Two new triterpenoid saponins from the flowers of Albizia lebbeck.

The chemical investigation of the fresh flowers of Albizia lebbeck (L.) Benth. (Fabaceae, Mimosoideae) led to the isolation of two new echinocystic acid saponins. They were isolated by using chromatographic methods and their structures were elucidated by detailed 1H and 13C NMR spectral data including 2 D-NMR (COSY, HSQC, HMBC and APT) spectroscopic techniques, high-resolution electrospray ionization mass spectrometry (HRESIMS) and acid hydrolysis. Their structures were established as 16-hydroxy-3-[[O-ß-D-xylopyranosyl-(1→2)-O-α-L-arabinopyranosyl-(1→6)-2-(acetylamino)-2-deoxy-ß-D-glucopyranosyl]oxy]-(3ß,16α)-olean-12-en-28-oic acid O-6-deoxy-α-L-mannopyranosyl-(1→4)-O-6-deoxy-α-L-mannopyranosyl-(1→2)-ß-D-glucopyranosyl ester (1) and 16-hydroxy-3-[[O-ß-D-xylopyranosyl-(1→2)-O-α-L-arabinopyranosyl-(1→6)-2-(acetylamino)-2-deoxy-ß-D-glucopyranosyl]oxy]-(3ß,16α)-olean-12-en-28-oic acid 6-O-[(2S,3R,4R)-tetrahydro-3-hydroxy-4-(hydroxymethyl)-2-furanyl]-ß-D-glucopyranosyl ester (2). Additionally, the permeability property and the capacity of interaction with biological membranes of compounds 1 and 2 were investigated.

Structural characterization of a complex triterpenoid saponin from Albizia lebbeck and investigation of its permeability property and supramolecular interactions with membrane constituents.

As part of the ongoing efforts in discovering potentially bioactive natural products from medicinal plants, the present study was conducted to isolate a new complex triterpenoid saponin from the barks of Albizia lebbeck. It was isolated by using chromatographic methods and its structural elucidation was performed using detailed analyses of 1H and 13C NMR spectra including 2D-NMR (COSY, TOCSY, HSQC and HMBC) spectroscopic techniques, high-resolution electrospray ionization mass spectrometry (HRESIMS) analysis and chemical conversions. Its structure was established as 21-[[(2E,6S)-6-[6-deoxy-4-O-[(2E,6S)-6-hydroxy-2-(hydroxymethyl)-6-methyl-1-oxo-2,7-octadienyl]-[(ß-d-glucopyranosyl)oxy]-2-(hydroxymethyl)-6-methyl-1-oxo-2,7-octadienyl]-[(ß-d-glucopyranosyl)oxy]-2,6-dimethyl-1-oxo-2,7-octadienyl]oxy]-16-hydroxy-3-[[O-ß-d-xylopyranosyl-(1 → 2)-O-α-l-arabinopyranosyl-(1 → 6)-2-(acetylamino)-2-deoxy-ß-d-glucopyranosyl]oxy]-(3ß,16α,21ß)-olean-12-en-28-oic acid O-α-l-arabinofuranosyl-(1 → 4)-O-[ß-d-glucopyranosyl-(1 → 3)]-O-6-deoxy-α-l-mannopyranosyl-(1 → 2)-ß-d-glucopyranosyl ester (1). Additionally, this study aimed to investigate the permeability property of 1, its activity on membrane integrity and supramolecular interactions with cellular constituents using in vitro experimental models.

Substrate and temperature on germination and performance of Albizia niopoides Benth. Seedlings / Substrato e temperatura na germinação e desempenho de plântulas de Albizia niopoides

This study aimed to determine the most appropriate conditions of temperature and substrate for germination and performance of A. niopoides seedlings. The design was completely randomized in 10x7 factorial arrangement (eight constant temperatures of 5, 10, 15, 20, 25, 30, 35, and 40°C and two alternating temperatures of 20-30 and 25-35°C, and seven substrates, that are paper towel, vermiculite, sand, blotting paper, coir dust, sugarcane bagasse, and tropstrato®) with four replicates of 25 seeds. The following variables were analyzed: (1) germination percentage; (2) germination speed index; (3) shoot length and primary root; and (4) dry weight of shoot and of root system. There was a significant interaction among the tested factors for all variables. The A. niopoides seeds germinate under a wide temperature range, with minimum and maximum limit at 10 and 40°C temperatures, respectively. Alternating temperatures of 20-30 and 25-35°C with sand substrate are ideal conditions for germination and performance of A. niopoides seedlings.

Objetivou-se com a realização deste estudo determinar as condições mais adequadas de temperatura e substrato para germinação e desempenho de plântulas de Albizia niopoides. O delineamento utilizado foi o inteiramente casualizado em arranjo fatorial 10x7 (oito temperaturas constantes de 5, 10, 15, 20, 25, 30, 35 e 40°C e duas alternadas 20-30 e 25-35°C e sete substratos: papel tolha, vermiculita, areia, papel mata borrão, pó de coco, bagaço de cana e tropstrato®), com quatro repetições de 25 sementes. Foram analisadas as seguintes variáveis: percentagem de germinação, índice de velocidade de germinação, comprimento de parte aérea e raiz primária e massa seca da parte aérea e do sistema radicular. Houve interação significativa dos fatores testados para as todas as variáveis analisadas. As sementes de A. niopoides germinam em uma ampla faixa de temperatura, com limite mínimo e máximo nas temperaturas de 10 e 40°C, respectivamente. As temperaturas alternadas de 20-30°C e 25-35 °C com o substrato areia são condições ideais para germinação e desempenho de plântulas de A. niopoides.

Substrate and temperature on germination and performance of Albizia niopoides Benth. Seedlings / Substrato e temperatura na germinação e desempenho de plântulas de Albizia niopoides

This study aimed to determine the most appropriate conditions of temperature and substrate for germination and performance of A. niopoides seedlings. The design was completely randomized in 10x7 factorial arrangement (eight constant temperatures of 5, 10, 15, 20, 25, 30, 35, and 40°C and two alternating temperatures of 20-30 and 25-35°C, and seven substrates, that are paper towel, vermiculite, sand, blotting paper, coir dust, sugarcane bagasse, and tropstrato®) with four replicates of 25 seeds. The following variables were analyzed: (1) germination percentage; (2) germination speed index; (3) shoot length and primary root; and (4) dry weight of shoot and of root system. There was a significant interaction among the tested factors for all variables. The A. niopoides seeds germinate under a wide temperature range, with minimum and maximum limit at 10 and 40°C temperatures, respectively. Alternating temperatures of 20-30 and 25-35°C with sand substrate are ideal conditions for germination and performance of A. niopoides seedlings.(AU)

Objetivou-se com a realização deste estudo determinar as condições mais adequadas de temperatura e substrato para germinação e desempenho de plântulas de Albizia niopoides. O delineamento utilizado foi o inteiramente casualizado em arranjo fatorial 10x7 (oito temperaturas constantes de 5, 10, 15, 20, 25, 30, 35 e 40°C e duas alternadas 20-30 e 25-35°C e sete substratos: papel tolha, vermiculita, areia, papel mata borrão, pó de coco, bagaço de cana e tropstrato®), com quatro repetições de 25 sementes. Foram analisadas as seguintes variáveis: percentagem de germinação, índice de velocidade de germinação, comprimento de parte aérea e raiz primária e massa seca da parte aérea e do sistema radicular. Houve interação significativa dos fatores testados para as todas as variáveis analisadas. As sementes de A. niopoides germinam em uma ampla faixa de temperatura, com limite mínimo e máximo nas temperaturas de 10 e 40°C, respectivamente. As temperaturas alternadas de 20-30°C e 25-35 °C com o substrato areia são condições ideais para germinação e desempenho de plântulas de A. niopoides.(AU)

Albizia lebbeck Seed Coat Proteins Bind to Chitin and Act as a Defense against Cowpea Weevil Callosobruchus maculatus.

The seed coat is an external tissue that participates in defense against insects. In some nonhost seeds, including Albizia lebbeck, the insect Callosobruchus maculatus dies during seed coat penetration. We investigated the toxicity of A. lebbeck seed coat proteins to C. maculatus. A chitin-binding protein fraction was isolated from seed coat, and mass spectrometry showed similarity to a C1 cysteine protease. By ELM program an N-glycosylation interaction motif was identified in this protein, and by molecular docking the potential to interact with N-acetylglucosamine (NAG) was shown. The chitin-binding protein fraction was toxic to C. maculatus and was present in larval midgut and feces but not able to hydrolyze larval gut proteins. It did not interfere, though, with the intestinal cell permeability. These results indicate that the toxicity mechanism of this seed coat fraction may be related to its binding to chitin, present in the larvae gut, disturbing nutrient absorption.

Evaluation of some pharmacological activities of Budmunchiamine - A isolated from Albizia amara.

The present investigations were aimed to evaluate the antimicrobial and antioxidant efficacies of budmunchiamine-A (BUA) of Albizia amara . The activity-guided isolation leaded to isolate the bioactive compound budmunchiamine-A from alkaloid extract of A. amara . The budmunchiamine-A showed significant broad-spectrum antimicrobial activity with zone of inhibition (ZOI), minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) values varied from 7.3 to 24.5 mm, 0.95 to 62.5 µg/mL, and 1.9 to 250 µg/mL, respectively. The budmunchiamine-A exhibited moderate antioxidant activity with inhibitory concentration 50% (IC 50 ) value of 400 µg/mL in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and percent inhibition of ß-carotene/linoleic acid was 67.8%. The results suggest the possible use of budmunchiamine-A as a molecular entity for drug development in pharmaceutical industry.

Evaluation of some pharmacological activities of Budmunchiamine - A isolated from Albizia amara

The present investigations were aimed to evaluate the antimicrobial and antioxidant efficacies of budmunchiamine-A (BUA) of Albizia amara. The activity-guided isolation leaded to isolate the bioactive compound budmunchiamine-A from alkaloid extract of A. amara. The budmunchiamine-A showed significant broad-spectrum antimicrobial activity with zone of inhibition (ZOI), minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) values varied from 7.3 to 24.5 mm, 0.95 to 62.5 μg/mL, and 1.9 to 250 μg/mL, respectively. The budmunchiamine-A exhibited moderate antioxidant activity with inhibitory concentration 50% (IC50) value of 400 μg/mL in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and percent inhibition of β-carotene/linoleic acid was 67.8%. The results suggest the possible use of budmunchiamine-A as a molecular entity for drug development in pharmaceutical industry.

Evaluation of some pharmacological activities of Budmunchiamine - A isolated from Albizia amara

The present investigations were aimed to evaluate the antimicrobial and antioxidant efficacies of budmunchiamine-A (BUA) of Albizia amara. The activity-guided isolation leaded to isolate the bioactive compound budmunchiamine-A from alkaloid extract of A. amara. The budmunchiamine-A showed significant broad-spectrum antimicrobial activity with zone of inhibition (ZOI), minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) values varied from 7.3 to 24.5 mm, 0.95 to 62.5 μg/mL, and 1.9 to 250 μg/mL, respectively. The budmunchiamine-A exhibited moderate antioxidant activity with inhibitory concentration 50% (IC50) value of 400 μg/mL in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and percent inhibition of β-carotene/linoleic acid was 67.8%. The results suggest the possible use of budmunchiamine-A as a molecular entity for drug development in pharmaceutical industry.(AU)

[Growth modeling of Albizia niopoides (Mimosaceae) using dendrochronological methods]. / Modelación del crecimiento de Albizia niopoides (Mimosaceae) por métodos dendrocronológicos.

The annual growth rings in tropical trees are fairly common, but their study is relatively recent. Growth rings were found in trees of Albizia niopoides from the Porce River Canyon, Central Cordillera of the Colombian Andes. A total of 33 cross-sections were collected from trees distributed throughout the study area from 664-870masl. Cross-dating, spaguetti plot and 14C analyses were used to demonstrate ring annuality, assuming as hypothesis that these are real annual growth rings. A combination of descriptive analysis of time series (smoothing and pre-whitening) to filter climate noise and nonlinear regression with weighted residuals was used to fit the diameter to Korfs growth model, in which the coefficient of determination reaches values close to 100%. The positive residual autocorrelation of order 1, although not significant, is explained by the existence of energy reserves in the stem and by the accumulation of diameter increments required for the construction of the diameter growth model. The current and mean annual maximum increment rates are 1.03 and 0.94cm/year at ages 18 and 46 years old, respectively. These trees are classified within the group of fast growing species which can reach a cut diameter of over 50cm in approximately 52 years.

Modelación del crecimiento de Albizia niopoides (Mimosaceae) por métodos dendrocronológicos / Growth modeling of Albizia niopoides (Mimosaceae) using dendrochronological methods

The annual growth rings in tropical trees are fairly common, but their study is relatively recent. Growth rings were found in trees of Albizia niopoides from the Porce River Canyon, Central Cordillera of the Colombian Andes. A total of 33 cross-sections were collected from trees distributed throughout the study area from 664-870masl. Cross-dating, spaguetti plot and 14C analyses were used to demonstrate ring annuality, assuming as hypothesis that these are real annual growth rings. A combination of descriptive analysis of time series (smoothing and prewhitening) to filter climate noise and nonlinear regression with weighted residuals was used to fit the diameter to Korf´s growth model, in which the coefficient of determination reaches values close to 100%. The positive residual autocorrelation of order 1, although not significant, is explained by the existence of energy reserves in the stem and by the accumulation of diameter increments required for the construction of the diameter growth model. The current and mean annual maximum increment rates are 1.03 and 0.94cm/year at ages 18 and 46 years old, respectively. These trees are classified within the group of fast growing species which can reach a cut diameter of over 50cm in approximately 52 years.

El crecimiento anual de los anillos en los árboles de los bosques tropicales es bastante frecuente y se evidenció en los árboles de Albizia niopoides, que crecen en el cañón del río Porce, cordillera Central de los Andes colombianos. Se recolectaron 33 secciones transversales de árboles entre 664-870msnm, cortadas a partir de 1.3m de altura del suelo. La hipótesis sobre una periodicidad anual en los anillos fue demostrada mediante cofechado, spaguetti plots y análisis de C14. Para ajustar el diámetro al modelo de crecimiento de Korf se empleó una combinación de análisis descriptivo de series de tiempo (suavizado y pre-whitening); para filtrar el ruido climático y regresión no lineal con residuales ponderados, se alcanzó un coeficiente de determinación cercano al 100%. A pesar de no ser significativa, la autocorrelación serial positiva de orden 1, se explica por la existencia de reservas de energía en el tronco y por la acumulación de los incrementos del diámetro requeridos para la construcción del modelo de crecimiento. Las tasas de crecimiento corriente y medio máximos fueron 1.03 y 0.94cm/año a las edades de 18 y 46 años, respectivamente. Albizia niopoides se clasifica dentro del grupo de rápido crecimiento, las cuales pueden alcanzar diámetros de corta de más de 50cm en aproximadamente 52 años.

Identification of Albizia lebbeck seed coat chitin-binding vicilins (7S globulins) with high toxicity to the larvae of the bruchid Callosobruchus maculatus

Seed coat is a specialized maternal tissue that interfaces the embryo and the external environment during embryogenesis, dormancy and germination. In addition, it is the first defensive barrier against penetration by pathogens and herbivores. Here we show that Albizia lebbeck seed coat dramatically compromises the oviposition, eclosion and development of the bruchid Callosobruchus maculatus. Dietary supplementation of bruchid larvae with A. lebbeck seed coat flour causes severe weight loss and reduces survival. By means of protein purification, mass spectrometry and bioinformatic analyses, we show that chitin-binding vicilins are the main source of A. lebbeck tegumental toxicity to C. maculatus. At concentrations as low as 0.1 percent, A. lebbeck vicilins reduce larval mass from 8.1 ± 1.7 (mass of control larvae) to 1.8 ± 0.5 mg, which corresponds to a decrease of 78 percent. Seed coat toxicity constitutes an efficient defense mechanism, hindering insect predation and preventing embryo damage. We hypothesize that A. lebbeck vicilins are good candidates for the genetic transformation of crop legumes to enhance resistance to bruchid predation.

Identification of Albizia lebbeck seed coat chitin-binding vicilins (7S globulins) with high toxicity to the larvae of the bruchid Callosobruchus maculatus.

Seed coat is a specialized maternal tissue that interfaces the embryo and the external environment during embryogenesis, dormancy and germination. In addition, it is the first defensive barrier against penetration by pathogens and herbivores. Here we show that Albizia lebbeck seed coat dramatically compromises the oviposition, eclosion and development of the bruchid Callosobruchus maculatus. Dietary supplementation of bruchid larvae with A. lebbeck seed coat flour causes severe weight loss and reduces survival. By means of protein purification, mass spectrometry and bioinformatic analyses, we show that chitin-binding vicilins are the main source of A. lebbeck tegumental toxicity to C. maculatus. At concentrations as low as 0.1%, A. lebbeck vicilins reduce larval mass from 8.1 ± 1.7 (mass of control larvae) to 1.8 ± 0.5 mg, which corresponds to a decrease of 78%. Seed coat toxicity constitutes an efficient defense mechanism, hindering insect predation and preventing embryo damage. We hypothesize that A. lebbeck vicilins are good candidates for the genetic transformation of crop legumes to enhance resistance to bruchid predation.

Cytotoxic oleanane-type saponins from Albizia inundata.

Bioassay-guided fractionation of a CH(2)Cl(2)-MeOH extract of the aerial parts of Albizia inundata resulted in the isolation of two new natural oleanane-type triterpene saponins {3-O-[α-L-arabinopyranosyl(1→6)]-2-acetamido-2-deoxy-ß-D-glucopyranosyl oleanolic acid (1) and 3-O-[α-L-arabinopyranosyl(1→2)-α-L-arabinopyranosyl(1→6)]-2-acetamido-2-deoxy-ß-D-glucopyranosyl acacic acid lactone (2)} along with seven known saponins {3-O-[α-L-arabinopyranosyl(1→6)]-2-acetamido-2-deoxy-ß-D-glucopyranosyl echinocystic acid (3), 3-O-[ß-D-xylopyranosyl (l→2)-α-L-arabinopyranosyl(l→6)]-2-acetamido-2-deoxy-ß-D-glucopyranosyl acacic acid lactone (concinnoside D) (4), 3-O-[ß-D-glucopyranosyl(l→2)]-ß-D-glucopyranosyl oleanolic acid (5), 3-O-[α-L-arabinopyranosyl(1→2)-α-L-arabinopyranosyl(l→6)]-ß-D-glucopyranosyl oleanolic acid (6), 3-O-[ß-D-xylopyranosyl(1→2)-α-L-arabinopyranosyl(l→6)]-ß-D-glucopyranosyl oleanolic acid (7), 3-O-[α-L-arabinopyranosyl(l→2)-α-L-arabinopyranosyl(1→6)-[ß-D-glucopyranosyl(l→2)]-ß-D-glucopyranoside echinocystic acid (8), and 3-O-[ß-D-xylopyranosyl(l→2)-α-L-arabinopyranosyl(1→6)-[ß-D-glucopyranosyl(l→2)]-ß-D-glucopyranoside echinocystic acid (9)}. The structures of 1 and 2 were established on the basis of extensive 2D NMR ((1)H-(1)H COSY or DQF-COSY, HSQC, HMBC, TOCSY, and HSQC-TOCSY) spectroscopic, ESIMS, and chemical methods. Saponins 1, 3, 6, and 7 showed cytotoxicity against human head and neck squamous cells (JMAR, MDA1986) and melanoma cells (B16F10, SKMEL28) with IC(50) values in the range 1.8-12.4 µM, using the MTS assay.

Influence of binder type and process parameters on the compression properties and microbial survival in diclofenac tablet formulations

The influence of binder type and process parameters on the compression properties and microbial survival in diclofenac tablet formulations were studied using a novel gum from Albizia zygia. Tablets were produced from diclofenac formulations containing corn starch, lactose and dicalcium phosphate. Formulations were analyzed using the Heckel and Kawakita plots. Determination of microbial viability in the formulations was done on the compressed tablets of both contaminated and uncontaminated tablets prepared from formulations. Direct compression imparted a higher plasticity on the materials than the wet granulation method. Tablets produced by wet granulation presented with a higher crushing strength than those produced by the direct compression method. Significantly higher microbial survival (p< 0.05) was obtained in formulations prepared by direct compression. The percent survival of Bacillus subtilis spores decreased with increase in binder concentration. The study showed that Albizia gum is capable of imparting higher plasticity on materials and exhibited a higher reduction of microbial contaminant in the formulations. The direct compression method produced tablets of reduced viability of microbial contaminant.

A influência do tipo de ligante e os parâmetros do processo de propriedades de compressão e sobrevivência microbiana em comprimidos de diclofenaco foram estudados utilizando uma nova goma de Albizia zygia. Os comprimidos foram produzidos a partir de formulações de diclofenaco contendo amido de milho, lactose e fosfato bicálcico. As formulações foram analisadas usando os gráficos de Heckel e Kawakita. A determinação da viabilidade microbiana nas formulações foi feita nos comprimidos contaminados e não contaminados preparados a partir de formulações. A compressão direta confere maior plasticidade dos materiais do que o método de granulação úmida. Comprimidos produzidos por granulação úmida apresentaram maior força de esmagamento do que aqueles produzidos pelo método de compressão direta. Observou-se sobrevivência significativamente maior (p<0,05) em formulações preparadas por compressão direta. A sobrevivência percentual dos esporos de Bacillus subtilis diminuiu com o aumento da concentração do agregante. O estudo mostrou que a goma de Albizia é capaz de conferir maior plasticidade aos materiais e apresentou maior redução da contaminação microbiana nas formulações. O método de compressão direta produziu comprimidos com viabilidade reduzida de contaminantes microbianos.

Gene flow among established Puerto Rican populations of the exotic tree species, Albizia lebbeck.

We estimate gene flow and patterns of genetic diversity in Albizia lebbeck, an invasive leguminous tree in the dry forest of southwestern Puerto Rico. Genetic diversity estimates calculated for 10 populations of 24 trees each indicated that these populations may have been formed from multiple introductions. The presence of unique genotypes in the northernmost populations suggests that novel genotypes are still immigrating into the area. This combination of individuals from disparate locations led to high estimates of genetic diversity (He = 0.266, P = 0.67). Indirect estimates of gene flow indicate that only 0.69 migrants per generation move between populations, suggesting that genetic diversity within populations should decrease due to genetic drift. Since migration-drift equilibrium was not found, however, this estimate needs to be viewed with caution. The regular production of pods in this outcrossing species (tm = 0.979) indicates that sufficient outcross pollen is received to insure successful reproduction. Direct estimates of gene flow indicate that between 44 and 100% of pollen received by trees in four small stands of trees (n < 11) was foreign. The role of gene flow in facilitating the spread of this invasive plant species is discussed.

Calcium mobilization as the endothelium-independent mechanism of action involved in the vasorelaxant response induced by the aqueous fraction of the ethanol extract of Albizia inopinata G. P. Lewis (AFL) in the rat aorta.

In a previous work, we demonstrated that, in normotensive rats, AFL induced a marked hypotension due to a decrease in total peripheral resistances (TPR), partially secondary to the release of NO by the endothelium. NO did not, however, account for the total vasodilation produced by AFL in these rats. The aim of this study was to determine the involvement of the intracellular calcium mobilization in the vasorelaxant action induced by AFL in the rat aorta. In aorta of normotensive rats AFL (10, 20, 40 and 80 microg/ml) inhibited the sustained contractions induced by KCl (80 and 30 mM) and phenylephrine (Phe, 1 microM) with similar IC50 values (54 +/- 6, 52 +/- 4 and 65 +/- 4 microg/ml, respectively). The relaxing response induced by AFL against Phe-induced contractions was modified significantly by the endothelium removal (IC50 = 132 +/- 23 and 65 +/- 4 microg/ml, endothelium removed and intact endothelium aortic rings, respectively). Nevertheless, removal of the endothelium did not significantly change IC50 values when KCl (30 and 80 mM) was used as the contractile agent. The inhibitory effect induced by AFL on high (64.5 mM) K+-induced contraction was potentiated slightly (p < 0.05) by the decrease (from 2.5 to 0.3 mM, Ca2+) and attenuated by the increase (from 2.5 to 7.5 mM Ca2+) in the external [Ca2+]. In addition, in aortas from normotensive rats, AFL antagonized transient contractions induced in Ca2+-free media induced by 1 microM noradrenaline in a concentration-dependent manner, but not those induced by 20 mM caffeine. It is suggested that the remaining vasodilator effect of AFL in normotensive rats is probably due to an inhibition of Ca2+ influx and/or inhibition of intracellular Ca2+ mobilization from the noradrenaline-sensitive stores.