solvent extraction with ethyl acetate (EtOAc), accelerated solvent extraction (ASE), ultrasonic assisted extraction (UAE) and soxhlet extraction (Sox). The EtOAc and SCE extraction methods resulted in BA-enriched extracts, with BA concentrations of 6.78 ± 0.2 and 5.54 ± 0.2 mg/g extract, respectively, as determined by HPLC-APCI-MS. The bioactivity of the BA-enriched extracts was compared in the elevated plus maze (EPM), a validated rodent anxiety behaviour assay. Rats orally administered a 75 mg/kg dose of SCE extract exhibited anxiolysis as compared with vehicle controls, with a 50% increase in the percent time spent in the open arms, a 73% increase in unprotected head dips and a 42% decrease in percent time spent in the closed arms. No significant differences were observed between the SCE and EtOAc extracts for these measures, but the animals dosed with SCE extract had significantly more unprotected head dips than those dosed with the EtOAc extract. The SCE extract demonstrated a dose-response in the EPM, with a trend toward decreased anxiety at 25 mg/kg, and significant anxiolysis was only observed at 75 mg/kg dose. This study demonstrates that SCE can be used to generate a betulinic acid-enriched extract with significant anxiolysis in vivo. Further, the study provides a scientific basis for the ethnobotanical use of this traditional medicine and a promising lead for a natural health product to treat anxiety.

Ethnopharmacology of Q'eqchi' Maya antiepileptic and anxiolytic plants: effects on the GABAergic system.

ETHNOPHARMACOLOGICAL RELEVANCE: The Q'eqchi' Maya possess a large selection of plants to treat neurological disorders, including epilepsy and susto (fright), a culture-bound illness related to anxiety disorders. AIM OF THE STUDY: To investigate the activity of antiepileptic and anxiolytic plants in the GABAergic system, and determine if there is a pharmacological basis for plant selection. MATERIALS AND METHODS: Ethanol extracts of 34 plants were tested in vitro for their ability to inhibit GABA-transaminase (GABA-T) or bind to the GABA(A)-benzodiazepine (BZD) receptor, two principal drug targets in epilepsy and anxiety. Pharmacological activity was correlated with relative frequency of use, based on informant consensus. RESULTS: Ten plants showed greater than 50% GABA-T inhibition at 1mg/ml, while 23 showed greater than 50% binding to the GABA(A)-BZD receptor at 250 microg/ml. Piperaceae, Adiantaceae and Acanthaceae families were highly represented and active in both assays. There was a significant positive correlation between GABA-T inhibition and relative frequency of use for epilepsy, and an even stronger correlation between GABA(A) binding and relative frequency of use for susto (fright). CONCLUSIONS: Clearly, Q'eqchi' traditional knowledge of antiepileptic and anxiolytic plants is associated with the use of pharmacologically active plants. Based on the evidence, it is suggested that the mechanism of action for some traditionally used plants may be mediated through the GABAergic system.

Cuatro compuestos nuevos del extracto no polar de la planta Amyris brenesii (Rutaceae) de Costa Rica

Four new compounds from the non-polar extract of the plant Amyris brenesii (Rutaceae) from Costa Rica. Fractionation of a non polar extract of the aerial parts of Amyris brenesii collected in Río Cuarto, Grecia, Costa Rica has resulted in the isolation of four new compounds, 6-hidroxy-6-O-(3-hidroxymethyl-3methylalyl)-angelicin 1, 6-(N-acetyl-2-etanamin)-2,2-dimethyl-2H-cromen 2, the lignan 2,5-dehidrohinokinin 3 and N-acetyl-O-(geranyl)-tiramine 4. In addition, we isolated six previously known compounds: the lignans hinokinin 5 and Justicidin E 6, the coumarins scopoletin 7 and marmesin 8, 24-moretenoic acid 9, and the nitrogen compound O-(3,3-dimethylalyl)-halfordinol 10. All the separations were done with chromatographic techniques and the structures were elucidated by using 1D and 2D NMR techniques. Rev. Biol. Trop. 56 (3): 1043-1052. Epub 2008 September 30.

El estudio fitoquímico de las partes aéreas de Amyris brenesii (Rutaceae) recolectadas en Río Cuarto, Grecia, Alajuela (Costa Rica) mostró la presencia de cuatro nuevos compuestos: la 6-hidroxi-6-O-(3-hidroximetil-3-metilalil)angelicina 1, el 6-(N-acetil-2-etanamin)-2,2-dimetil-2Hcromeno 2, el lignano 2,5-deshidrohinokinina 3 y la N-acetil-O-(geranil)-tiramina 4. Adicionalmente se aislaron los lignanos hinokinina 5, y justicidina E 6, las cumarinas escopoletina 7 y marmesina 8, el ácido 24-moretenoico 9 y el O-(3,3-dimetilalil)-halfordinol 10. Las separaciones se llevaron a cabo mediante la aplicación de técnicas cromatográficas y la elucidación de las estructuras se realizó con la ayuda de técnicas espectroscópicas de Resonancia Magnética Nuclear (RMN) de una y dos dimensiones.

[Four new compounds from the non-polar extract of the plant Amyris brenesii (Rutaceae) from Costa Rica]. / Cuatro compuestos nuevos del extracto no polar de la planta Amyris brenesii (Rutaceae) de Costa rica.

Fractionation of a non polar extract of the aerial parts of Amyris brenesii collected in Rio Cuarto, Grecia, Costa Rica has resulted in the isolation of four new compounds, 6-hidroxy-6-O-(3-hidroxymethyl-3-methylalyl)-angelicin 1, 6-(N-acetyl-2-etanamin)-2,2-dimethyl-2H-cromen 2, the lignan 2,5-dehidrohinokinin 3 and N-acetyl-O-(geranyl)-tiramine 4. In addition, we isolated six previously known compounds: the lignans hinokinin 5 and Justicidin E 6, the coumarins scopoletin 7 and marmesin 8, 24-moretenoic acid 9, and the nitrogen compound O-(3,3-dimethylalyl)-halfordinol 10. All the separations were done with chromatographic techniques and the structures were elucidated by using 1D and 2D NMR techniques.

Edema induced by Bothrops asper (Squamata: Viperidae) snake venom and its inhibition by Costa Rican plant extracts

We tested the capacity of leaf (Urera baccifera, Loasa speciosa, Urtica leptuphylla, Chaptalia nutans, and Satureja viminea) and root (Uncaria tomentosa) extracts to inhibit edema induced by Bothrops asper snake venom. Edema-forming activity was studied plethysmographically in the rat hind paw model. Groups of rats were injected intraperitoneally with various doses of each extract and, one hour later, venom was injected subcutaneously in the right hind paw. Edema was assessed at various time intervals. The edematogenic activity was inhibited in those animals that received an injection U. tomentosa, C. nutans or L. speciosa extract. The extract of U. baccifera showed a slight inhibition of the venom effect. Extract from S. viminea and, to a lesser extent that of U. leptuphylla, induced a pro-inflammatory effect, increasing the edema at doses of 250 mg/kg at one and two hours.

Se investigó la capacidad de los extractos de las hojas de Urera baccifera, Loasa speciosa, Urtica leptuphylla, Chaptalia nutans, Satureja viminea y de la raíz de Uncaria tomentosa para inhibir el edema inducido por el veneno de Bothrops asper por métodos pletismométricos. Los grupos de ratas fueron inyectados intraperitonealmente con varias dosis de cada extracto y una hora mas tarde se inyectó veneno por vía subcutánea en la pata trasera derecha de la rata. Se evaluó el edema en distintos intervalos de tiempo. Los resultados muestran que la actividad edematogénica fue inhibida en los animales que recibieron los extractos de raíz de U. tomentosa, hojas de C. nutans y L. speciosa. Los extractos de hojas de U. baccifera mostraron leve inhibición del efecto del veneno. El extracto de hojas de S. viminea y en menor grado el de U. leptuphylla indujeron un efecto pro inflamatorio.

Edema induced by Bothrops asper (Squamata: Viperidae) snake venom and its inhibition by Costa Rican plant extracts.

We tested the capacity of leaf (Urera baccifera, Loasa speciosa, Urtica leptuphylla, Chaptalia nutans, and Satureja viminea) and root (Uncaria tomentosa) extracts to inhibit edema induced by Bothrops asper snake venom. Edema-forming activity was studied plethysmographically in the rat hind paw model. Groups of rats were injected intraperitoneally with various doses of each extract and, one hour later, venom was injected subcutaneously in the right hind paw. Edema was assessed at various time intervals. The edematogenic activity was inhibited in those animals that received an injection U. tomentosa, C. nutans or L. speciosa extract. The extract of U. baccifera showed a slight inhibition of the venom effect. Extract from S. viminea and, to a lesser extent that of U. leptuphylla, induced a pro-inflammatory effect, increasing the edema at doses of 250 mg/kg at one and two hours.

Analysis of Piperaceae germplasm by HPLC and LCMS: a method for isolating and identifying unsaturated amides from Piper spp extracts.

A method for extraction and high performance liquid chromatography-mass spectrometer (HPLC-MS) analysis of the medicinally important genus Piper (Piperaceae) was developed. This allows for a rapid and accurate measure of unsaturated amides, or piperamides, in black pepper, Piper nigrum L., and in wild species from Central America. Reflux extraction provided the highest recovery of piperine (>80%) from leaf and peppercorn material. HPLC analysis using a binary gradient of acetonitrile and water separated the major amide peaks between 5 and 12 min. Atmospheric pressure chemical ionization (APCI)-MS improved the detection limit to 0.2 ng, 10-fold below the 2 ng limit of the HPLC-diode array detector (DAD) based on linear standard curves between 0.1 and 250 microg/mL (R2 = 0.999). The HPLC-MS method identified pellitorine, piperylin, 4,5-dihydropiperlonguminine, piperlonguminine, 4,5-dihydropiperine, piperine, and pipercide. The biological activity of six Costa Rican Piper species assessed by mosquito larval bioassays correlated well with piperamide content.