High antiplasmodial activity of novel plasmepsins I and II inhibitors

The aim of this study was to develop new antiplasmodial compounds acting through distinct mechanisms during both the liver and the blood stages of the parasite life cycle. Compounds were designed on the basis of the "double-drug" approach: primaquine, which has been linked to statine-based inhibitors of plasmepsins (PLMs), the plasmodial aspartic proteases involved in degradation of hemeoglobin. The compounds were tested in vitro for anti-PLM I/PLM II activities and against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of P. falciparum. An antiplasmodial activity (IC50) as low as 0.1 M was obtained, an excellent improvement in comparison with inhibitors previously reported (IC50 = 2-20 M). The killing activity was equally directed against both P. falciparum strains and was correlated to lipophilicity (calculated as ALogP), for all compounds but one (9). All compounds inhibited PLM I and PLM II in the nanomolar range (Ki = 1-700 nM). The most promising compounds (2, 6, 10) were not cytotoxic against human fibroblasts at 100 M and were highly selective for PLMs vs human cathepsin

Dietary polyphenols and regulation of gelatinases expression and activity

The interaction of cells with the extracellular matrix (ECM) is critical for normal development and function of organisms. The matrix metalloproteinases (MMPs) are a family of Zn++ and Ca++ dependent endopeptidases, which are key mediators of the ECM remodelling. In particular, the gelatinases (MMP-2 and \u20139) are abundantly expressed in various malignant tumors, play an active role in angiogenesis, and may influence also the process of atherosclerotic lesion formation. The turnover and remodelling of ECM must be tightly regulated since uncontrolled proteolysis contributes to abnormal development and to the generation of many pathological conditions characterized by either excessive degradation or lack of degradation of ECM components. In the recent years, much consideration has been given to the role of diet in preventing degenerative diseases such as cancer and cardiovascular diseases. Dietary constituents have been shown to be able to affect gelatinases activity. This review will focus on the effect of dietary polyphenolic components on gelatinases expression and activity

Chamomile infusions inhibit proteases involved in gastric inflammation

Chamomile, prepared with dried flowers from Matricaria recutita L., is one of the most commonly consumed herbal tea. The drug is used for the treatment of gastrointestinal complaints (minor spasms, epigastric distensions, gastritis and gastric inflammation). Several classes of bio-active compounds have been identified in the extracts of chamomile including phenolic acids, coumarins, and flavonoids such as the glycosides of apigenin, quercetin, patuletin, luteolin and several derivatives. Several studies showed that chamomile infusions possess a protective effect on gastritis and gastric ulcer, but the mechanisms involved in this effect are not completely established. Matrix metalloproteinases (MMPs) and neutrophils elastase (NE) are proteases that degrade extracellular matrix in physiological and pathological conditions. Since MMP-9 and NE are involved in gastric inflammation, the aim of this work was the evaluation of the effects of chamomile infusions of dried capitula (CFI) and sifted (SFI) flowers on MMP-9 and NE, and the identification of the compounds responsible for the observed effect. Each infusion was analyzed by LC-MS/MS in order to verify whether compositional differences affected biological activity. Analysis of CFI and SFI by LC-MS showed a complex profile. The compounds unequivocally identified by LC-MS/MS were the flavonoids apigenin-7-O-glucoside (api7glu), luteolin-7-O-glucoside (lut7glu), patuletin-7-O-glucoside (pat7glu) and hyperoside (hyp). Api7glu was more abundant in CFI than in SFI whereas the opposite was for lut7glu (5.2 \ub5g/ml vs 8.1 \ub5g/ml). Pat7glu was the most abundant in both the infusions, whereas Hyp was the lowest. CFI and SFI inhibited enzymatic activity of MMP-9 catalytic domain in a concentration-dependent manner. At 1500 microg/ml the inhibition was 28 % and 55 % for CFI and SFI, respectively. Api7glu and lut7glu (10 \ub5M) showed an inhibitory activity of 40% and 30%, respectively, demonstrating their contribute to the effect of the infusions. The inhibitory effect of CFI and SFI was confirmed on MMP-9 released by human adenocarcinoma cells (AGS cells). CFI was able to inhibit MMP-9 secretion from AGS cells (85% at 1500 microg/ml). The inhibitory effect of the infusions on NE was also tested. Concentration-response curves were performed and IC50 of CFI and SFI on NE were 369.2 microg/ml and 536.7 microg/ml, respectively. The individual compounds that showed an inhibitory effect on NE activity were api7glu (IC50 74.3 microM), lut7glu (IC50 8.6 microM), pat7glu (IC50 10.4 microM), and chlorogenic acid (IC50 31.3 microM). In conclusion, the present study shows some biochemical mechanism of action for the effect of chamomile infusions and supports the use of chamomile in the treatment of gastrointestinal inflammation

Minor polar components of extra-virgin olive oil: inhibition of NF-kB activity at gastric level

The transcription factor modulates the expression of several genes involved in the inflammatory process and several studies demonstrate its involvement in gastritis and peptic ulcer, in the presence or not of the bacterium H. pylori infection. In vivo studies show that the minor polar components of extra-virgin olive oil possess anti-inflammatory properties. The aim of this work was to investigate the mechanism of action of the anti-inflammatory effect, by evaluating whether the phenolic extract from extra-virgin olive oil inhibited the NF-kB driven transcription in gastric adenocarcinoma cells (AGS), and assessing the contribution of individual phenols. Phenolic extracts were obtained from two commercial extra-virgin olive oils, respectively from Italy and Spain, by extraction with MeOH:H2O, 80:20 v:v, and quantified with Folin-Ciocalteu assay. Secoiridoids (oleuropein aglycone and ligustroside aglycone) were analyzed by LC-MS, and phenolic alcohols (tyrosol and hydroxytyrosol) by GC-MS. AGS cells were transfected with luciferase reporter plasmid and stimulated with TNF as a pro-inflammatory agent. Both phenolic extracts inhibited NF-kB activity in a concentration-dependent manner (IC50 of 0.86 \ub1 0.12 \u3bcg/ml, and 1.28 \ub1 0.13 \u3bcg/ml for the Italian and Spanish oil respectively; mean + sd). Secoiridoids represented over the 70% of the phenolic extracts while phenolic alcohols corresponded to less than 7%. All individual compounds inhibited the activity of NF-kB in a statistically significant manner (-50% at 10 \u3bcM). Since hydroxytyrosol is catabolized to homovanillic alcohol by catechol-o-methyltransferase in humans, the effect of the later was also evaluated even if not present in the oil. The inhibitory effect reached statistically significant values at 1 \u3bcM. The effect of the two phenolic extracts was then compared to the reconstituted mixtures of the four constituents. Ligustroside aglycone and oleuropein aglycone, not commercially available, were prepared from their glucosides by hydrolysis with \u3b2-glucosidase. Reconstituted mixtures had an inhibitory effect comparable to that of the total phenolic extracts, indicating that the compounds are the main responsible of the activity of the extract. In conclusion, the results of this study suggest that the anti-inflammatory effect of the polar fraction of extra-virgin olive oil may be mediated by the inhibition of NF-kB activity. The concentration at which the phenolic extracts are active are compatible with a consumption of 30-50 g/die of oil, as estimated in the Mediterranean dietary regimen. Secoiridoids and phenolic alcohols, as well as homovanillic alcohol, contribute to anti-inflammatory action in gastric cells

Inibizione dell\u2019attivit\ue0 e dell\u2019espressione genica della metalloproteasi-9 da parte di Onopordum illyricum L. (Asteracee)

Le metalloproteasi costituiscono una famiglia di enzimi proteolitici, coinvolti nel rimodellamento della matrice extracellulare. La sovraespressione della metalloproteasi-9 (MMP-9) conduce alla degradazione eccessiva della matrice extracellulare associata a tumori maligni, dove svolge un ruolo chiave nell\u2019angiogenesi e nell\u2019invasivit\ue0 tumorale, e influenza il processo di formazione e progressione della placca aterosclerotica. Alla dieta \ue8 stato attribuito un ruolo protettivo verso l\u2019insorgenza di patologie, come tumori e malattie cardiovascolari. L\u2019Onopordum illyricum L. \ue8 una pianta edibile, consumata in Sardegna come verdura, i cui capolini e giovani scapi sono mangiati crudi in insalata. Lo scopo di questo studio \ue8 stato quello di valutare l\u2019effetto di estratti e frazioni ottenuti dalle foglie e dagli scapi florali di O. illyricum sulla attivit\ue0, secrezione ed espressione genica della MMP-9. L\u2019attivit\ue0 e la secrezione della MMP-9 da parte di macrofagi peritoneali murini sono state valutate mediante zimografia; l\u2019attivit\ue0 del promotore della MMP-9 \ue8 stata valutata mediante trasfezione transiente in cellule CV-1. Sono stati saggiati l\u2019estratto acquoso, l\u2019estratto n-butanolico (n-BuOH) e l\u2019onopordopicrina, un lattone sesquiterpenico presente in elevate quantit\ue0 nella pianta, valutati nell\u2019intervallo di concentrazione 1-50 \ub5g/ml. L\u2019estratto acquoso inibiva debolmente l\u2019attivit\ue0 della MMP-9 (-10 e -20% a 25 e 50 \ub5g/ml, rispettivamente), mentre era inattivo sulla secrezione dell\u2019enzima. L\u2019onopordopicrina era invece inattiva su entrambi i parametri, anche alla pi\uf9 alta concentrazione saggiata (25 \ub5M). L\u2019estratto n-BuOH non aveva effetto sull\u2019attivit\ue0, mentre inibiva del 53% la secrezione della MMP-9 alla concentrazione 50 \ub5g/ml. Un effetto analogo a quello sulla secrezione era osservato sull\u2019attivit\ue0 del promotore della MMP-9: tale estratto esibiva un\u2019inibizione significativa e concentrazione dipendente dell\u2019attivit\ue0 del promotore della MMP-9, raggiungendo il 64% di inibizione alla concentrazione 50 \ub5g/ml. Allo scopo di identificare i principi attivi, l\u2019estratto n-BuOH \ue8 stato frazionato per cromatografia su Sephadex LH20, ottenendo otto frazioni. L\u2019attivit\ue0 inibitoria si concentrava principalmente nella frazione 1, che non aveva effetto sull\u2019attivit\ue0, ma inibiva la secrezione della MMP-9 (-49.1% a 50 \ub5g/ml). Tale effetto era accompagnato dall\u2019inibizione significativa e concentrazione dipendente dell\u2019attivit\ue0 del promotore della MMP-9 (IC50 \ub1 SD 12.2 \ub1 1.34 \ub5g/ml). Sono in corso esperimenti al fine di determinare se l\u2019inibizione dell\u2019attivit\ue0 del promotore della MMP-9 possa essere ascrivibile alla riduzione della trascrizione genica guidata dal fattore di trascrizione NF-kB. I risultati riportati supportano l\u2019ipotesi che l\u2019apporto di micronutrienti attraverso la dieta possa contribuire alla prevenzione di patologie, quali tumori e aterosclerosi, associate all\u2019eccessiva degradazione della matrice extracellulare

Potent inhibition of human phosphodiesterase-5 by icariin derivatives

Plant extracts traditionally used for male impotence (Tribulus terrestris, Ferula hermonis, Epimedium breVicornum, Cinnamomum cassia), and the individual compounds cinnamaldehyde, ferutinin, and icariin, were screened against phosphodiesterase-5A1 (PDE5A1) activity. Human recombinant PDE5A1 was used as the enzyme source. Only E. breVicornum extract (80% inhibition at 50 μg/mL) and its active principle icariin (1) (IC50 5.9 μM) were active. To improve its inhibitory activity, 1 was subjected to various structural modifications. Thus, 3,7-bis(2-hydroxyethyl)icaritin (5), where both sugars in 1 were replaced with hydroxyethyl residues, potently inhibited PDE5A1 with an IC50 very close to that of sildenafil (IC50 75 vs 74 nM). Thus, 5 was 80 times more potent than 1, and its selectivity versus phosphodiesterase-6 (PDE6) and cyclic adenosine monophosphate-phosphodiesterase (cAMP-PDE) was much higher in comparison with sildenafil. The improved pharmacodynamic profile and lack of cytotoxicity on human fibroblasts make compound 5 a promising candidate for further development

Inhibition of metalloproteinase-9 activity and gene expression by polyphenolic compounds isolated from the bark of Tristaniopsis calobuxus (Myrtaceae)

Excessive breakdown of extracellular matrix by metalloproteinases (MMPs) occurs in many pathological conditions, and thus inhibition of MMP activity might have therapeutic potential. The methanolic extract and the identified compounds from the bark of Tristaniopsis calobuxus Brongniart & Gris (Myrtaceae) were tested on the activity, production, and gene expression of MMP-9. The extract produced a concentration-dependent inhibition (50-95% at 10-50 \u3bcg/ml) of MMP-9 activity. The inhibitory activity was retained in the ethyl acetate-soluble fraction (50-95% inhibition at 10-50 \u3bcg/ml) which also reduced the release of MMP-9 by mouse peritoneal macrophages up to 80%. In the ethyl acetate-soluble fraction, two active fractions, 5A and 5B were identified. HPLC-MS and NMR analyses of these fractions indicated the presence of gallocatechin, ellagic acid, and its glycoside derivatives. Since the absolute configuration of gallocatechin was not determined, in the next experiments both (+)-gallocatechin (2R,3S) and (-)-gallocatechin (2S,3R) were tested, and (-)-epigallocatechin (2R,3R) was included for comparison. 5A and 5B inhibited MMP-9 secretion, an observation which correlated with the decrease of MMP-9 promoter activity and the downregulation of mRNA levels. All compounds decreased MMP-9 mRNA levels and secretion. Ellagic acid, (+)-gallocatechin and (-)-epigallocatechin, but not (-)-gallocatechin inhibited promoter-driven transcription. Thus configuration at C2 (R) of the flavanol seem to be critical for the interaction with the promoter