Biochemical characterization of the venom of Central American scorpion Didymocentrus krausi Francke, 1978 (Diplocentridae) and its toxic effects in vivo and in vitro.

Venoms of medically important scorpions from Buthidae family have been intensively studied, in contrast to non-buthid venoms, for which knowledge is scarce. In this work, we characterized the venom of a Diplocentridae species, Didymocentrus krausi, a small fossorial scorpion that inhabits the Tropical Dry Forest of Central America. D. krausi venom soluble fraction contains proteases with enzymatic activity on gelatin and casein. Mass spectrometry and venomic analysis confirmed the presence of elastase-like, cathepsin-O-like proteases and a neprilysin-like metalloproteinase. We did not detect phospholipase A2, C or D, nor hyaluronidase activity in the venom. By homology-based venom gland transcriptomic analysis, NDBPs, a ß-KTx-like peptide, and other putative toxin transcripts were found, which, together with a p-benzoquinone compound present in the venom, could potentially explain its direct hemolytic and cytotoxic effects in several mammalian cell lines. Cytotoxicity of D. krausi venom was higher than the effect of venoms from two buthid scorpion species distributed in Costa Rica, Centruroides edwardsii and Tityus pachyurus. Even though D. krausi venom was not lethal to mice or crickets, when injected in mouse gastrocnemius muscle at high doses it induced pathological effects at 24 h, which include myonecrosis, weak hemorrhage, and inflammatory infiltration. We observed an apparent thrombotic effect in the skin blood vessels, but no in vitro fibrinogenolytic activity was detected. In crickets, D. krausi venom induced toxicity and paralysis in short periods of time.

In vitro studies to evaluate the wound healing properties of Calendula officinalis extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Calendula officinalis (pot marigold) flower extracts have a long-lasting tradition in ethnopharmacology. Currently, the European Medicines Agency (EMA) has approved its lipophilic and aqueous alcoholic extracts as traditional medicinal products for the treatment of minor inflammation of the skin and as an aid in the healing of minor wounds. AIM OF THE STUDY: The purpose of this study was to analyse the molecular mechanism of the wound healing effects of Calendula extracts, which may reflect the phytomedicines currently used in the market. MATERIALS AND METHODS: The effect of three different extracts from Calendula flowers (n-hexanic, ethanolic, aqueous) on the inflammatory phase of wound healing was studied in human immortalized keratinocytes and human dermal fibroblasts. An electrophoretic mobility shift assay on NF-κB-DNA binding, qRT-PCR and ELISA experiments were performed. The effect of Calendula extracts on the new tissue formation phase of wound healing was evaluated by studying the migratory properties of these extracts, triterpene mixtures and single compounds in human immortalized keratinocytes using the scratch assay. Finally, the effect of the extracts on the formation of granulation tissue in wound healing was studied using bacterial collagenase isolated from Clostridium histolyticum and the determination of soluble collagen in the supernatant of human dermal fibroblasts. RESULTS: The n-hexanic and the ethanolic extracts from Calendula flowers influence the inflammatory phase by activating the transcription factor NF-κB and by increasing the amount of the chemokine IL-8, both at the transcriptional and protein level, in human immortalized keratinocytes. The migration of the keratinocytes during the new tissue formation phase was only marginally influenced in the scratch assay. However, it can be assumed that the granulation tissue was affected, as the ethanolic extract inhibited the activity of collagenase in vitro and enhanced the amount of collagen in the supernatant of human dermal fibroblasts. CONCLUSIONS: Our results contribute to a better understanding of the wound healing properties of the traditional medicinal plant Calendula officinalis. However, further studies are necessary to evaluate which of its known constituents are responsible for these effects. Triterpenes seem to play only a marginal role, but carotene and xanthophyll derivatives should garner more attention in future studies.

Isolation of Flavonoids from Deguelia duckeana and Their Effect on Cellular Viability, AMPK, eEF2, eIF2 and eIF4E.

Preparations of Deguelia duckeana, known in Brazil as timbó, are used by indigenous people to kill fish. Reinvestigation of its extracts resulted in the isolation and identification of 11 known flavonoids identified as 3,5,4'-trimethoxy-4-prenylstilbene (1), 4-methoxyderricidine (2), lonchocarpine (3), 4-hydroxylonchocarpine (4), 4-methoxylonchocarpine (5), 5-hydroxy-4',7-dimethoxy-6-prenylflavanone (6), 4'-hydroxyisolonchocarpine (7), 4'-methoxyisolonchocarpine (8), 3',4',7-trimethoxyflavone (9), 3',4'-methylenedioxy-7-methoxyflavone (10), and 2,2-dimethyl-chromone-5,4'-hydroxy-5'-methoxyflavone (11). Except for 1, 3, and 4 all of these flavonoids have been described for the first time in D. duckeana and the flavanone 6 for the first time in nature. Compounds 2, 3, 4, 7, 9, and 10 were studied for their potential to induce cell death in neuronal SK-N-SH cells. Only the chalcone 4 and the flavanone 7 significantly induced lactate dehydrogenase (LDH) release, which was accompanied by activation of caspase-3 and impairment of energy homeostasis in the MTT assay and may explain the killing effect on fish. Interestingly, the flavone 10 reduced cell metabolism in the MTT assay without inducing cytotoxicity in the LDH assay. Furthermore, the flavonoids 2, 3, 4, 7, and 10 induced phosphorylation of the AMP-activated protein kinase (AMPK) and the eukaryotic elongation factor 2 (eEF2). The initiation factor eIF4E was dephosphorylated in the presence of these compounds. The initiation factor eIF2alpha was not affected. Further studies are needed to elucidate the importance of the observed effects on protein synthesis and potential therapeutic perspectives.

Mastering analytical challenges for the characterization of pentacyclic triterpene mono- and diesters of Calendula officinalis flowers by non-aqueous C30 HPLC and hyphenation with APCI-QTOF-MS.

Pentacyclic triterpene mono- and diesters have been isolated from Calendula officinalis flowers. GC-MS, APCI-Exactive Orbitrap HR-MS and NMR allowed to identify the triterpene skeleton in various samples (different triterpene mixtures from Calendula n-hexane extract). NMR provided evidence that triterpene diesters are present in the samples as well. However, the corresponding quasi-molecular ions could not be detected by APCI-Exactive Orbitrap HR-MS. Instability of triterpene diesters and loss of a fatty acid residue, respectively, in the ion-source made their MS detection challenging. Thus, a set of new APCI-QTOF-MS methods (using the TripleTOF 5600+ mass spectrometer) were developed which made it eventually possible to solve this problem and confirm the diester structures by MS via quasi-molecular ion [M+H](+) detection. Direct infusion APCI-QTOF MS experiments in MS/MS high sensitivity scan mode with low collision energy and multi-channel averaging acquisition (MCA) allowed the detection of quasi-molecular ions of triterpene diesters for the first time and unequivocally confirmed the presence of faradiol 3,16-dimyristate and -dipalmitate, as well as the corresponding mixed diesters faradiol 3-myristate,16-palmitate and faradiol 3-palmitate,16-myristate. Preferential loss of the fatty acid in 16-position made it possible to distinguish the mixed diesters by MS/MS spectra. Their chromatographic separations turned out to be challenging due to their bulkiness and extended molecular dimensions. However, separation could be achieved by an uncommon non-aqueous RPLC mode with an in-house synthesized C30 phase. Finally, two (U)HPLC-APCI-QTOF-MS methods with C18- and C30-based non-aqueous RPLC provided suitable, sensitive assays to monitor the presence of monoesters and diesters of various triterpenes (faradiol, maniladiol, arnidiol, arnitriol A and lupane-3ß,16ß,20-triol esters) in the n-hexane extract of C. officinalis with high mass resolution and good mass accuracy.

Changing Biosynthetic Profiles by Expressing bldA in Streptomyces Strains.

Recently we described an unusual way of activating a cryptic gene cluster when we explored the origin of the bald phenotype of Streptomyces calvus. Complementation of S. calvus with a correct copy of bldA restored sporulation and additionally promoted production of a new natural products. In this study we report on the expression of bldA in several Streptomyces strains that have been described as "poorly sporulating" strains. In seven out of 15 cases, HPLC profiling revealed the production of new compounds, and in two cases the overproduction of known compounds. Two compounds were isolated and their structures were determined.

Discovery of Tricyclic Clerodane Diterpenes as Sarco/Endoplasmic Reticulum Ca(2+)-ATPase Inhibitors and Structure-Activity Relationships.

Tricyclic clerodane diterpenes (TCDs) are natural compounds that often show potent cytotoxicity for cancer cells, but their mode of action remains elusive. A computationally based similarity search (CDRUG), combined with principal component analysis (ChemGPS-NP) and docking calculations (GOLD 5.2), suggested TCDs to be inhibitors of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, which is also the target of the sesquiterpene lactone thapsigargin. Biochemical studies were performed with 11 TCDs on purified rabbit skeletal muscle sarcoplasmic reticulum membranes, which are highly enriched with the SERCA1a isoform. Casearborin D (2) exhibited the highest affinity, with a KD value of 2 µM and giving rise to complete inhibition of SERCA1a activity. Structure-activity relationships revealed that functionalization of two acyl side chains (R1 and R4) and the hydrophobicity imparted by the aliphatic chain at C-9, as well as a C-3,C-4 double bond, play crucial roles for inhibitory activity. Docking studies also suggested that hydrophobic interactions in the binding site, especially with Phe256 and Phe834, may be important for a strong inhibitory activity of the TCDs. In conclusion, a novel class of SERCA inhibitory compounds is presented.

Four new flavonol glycosides from the leaves of Brugmansia suaveolens.

Four new flavonol glycosides were isolated from the leaves of Brugmansia suaveolens: kaempferol 3-O-ß-D-glucopyranosyl-(1'''→2'')-O-α-L-arabinopyranoside (1), kaempferol 3-O-ß-D-glucopyranosyl-(1'''→2'')-O-α-L-arabinopyranoside-7-O-i-D-gluco-pyranoside (2), kaempferol 3-O-ß-D-[6'''-O-(E-caffeoyl)]-glucopyranosyl-(1'''→2'')-O-α-l-arabinopyranoside-7-O-ß-D-glucopyranoside (3), and kaempferol 3-O-ß-D-[2'''-O-(E-caffeoyl)]-glucopyranosyl-(1'''→2'')-O-α-l-arabinopyranoside-7-O-ß-D-glucopyranoside (4). The structure elucidation was performed by MS, 1D and 2D NMR analyses.

Cytotoxic clerodane diterpenes from Zuelania guidonia.

The leaves of Zuelania guidonia yielded eight new clerodane diterpenes, namely, zuelaguidins A-H (1-8), and the known clerodane diterpene esculentin A (9). Some of these structures contained a 3,6-dihydro-1,2-dioxin moiety. The new compounds were isolated and identified using 1D- and 2D-NMR experiments. All compounds were evaluated for cytotoxicity against the CCRF-CEM (human acute lymphocytic leukemia), CEM-ADR5000 (human acute lymphocytic leukemia resistant to doxorubicin), and MIA-PaCa-2 (human pancreatic carcinoma) cell lines as well as for their selectivity against peripheral blood mononuclear cells from healthy human subjects. Zuelaguidins B, C, and E were the most potent compounds against the CCRF-CEM cell line, with IC50 values ranging from 1.6 to 2.5 µM.

Efecto in vitro de los terpenos lupeol y casearina G sobre células sanguíneas y tumorales / In vitro effect of lupeol and casearin G on peripheral blood mononuclear and tumor cells

Background: The rainforest is an important source of natural compounds with therapeutic properties. Although there are many anti-inflammatory and antineoplastic drugs available to the clinician, there is an ongoing need for new therapeutic drugs with fewer serious adverse effects. Aim: To evaluate the in vitro cytotoxic effects of lupeol and casearin G on tumor cells, on phagocytic activity and nitric oxide (NO) production by blood mononuclear cells. Material and Methods: The cytotoxic effect of these compounds on cell lines MCF-7 (human breast adenocarcinoma) and PC-3 (human prostate cancer) was measured by a colorimetric assay (MTS/PMS) and the sulphorhodamine B assay. Peripheral blood mononuclear cells were obtained from eight healthy volunteers. The effect of these compounds on nitric oxide (NO) production was measured using the Griess reaction. Their effect on phagocytic activity of PBMC was also evaluated. Results: Lupeol (≥ 2 mM) resulted in a reduction of both the phagocytic index and the percentage of phagocytic monocytes and macrophages. Treatment of monocytes/macrophages with lupeol (72 µM) and casearin G (4 µM) reduced the production of NO. Neither lupeol (< 969 µM) nor casearin G (< 55 µM) had cytotoxic effects on PBMC. Casearin G showed both cytotoxic (IC50, LC50) and cytostatic (GI50) effects against tumor cells, PC-3 (IC50 = 12.5 µM; GI50 = 13.3 µM; LC50 = 51.9 µM) and MCF-7 (IC50 = 112.8 µM; GI50 = 11.8 µM; LC50 = 49.4 µM), as well as a hemolytic effect (≥ 182 µM). Conclusions: These observations indicate that lupeol and casearin G might be useful compounds in the preparation of anti-inflammatory drugs, whereas casearin G might be useful in the elaboration of antitumor drugs.

[In vitro effect of lupeol and casearin G on peripheral blood mononuclear and tumor cells]. / Efecto in vitro de los terpenos lupeol y casearina G sobre células sanguíneas y tumorales.

BACKGROUND: The rainforest is an important source of natural compounds with therapeutic properties. Although there are many anti-inflammatory and antineoplastic drugs available to the clinician, there is an ongoing need for new therapeutic drugs with fewer serious adverse effects. AIM: To evaluate the in vitro cytotoxic effects of lupeol and casearin G on tumor cells, on phagocytic activity and nitric oxide (NO) production by blood mononuclear cells. MATERIAL AND METHODS: The cytotoxic effect of these compounds on cell lines MCF-7 (human breast adenocarcinoma) and PC-3 (human prostate cancer) was measured by a colorimetric assay (MTS/PMS) and the sulphorhodamine B assay. Peripheral blood mononuclear cells were obtained from eight healthy volunteers. The effect of these compounds on nitric oxide (NO) production was measured using the Griess reaction. Their effect on phagocytic activity of PBMC was also evaluated. RESULTS: Lupeol (≥ 2 mM) resulted in a reduction of both the phagocytic index and the percentage of phagocytic monocytes and macrophages. Treatment of monocytes/macrophages with lupeol (72 µM) and casearin G (4 µM) reduced the production of NO. Neither lupeol (< 969 µM) nor casearin G (< 55 µM) had cytotoxic effects on PBMC. Casearin G showed both cytotoxic (IC50, LC50) and cytostatic (GI50) effects against tumor cells, PC-3 (IC50 = 12.5 µM; GI50 = 13.3 µM; LC50 = 51.9 µM) and MCF-7 (IC50 = 112.8 µM; GI50 = 11.8 µM; LC50 = 49.4 µM), as well as a hemolytic effect (≥ 182 µM). CONCLUSIONS: These observations indicate that lupeol and casearin G might be useful compounds in the preparation of anti-inflammatory drugs, whereas casearin G might be useful in the elaboration of antitumor drugs.

Identification of new snake venom metalloproteinase inhibitors using compound screening and rational Peptide design.

The majority of snakebite envenomations in Central America are caused by the viperid species Bothrops asper, whose venom contains a high proportion of zinc-dependent metalloproteinases that play a relevant role in the pathogenesis of hemorrhage characteristic of these envenomations. Broad metalloproteinase inhibitors, such as the peptidomimetic hydroxamate Batimastat, have been shown to inhibit snake venom metalloproteinases (SVMP). However, the difficulty in having open public access to Batimastat and similar molecules highlights the need to design new inhibitors of SVMPs that could be applied in the treatment of snakebite envenomations. We have chosen the SVMP BaP1 as a model to search for new inhibitors using different strategies, that is, screening of the Prestwick Chemical Library and rational peptide design. Results from these approaches provide clues on the structural requirements for efficient BaP1 inhibition and pave the way for the design of new inhibitors of SVMP.

Structural and conformational analysis of proanthocyanidins from Parapiptadenia rigida and their wound-healing properties.

Structure elucidation and conformation analysis of four proanthocyanidins isolated from the bark of Parapiptadenia rigida were performed by two-dimensional NMR spectroscopy, HRESIMS, CD, and molecular mechanics (MM+) force field calculations. The known prodelphinidin, epigallocatechin-(4ß→8)-epigallocatechin-3-O-gallate (1) was accompanied by the new epigallocatechin-(4ß→8)-4'-O-methylgallocatechin (2), epicatechin-(4ß→8)-4'-O-methylgallocatechin (3), and (4α→8)-bis-4'-O-methylgallocatechin (4). Compound 4 was previously published but the earlier structure must presumably be revised to 4'-O-methylgallocatechin-(4α→8)-4'-O-methylepigallocatechin. Conformational studies showed the compact rotamer with B and E rings in quasi-equatorial orientations as the preferred conformation for compounds 1-3, whereas 4 consists of two stable rotamers, each with a quasi-equatorial orientation of ring B and E, respectively. The isolated compounds were studied for their wound-healing effects in a scratch assay and showed promising results.

Chemical constituents from Zanthoxylum setulosum (Rutaceae) / Costituyentes químicos de Zanthoxylum setulosum (Rutaceae)

Following our phytochemical studies of Costa Rican plants, in this work we report the isolation and identification of eight compounds from aerial parts of Zanthoxylum setulosum (Rutaceae). They were identified as the alkaloid skimmianine, the lignans savinin, kusunokinin, sesamin, syringaresinol and the isopentenyl ether of pluviatol, the amide aurantiamide acetate, and the triterpen lupeol. This is the first report of isolation of skimmianine from the leaves of Z. setulosum and its presence confirm that quinoline and benzophenanthridine alkaloids, can be considered as chemotaxonomic markers of this genus. All the isolated compounds were characterized by spectroscopic methods (including 1H-NMR, 13C-NMR, , HMQC, HMBC and NOESY) and comparison with the literature data.

Continuando con el estudio fitoquímico de plantas de Costa Rica, en este trabajo informamos el aislamiento e identificación de ocho compuestos de las partes aéreas de Zanthoxylum setulosum (Rutaceae). Los compuestos fueron identificados como el alcaloide skimmianina, los lignanos savinina, kusunokinina, sesamina, siringaresinol y el éter isopentílico del pluviatol, la amida conocida como acetato de aurantiamida, y el triterpeno lupeol. Este es el primer informe del aislamiento de skimmianina en las hojas de Z. setulosum, lo cual confirma que alcaloides quinolínicos y benzofenantridinicos pueden ser considerados marcadores quimiotaxonómicos en éste género. La estructura de los compuestos aislados fue caracterizada por métodos espectroscópicos (incluyendo 1HNMR, 13C-NMR, HMQC, HMBC y NOESY) y comparación con datos de la literatura.

Catechin derivatives from Parapiptadenia rigida with in vitro wound-healing properties.

Analysis of the ethanolic extract of the bark from Parapiptadenia rigida resulted in the isolation of the new catechin derivatives 4',3''-di-O-methylapocynin-D (10), 4',3''-di-O-methylapocynin-B (11), epigallocatechin-3-O-ferulate (8), and 4'-O-methylepigallocatechin-3-O-ferulate (9) and the catechins 4'-O-methylepigallocatechin-3-O-gallate (6) and 4'-O-methylepicatechin-3-O-gallate (7). These compounds, isolated for the first time from a natural source, are accompanied by the five known catechins 4'-O-methylgallocatechin (1), 4'-O-methylepigallocatechin (2), 3'-O-methylepicatechin (3), epigallocatechin-3-O-gallate (4), and epicatechin-3-O-gallate (5). Compounds 5 and 7 displayed promising wound-healing effects in a scratch assay. Some of the catechin derivatives showed inhibitory effects on NF-κB DNA binding and p38α MAPK activity.

Functions of genes and enzymes involved in phenalinolactone biosynthesis.

Phenalinolactones are novel terpene glycoside antibiotics produced by Streptomyces sp. Tü6071. Inactivation of three oxygenase genes (plaO2, plaO3 and plaO5), two dehydrogenase genes (plaU, plaZ) and one putative acetyltransferase gene (plaV) led to the production of novel phenalinolactone derivatives (PL HS6, PL HS7, PL HS2 and PL X1). Furthermore, the exact biosynthetic functions of two enzymes were determined, and their in vitro activities were demonstrated. PlaO1, an Fe(II)/alpha-ketoglutarate-dependent dioxygenase, is responsible for the key step in gamma-butyrolactone formation, whereas PlaO5, a cytochrome P450-dependent monooxygenase, catalyses the 1-C-hydroxylation of phenalinolactone D. In addition, stable isotope feeding experiments with biosynthetic precursors shed light on the origin of the carbons in the gamma-butyrolactone moiety.

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.

Anticancer activities of two sesquiterpene lactones, millerenolide and thieleanin isolated from Viguiera sylvatica and Decachaeta thieleana.

The present study was carried out in order to examine the anticancer properties of two sesquiterpene lactones, millerenolide and thieleanin, isolated from Viguiera sylvatica and Decachaeta thieleana, against cell lines in vitro, and on the growth B16/BL6 melanoma tumors in C57BL/6 mice. Millerenolide and thieleanin showed a similar pattern of cytotoxicity with the greatest effect on viability being evident with A549 human lung cancer cells (IC(50) - 40 and 32 microM respectively), and with the 3T3/HER2 cell line which are 3T3 mouse fibroblasts transfected with the HER2 oncogene (IC(50) - 16 and 28 microM respectively). The parent 3T3 cells and the B16/BL6 mouse melanoma cells were less sensitive to these compounds, with thieleanin showing an IC(50) with B16/BL6 greater than the highest dose tested (203 microM). Treatment with millerenolide (8 mg/kg, i.p. on days 0, 2 and 4 post-inoculation) significantly inhibited the growth of subcutaneous B16/BL6 tumors in C57BL/6 mice, (50% inhibition at day 25, P=0.015), as well as retarding the appearance of detectable tumor (millerenolide - day 15.2+/-0.4 vs control - day 12.8+/-0.5, mean+/-SEM, P=0.011). In contrast, treatment with thieleanin (8 mg/kg every other day up to the day of kill) neither retarded the appearance of the tumor nor its growth.

[Millerenolide induces suppressive activity on human peripheral blood mononuclear cells]. / Actividad supresora del millerenólido sobre células mononucleares de sangre periférica humana.

BACKGROUND: Natural products are used in the production of therapeutic drugs due to their wide diversity and excellent adaptability to biological structures. Sesquiterpene lactones are the active constituents of several plants from the Asteraceae family. AIM: To assess the in vitro effect of a sesquiterpene lactone (millerenolide). MATERIAL AND METHODS: The drug effect was assessed measuring the proliferation of lymphocytes using the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonylJ-2H-tetrazolium hydroxide (XTT) technique. Changes on the cell cycle were analyzed on a FACSort flow cytometer The effect of millerenolide on the production of nitric oxide (NO) by macrophages was evaluated using the Griess reagent. Additionally, phagocytosis of latex particles and nitroblue tetrazolium (NBT) reduction by macrophages were evaluated microscopically. RESULTS: Treatment of human peripheral blood mononuclear cells (PBMC) with millerenolide decreases the proliferation of lymphocytes, decreases the percentage of cells in S, and G2/M phases, and increases the proportion of cells in GO/Gl phase. Treatment of macrophages with millerenolide, reduces the production of NO, the phagocytic capacity and the number of cells able to reduce NBT. Cytotoxic effects of the lactone on human PBMC were only observed when the concentration was increased to 6 microg/ml. CONCLUSIONS: Millerenolide could be considered as a potential therapeutic agent with immunosuppressive properties.

Neural networks as valuable tools to differentiate between sesquiterpene lactones' inhibitory activity on serotonin release and on NF-kappaB.

Sesquiterpene lactones are the active components of a variety of medicinal plants from the Asteraceae family. They possess biological activities such as the inhibition of NF-kappaB and the release inhibition of the vasoactive serotonin. On the basis of a data set of 54 SLs, we report the development of a quantitative model for the prediction of serotonin release inhibition. Comparing this model with a previous investigation of the target NF-kappaB, structural features necessary for specific compounds could be acquired. Atomic properties encoded by radial distribution function and molecular surface potentials encoded by autocorrelation were used as descriptors. Whereas some descriptors describe the structural requirements for both activities, other descriptors can be used to decide whether an SL is more active to NF-kappaB or to serotonin release. Again, counter propagation neural networks proved to be a valuable tool to establish structure-activity relationships that are necessary for the search for and optimization of lead structures.

Actividad supresora del millerenólido sobre células mononucleares de sangre periférica humana / Millerenolide induces suppressive activity on human peripheral blood mononuclear cells

Background: Natural products are used in the production of therapeutic drugs due to their wide diversity and excellent adaptability to biological structures. Sesquiterpene ¡aciones are the active constituents of several plants from the Asteraceae family. Aim: To assess the in vitro effect of a sesquiterpene lactone (millerenolide). Material and methods: The drug effect was assessed measuring the proliferation of lymphocytes using the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonylJ-2H-tetrazolium hydroxide (XTT) technique. Changes on the cell cycle were analyzed on a FACSort flow cytometer The effect of millerenolide on the production of nitric oxide (NO) by macrophages was evaluated using the Griess reagent. Additionally, phagocytosis of latex particles and nitroblue tetrazolium (NBT) reduction by macrophages were evaluated microscopically. Results: Treatment of human peripheral blood mononuclear cells (PBMC) with millerenolide decreases the proliferation of lymphocytes, decreases the percentage of cells in S, and G2/Mphases, and increases the proportion of cells in GO/Gl phase. Treatment of macrophages with millerenolide, reduces the production of NO, the phagocytic capacity and the number of cells able to reduce NBT. Cytotoxic effects of the lactone on human PBMC were only observed when the concentration was increased to 6 fig/ml. Conclusions: Millerenolide could be considered as a potential therapeutic agent with immunosuppressiveproperties.