Breast conserving surgery in combination with intraoperative radiotherapy after previous external beam therapy: an option to avoid mastectomy.

PURPOSE: Mastectomy is the standard procedure in patients with in-breast tumor recurrence (IBTR) or breast cancer after irradiation of the chest due to Hodgkin's disease. In certain cases a second breast conserving surgery (BCS) in combination with intraoperative radiotherapy (IORT) is possible. To date, data concerning BCS in combination with IORT in pre-irradiated patients are limited. This is the first pooled analysis of this special indication with a mature follow-up of 5 years. METHODS: Patients with IBTR after external beam radiotherapy (EBRT; treated in two centers) for breast cancer were included. Patients with previous EBRT including the breast tissue due to other diseases were also included. IORT was performed with the Intrabeam™-device using low kV X-rays. Clinical data including outcome for all patients and toxicity for a representative cohort (LENT-SOMA scales) were obtained. Statistical analyses were done including Kaplan-Meier estimates for local recurrence, distant metastasis and overall survival. RESULTS: A total of 41 patients were identified (39 patients with IBTR, 2 with Hodgkin`s disease in previous medical history). Median follow-up was 58 months (range 4-170). No grade 3/4 acute toxicity occurred within 9 weeks. Local recurrence-free survival rate was 89.9% and overall survival was 82.7% at 5 years. Seven patients developed metastasis within the whole follow-up. CONCLUSIONS: BCS in combination with IORT in IBTR in pre-irradiated patients is a feasible method to avoid mastectomy with a low risk of side effects and an excellent local control and good overall survival.

Spirostanol saponins and esculin from Rusci rhizoma reduce the thrombin-induced hyperpermeability of endothelial cells.

Rusci rhizoma extracts are traditionally used against chronic venous disorders (CVD). To determine the effect of its secondary plant metabolites on the endothelium, phenolic compounds and saponins from Butcher's broom were isolated from a methanolic extract, and their activity on the thrombin-induced hyperpermeability of human microvascular endothelial cells (HMEC-1) was investigated in vitro. In addition to the six known spirostanol saponins deglucoruscin (5), 22-O-methyl-deglucoruscoside (6), deglucoruscoside (7), ruscin (8), ruscogenin-1-O-(α-l-rhamnopyranosyl-(1→2)-ß-d-galactopyranoside (9) and 1-O-sulpho-ruscogenin (10), three new spirostanol derivatives were isolated and identified: 3'-O-acetyl-4'-O-sulphodeglucoruscin (1), 4'-O-(2-hydroxy-3-methylpentanoyl)-deglucoruscin (2) and 4'-O-acetyl-deglucoruscin (3). Furthermore, the coumarin esculin (4), which is also prominently present in other medicinal plants used in the treatment of CVD, was isolated for the first time from Rusci rhizoma. Five of the isolated steroid derivatives (2, 5, 8, 9 and 10) and esculin (4) were tested for their ability to reduce the thrombin-induced hyperpermeability of endothelial cells in vitro, and the results were compared to those of the aglycone neoruscogenin (11). The latter compound showed a slight but concentration-dependent reduction in hyperpermeability to 71.8% at 100µM. The highest activities were observed for the spirostanol saponins 5 and 8 and for esculin (4) at 10µM, and these compounds resulted in a reduction of the thrombin-induced hyperpermeability to 41.9%, 42.6% and 53.3%, respectively. For 2, 5 and 8, the highest concentration tested (100µM) resulted in a drastic increase of the thrombin effect. The effect of esculin observed at a concentration of 10µM was diminished at 100µM. These in vitro data provide insight into the pharmacological mechanism by which the genuine spirostanol saponins and esculin can contribute to the efficacy of Butcher's broom against chronic venous disorders.

Novel neurological and immunological targets for salicylate-based phytopharmaceuticals and for the anti-depressant imipramine.

Inflammatory processes are increasingly recognised to contribute to neurological and neuropsychatric disorders such as depression. Thus we investigated whether a standardized willow bark preparation (WB) which contains among other constituents salicin, the forerunner of non-steroidal antiphlogistic drugs, would have an effect in a standard model of depression, the forced swimming test (FST), compared to the antidepressant imipramine. Studies were accompanied by gene expression analyses. In order to allocate potential effects to the different constituents of WB, fractions of the extract with different compositions of salicyl alcohol derivative and polyphenols were also investigated. Male Sprague Dawley rats (n=12/group) were treated for 14 days (p.o.) with the WB preparation STW 33-I (group A) and its fractions (FR) (groups FR-B to E) in concentrations of 30 mg/kg. The FRs were characterized by a high content of flavone and chalcone glycosides (FR-B), flavonoid glycosides and salicyl alcohol derivatives (FR-C), salicin and related salicyl alcohol derivatives (FR-D) and proanthocyanidines (FR-E). The tricyclic antidepressant imipramine (20 mg/kg) (F) was used as positive control. The FST was performed on day 15. The cumulative immobility time was significantly (p<0.05) reduced in group A (36%), group FR-D (44%) and by imipramine (16%) compared to untreated controls. RNA was isolated from peripheral blood. RNA samples (group A, group FR-D, and imipramine) were further analysed by rat whole genome microarray (Agilent) in comparison to untreated controls. Quantitative PCR for selected genes was performed. Genes (>2 fold, p<0.01), affected by WB and/or FR-D and imipramine, included both inflammatory (e.g. IL-3, IL-10) and neurologically relevant targets. Common genes regulated by WB, FR-D and imipramine were GRIA 2 ↓, SRP54 ↓, CYP26B ↓, DNM1L ↑ and KITLG ↓. In addition, the hippocampus of rats treated (27 d) with WB (15-60 mg/kg WB) or imipramine (15 mg/kg bw) showed a slower serotonin turnover (5-hydroxyindol acetic acid/serotonin (p<0.05)) depending on the dosage. Thus WB (30 mg/kg), its ethanolic fraction rich in salicyl alcohol derivatives (FR-D) (30 mg/kg) and imipramine, by being effective in the FST, modulated known and new targets relevant for neuro- and immunofunctions in rats. These findings contribute to our understanding of the link between inflammation and neurological functions and may also support the scope for the development of co-medications from salicylate-containing phytopharmaceuticals as multicomponent mixtures with single component synthetic drugs.

Pitfalls in cell culture work with xanthohumol.

Xanthohumol, the most abundant prenylated chalcone in hop (Humulus lupulus L.) cones, is well known to exert several promising pharmacological activities in vitro and in vivo. Among these, the chemopreventive, anti-inflammatory and anti-cancer effects are probably the most interesting. As xanthohumol is hardly soluble in water and able to undergo conversion to isoxanthohumol we determined several handling characteristics for cell culture work with this compound. Recovery experiments revealed that working with xanthohumol under cell culture conditions requires a minimal amount of 10% FCS to increase its solubility to reasonable concentrations (-50-75 micromol/l) for pharmacological in vitro tests. Additionally, more than 50% of xanthohumol can be absorbed to various plastic materials routinely used in the cell culture using FCS concentrations below 10%. In contrast, experiments using fluorescence microscopy in living cells revealed that detection of cellular intake of xanthohumol is hampered by concentrations above 1% FCS.

Contribution of flavonoids and catechol to the reduction of ICAM-1 expression in endothelial cells by a standardised Willow bark extract.

INTRODUCTION: A quantified aqueous Willow bark extract (STW 33-I) was tested concerning its inhibitory activity on TNF-α induced ICAM-1 expression in human microvascular endothelial cells (HMEC-1) and further fractionated to isolate the active compounds. RESULTS: At 50 µg/ml the extract, which had been prepared from Salix purpurea L., decreased ICAM-1 expression to 40% compared to control cells without showing cytotoxic effects. Further liquid-liquid partition revealed an ethyl acetate phase with potent reduction of ICAM-1 expression to 40% at 8 µg/ml. This fraction was comprehensively characterised by the isolation of flavanone aglyca and their corresponding glycosides, chalcone glycosides, salicin derivatives, cyclohexane-1,2-diol glycosides, catechol and trans-p-coumaric acid. All compounds were investigated for their activity on TNF-α induced ICAM-1 expression. The flavonoid and chalcone glycosides were not active up to 50 µM, whereas catechol and eriodictyol at the same concentration showed a significant reduction of ICAM-1 expression to 50% of control. Interestingly, other isolated flavanone aglyca like taxifolin, dihydrokaempferol and naringenin showed only weak or moderate inhibitory activity. Eriodictyol was a minor compound in the extract, whereas the catechol content in the extract (without excipients) reached 2.3%, determined by HPLC. One of the isolated cyclohexan-1,2-diol glucosides, 6'-O-4-hydroxybenzoyl-grandidentin, is a new natural compound. CONCLUSION: As catechol is quantitatively important in Willow bark extracts it can be concluded from the in vitro data that not only flavonoids and salicin derivatives, but also catechol can probably contribute to the anti-inflammatory activity of Willow bark extracts.

Prediction of adverse events by in vivo gene expression profiling exemplified for phytopharmaceuticals containing salicylates and the antidepressant imipramine.

BACKGROUND AND OBJECTIVE: Gene expression profiles of Sprague-Dawley (SD) rats treated with a standardized willow bark extract (WB), its salicin rich ethanol fraction (EtOH-FR) or the tricyclic antidepressant imipramine were evaluated for their potential to induce adverse events. Treatments had shown antidepressant-like effects. METHODS: Gene expression profiles (Agilent Whole Genome Array, n=4/group) obtained from the peripheral blood of male SD rats treated with WB (STW 33-I), EtOH-FR (30 mg/kg bw) or imipramine (20 mg/kg bw) were analysed comparatively by the Ingenuity Systems Programme, which allows to conduct model calculations of thresholds for theoretical potential adverse events (AE). RESULTS: The number of genes regulated by the three treatments were 1673 (WB), 117 (EtOH-FR) and 1733 (imipramine). The three treatments related to 47 disease clusters. The WB extract reached the threshold for a potential AE in one disease cluster (cardiac hypertrophy), whereas the EtOH-FR exceeded the threshold in 5 disease clusters (cardiac arteriopathy and stenosis, glomerular injury, pulmonary hypertension, alkaline phosphatase levels ⇑). Imipramine treatment hit 13 disease clusters: tachycardia, palpitation, myocardial infarction, arrhythmias, heart block, precipitation of congestive heart failure; urinary retention, altered liver functions. Those correspond to known potential adverse events. Glomerular injury and altered liver functions are part of the side effect profile of salicylic acid derivatives in agreement with the findings for the salicin rich EtOH-FR. CONCLUSION: There is no linear relationship between the number of constituents of a drug (preparation) and the number of different targets hit in a biological system on the gene expression level. Therefore, the number of genetic targets in a biological system does not necessarily increase with the complexity of the treatment corresponding to the non-linear behaviour of biological systems. Regarding gene expression levels AE of single treatments are not necessarily additive in combination treatments. The applied method appears to be an interesting screening tool for the prediction of potential AE. The phenomena that imipramine crossed the potential threshold for AEs several times whereas the WB extract did reach the threshold level only once, however not backed by clinical data for this AE, deserves to be further investigated. It questions the commonly assumed principle that substances with low number or without AE will have a poor efficacy.

Primary radiotherapy with or without chemotherapy in non-metastatic esophageal squamous cell carcinoma: a retrospective study.

The purpose of this study was to report the outcome of radio(chemo)therapy in the curative management of esophageal squamous cell carcinoma (ESCC). We retrospectively analyzed 163 patients with T1-T4, N0-1, M0 ESCC who were treated between January 1988 and December 2006 at the Technische Universität München. One hundred sixty patients were inoperable due to a poor performance status, comorbidities or locally advanced unresectable disease. External beam radiation therapy (EBRT) was performed with (n= 146) or without (n= 17) systemic chemotherapy. Fifty-four patients received an additional boost with intraluminal brachytherapy (IBT). Surviving patients were followed for a median of 72 months (range 10-173 months). The estimated overall survival (OS) at 2 and 5 years was 27 ± 4% and 11 ± 3%, respectively. Loco-regional recurrence at the primary site was observed in 29% of patients (n= 47). The recurrence-free survival (RFS) at 2 and 5 years was 24 ± 3% and 9 ± 2%, respectively. In multivariate analyses, the ECOG performance status (P= 0.004), 3D conformal (vs conventional) radiotherapy (P= 0.031) and continuous standard fractionation (vs split-course radiotherapy, P= 0.048) were associated with a better OS. Simultaneous chemotherapy (P= 0.49) or IBT (P= 0.31) had no significant impact on survival. Outcome for patients with ESCC is poor. Despite the very unfavorable patient selection (poor performance status, high rate of comorbidities, and advanced disease), long-term survival with radio(chemo)therapy was achieved in about 10% of patients. The introduction of modern treatment techniques/modalities (3D conformal planning/ continuous standard fractionation) might be associated with better outcomes.

Tea catechins' affinity for human cannabinoid receptors.

Among the many known health benefits of tea catechins count anti-inflammatory and neuroprotective activities, as well as effects on the regulation of food intake. Here we address cannabimimetic bioactivity of catechin derivatives occurring in tea leaves as a possible cellular effector of these functionalities. Competitive radioligand binding assays using recombinant human cannabinoid receptors expressed in Chem-1 and CHO cells identified (-)-epigallocatechin-3-O-gallate, EGCG (K(i)=33.6 microM), (-)-epigallocatechin, EGC (K(i)=35.7 microM), and (-)-epicatechin-3-O-gallate, ECG (K(i)=47.3 microM) as ligands with moderate affinity for type 1 cannabinoid receptors, CB1. Binding to CB2 was weaker with inhibition constants exceeding 50 microM for EGC and ECG. The epimers (+)-catechin and (-)-epicatechin exhibited negligible affinities for both CB1 and CB2. It can be concluded that central nervous cannabinoid receptors may be targeted by selected tea catechins but signaling via peripheral type receptors is less likely to play a major role in vivo.

Determining the cytotoxicity of catanionic surfactant mixtures on HeLa cells.

The cytotoxicity of commonly used synthetic surfactants and catanionic mixtures of those was evaluated using MTT on HeLa cells. The 50% inhibition concentration (IC(50)) for MTT reduction was calculated. The effect on chain length increase and inclusion of polyoxyethylene groups on the toxicity was tested on single surfactant systems. A general trend of increasing toxicity with increasing chain length and the presence of polyoxyethylene groups was observed. The measured IC(50) values of catanionic systems lie between those of participating surfactants. The increase in toxicity as the cationic surfactant is added to the anionic one is however not linear. A steep decrease of the IC(50) values (and therefore increase in the toxic properties) is observed immediately already at low concentrations of the cationic surfactants. This behavior is analogous to the enzyme activity in catanionic microemulsions.

Spontaneous formation of bilayers and vesicles in mixtures of single-chain alkyl carboxylates: effect of pH and aging and cytotoxicity studies.

We report the observation of bilayer fragments, some of which close to form vesicles, over a large range of pH at room temperature from mixtures of single-chain biocompatible commercially available nontoxic alkyl carboxylic surfactants after neutralization with HCl. The pH at which the morphological transitions occur is varied only by changing the ratio between two surfactants: the alkyloligoethyleneoxide carboxylate and sodium laurate. The effect of aging of the mixed surfactant systems in the pH region desired for dermatologic application (4.5 < pH < 7) is also studied. Finally, we show results of cytotoxicity studies on the surfactant mixtures.

Ex vivo/in vitro absorption of STW 5 (Iberogast) and its extract components.

To correlate the pharmacological effects of the fixed herbal combination STW 5 (Iberogast) containing nine extract components with its confirmed clinical efficacy, ex vivo/in vitro absorption tests were performed. For the investigation, the everted gut sac technique and, in a pilot study, the Caco-2-cell model were used. The absorption rate of the extracts was determined by measuring characteristic marker substances of each of the individual extracts using HPLC or GC techniques. The results allow us to conclude that the investigated substances from STW 5 possess a good bioavailability, which is in accordance with the rapid onset of the therapeutic efficacy and explains its known pharmacological effects and clinical efficacy in terms of multiple drug action and multi-target therapy, respectively.

The anthelmintic efficacy of five plant products against gastrointestinal trichostrongylids in artificially infected lambs.

Forty-eight helminth-free lambs were divided into eight groups (A-H) of six animals. Groups A-G were infected artificially with 10,000 third stage larvae of Haemonchus contortus and 20,000 third stage larvae of Trichostrongylus colubriformis, whereas group H remained uninfected. Thirty days post-infection the lambs were treated orally with a single dosage of one of the following products: group A with 3 mg/kg body weight (BW) of an aqueous ethanol extract (70%, v/v) of the seeds of Azadirachta indica A. Juss syn. Melia azedarach L. (Meliaceae); group B with 1 g/kg BW of a raw powder of the leaves of Ananas comosus (L.) Merr. (Bromeliaceae); group C with 0.3 mg/kg BW of an aqueous ethanol extract of a 1:1 mixture (g/g) of Vernonia anthelmintica (L.) Willd. (Asteraceae) seeds and Embelia ribes Burm (Myrsinaceae) fruits; group D with 183 mg/kg BW of an aqueous ethanol extract of the whole plants of Fumaria parviflora Lam. (Fumariaceae); group E with 28 mg/kg BW of an aqueous ethanol extract of the seeds of Caesalpinia crista L. (Caesalpiniaceae); group F with 25 mg/kg BW of pyrantel tartrate and group G with 50% ethanol. Group H remained untreated. Only the ethanol extract of F. parviflora caused a strong reduction of the faecal egg counts (100%) and a 78.2 and 88.8% reduction of adult H. contortus and T. colubriformis on day 13 post-treatment. The extract was as effective as the reference compound pyrantel tartrate. Therefore, the ethanol extract itself or single constituents of F. parviflora could be a promising alternative source of anthelmintic for the treatment of gastrointestinal trichostrongylids in small ruminants.

Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation.

The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV micrometers-1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At the highest LET value (16,300 keV micrometers-1) no significant repair is observed. These LET-dependencies are consistent with the current mechanistic model for radiation induced cataractogenesis which postulates that genomic damage to the surviving fraction of epithelial cells is responsible for lens opacification.

Studies on the antioxidative activity of phloroglucinol derivatives isolated from hypericum species.

Twenty-one phloroglucinol derivatives, belonging to 8 different carbon skeletons, were tested for their ability to influence the oxidative burst of polymorphonuclear cells (PMNs) after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). Results revealed a strong reduction of oxygen production by PMNs after stimulation with FMLP for the compounds ialibinone E ( 5), hyperguinone B ( 15) and hyperforin ( 21). The IC 50 values obtained were 2.5 microM ( 5), 3.3 microM ( 15) and 1.8 microM ( 21), respectively. Slight modifications of the substituents or variation of the stereochemistry resulted in a significant loss of activity. None of the active compounds showed antioxidative activity after stimulation with OZ. The influence of compounds 5, 15 and 21 on the production of oxygen radicals in an H 2 O 2 /horseradish peroxidase system was investigated and revealed potent activity only for compound 5 (IC 50 1 microM). The superoxide-scavenging properties of ialibinone E ( 5) and hyperguinone B ( 15) were additionally tested in a cytochrome c assay and only ialibinone E ( 5) was found to be significantly active at lower micromolar concentrations. Ialibinone E ( 5) was not active in a xanthine oxidase assay (urate formation) in concentrations up to 100 microM and its activity is therefore not attributable to the inhibition of this enzyme. It can be assumed that the activity of compounds 5 and 15 in the different cellular and enzymatic assays, is most likely caused by different and maybe specific mechanisms and cannot be explained by a radical scavenger activity alone. None of the active phloroglucinols showed cytotoxic effects against the PMNs.

Transport of alkamides from Echinacea species through Caco-2 monolayers.

To gain more insights into the human intestinal absorption of alkamides from Echinacea species, transport studies were performed with the human adenocarcinoma colonic cell line Caco-2 (ATCC) as a model to assess the epithelial transport of dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamides (1/ 2). 30 minutes after apical loading of 25 microg/ml 1/ 2, about 15 % of these alkamides were detectable on the basolateral side. Close monitoring of the transport during 6 hours revealed a nearly complete transport to the basolateral side after 4 hours and no significant metabolism was observable. Transport experiments performed at 4 degrees C showed only a slight decrease in transport, which is a strong hint that dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamides (1/ 2) cross biological membranes by passive diffusion. Nearly the same results were obtained after preincubation of the Caco-2 cells with lipopolysaccharides (LPS) or phorbol 12-myristate-13-acetate (PMA) to mimic an inflammatory status. These results support the assumption that the alkamides can be easily transported from the intestinum and hence may contribute to the in vivo effects of Echinacea preparations.

Absorption of dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides after oral application of Echinacea purpurea tincture.

Alkamides are suspected to contribute to the activity of Echinacea preparations. In preliminary experiments a quantification method for dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides in human blood has been developed by which it was possible to detect dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides in human blood after oral application of Echinacea purpurea mother tincture.

The influence of glutathione and cysteine levels on the cytotoxicity of helenanolide type sesquiterpene lactones against KB cells.

The biological activities of sesquiterpene lactones have been attributed to their reactivity with the cysteine residues of functional proteins forming covalent bonds via Michael type addition. In the present study we investigated the influence of different L-cysteine (cys) and glutathione (GSH) concentrations on the cytotoxicity of the sesquiterpene lactones (STLs) helenalin, 11alpha,13-dihydrohelenalin acetate and chamissonolide against KB cells. Due to the significantly higher reactivity of the alpha-methylene-gamma-lactone (ML) towards cys as compared with the cyclopentenone (CP) site at physiological pH, addition of 20, 50 and 100 molar equivalents of cys decreased the cytotoxicity of helenalin and chamissonolide, whereas the cytotoxicity of 11alpha,13-dihydrohelenalin acetate remained unaffected. In contrast, the influence of GSH addition on the cytotoxicity of 11alpha,13-dihydrohelenalin acetate depends on the concentration of GSH added. Concentration-effect curves obtained for chamissonolide and GSH resembled the decline in cytotoxicity after cys addition. Helenalin showed a biphasic shape of the concentration-effect curve for the 100:1 GSH/helenalin ratio resembling at higher doses the chamissonolide and in lower doses the 11alpha,13-dihydrohelenalin acetate curve at 50-fold excess. These results can be explained by the different reactivity and equilibrium conditions for thiol addition of the two reactive centers of bifunctional STLs in cellular test systems and verified a clear correlation between the different reactivity of their electrophilic centers and the observed biological effects in in-vitro cell systems.

Further phenols and polyacetylenes from the rhizomes of Atractylodes lancea and their anti-inflammatory activity.

From the rhizomes of Atractylodes lancea, 2-[(2'E)-3',7'-dimethyl-2',6'-octadienyl]-4-methoxy-6-methylphenol (1) was isolated as a new natural product. The compound showed strong inhibitory effects on 5-lipoxygenase (5-LOX) and cyclooxygenase-1 (COX-1), but exhibited only weak antioxidative activities [IC50 = 0.1 microM (5-LOX), 2 microM (COX-1), 9 microM (PMN/FMLP), 28 microM (PMNIOZ)]. Moreover, five new acetylenes were isolated and elucidated as (3Z,5E,11E)-tridecatriene-7,9-diynyl-1-O-(E)-ferulate (2), erythro-(1,3Z,11E)-tridecatriene-7,9-diyne-5,6-diyl diacetate (3), (1Z)-atractylodin (4), (1Z)-atractylodinol (5), (1Z)-acetylatractylodinol (6) plus the known (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate (7). Among the acetylenes, only 2 showed strong inhibition of 5-LOX and COX-1 activity (IC50 (5-LOX) = 3 microM, IC50 (COX-1) = 1 microM). In addition, the fatty acids linoleic acid, oleic acid and palmitic acid with previously established 5-LOX-/COX-1 inhibitory actions were identified as major constituents of the n-hexane extract and thus seem to contribute to the plant's in vitro activity.

Flavonoid, iridoid, and lignan glycosides from Putoria calabrica.

From the aerial parts of Putoria calabrica, two new flavonol triglycosides were isolated and their structures were elucidated as quercetin-3-O-[alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranoside]-7-O-beta-D-glucopyranoside (1, calabricoside A) and quercetin-3-O-[4' "-O-caffeoyl-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranoside]-7-O-beta-D-glucopyranoside (2, calabricoside B). Additionally, seven iridoid and three lignan glycosides were isolated and characterized. Radical scavenging activities of all compounds were determined by quantifying their effects on luminol-enhanced chemiluminescence in formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated human polymorphonuclear neutrophils (PMNs). Calabricoside A and B showed strong radical scavenging activity with IC(50) values of 0.25 and 0.3 microM, respectively.

Minor cytotoxic and antibacterial compounds from the rhizomes of Amomum aculeatum.

A new cytotoxic 1,7-dioxa-dispiro[5.1.5.2]pentadeca-9,12-dien-11-one derivative, aculeatin D, and a new alkenone, 5-hydroxy-hexacos-1-en-3-one, have been isolated as minor compounds from the rhizomes of Amomum aculeatum. Their structures have been determined mainly by NMR spectroscopy and mass spectrometry. Aculeatin D showed high cytotoxicity against the KB and the L-6 cell line with IC(50) of 0.38 microg/ml and 1 microg/ml, respectively. Additionally, it revealed remarkable activity against two Plasmodium falciparum strains, as well as against Trypanosoma brucei rhodesiense and Trypanosoma cruzi. 5-Hydroxy-hexacos-1-en-3-one exhibited neither cytotoxic nor antiprotozoal activity, whereas antibacterial testing against Bacillus cereus, Escherichia coli and Staphylococcus epidermidis showed moderate to strong activity for both compounds.