G.V. Galli

Ellagitannins of the fruit rind of pomegranate (Punica granatum) antagonize in vitro the host inflammatory response mechanisms involved in the onset of malaria

BackgroundThe sun-dried rind of the immature fruit of pomegranate (Punica granatum) is presently used as a herbal formulation (OMARIA, Orissa Malaria Research Indigenous Attempt) in Orissa, India, for the therapy and prophylaxis of malaria. The pathogenesis of cerebral malaria, a complication of the infection by Plasmodium falciparum, is an inflammatory cytokine-driven disease associated to an up-regulation and activity of metalloproteinase-9 and to the increase of TNF production. The in vitro anti-plasmodial activity of Punica granatum (Pg) was recently described. The aim of the present study was to explore whether the anti-malarial effect of OMARIA could also be sustained via other mechanisms among those associated to the host immune response.MethodsFrom the methanolic extract of the fruit rind, a fraction enriched in tannins (Pg-FET) was prepared. MMP-9 secretion and expression were evaluated in THP-1 cells stimulated with haemozoin or TNF. The assays were conducted in the presence of the Pg-FET and its chemical constituents ellagic acid and punicalagin. The effect of urolithins, the ellagitannin metabolites formed by human intestinal microflora, was also investigated.ResultsPg-FET and its constituents inhibited the secretion of MMP-9 induced by haemozoin or TNF. The effect occurred at transcriptional level since MMP-9 mRNA levels were lower in the presence of the tested compounds. Urolithins as well inhibited MMP-9 secretion and expression. Pg-FET and pure compounds also inhibited MMP-9 promoter activity and NF-kB-driven transcription.ConclusionsThe beneficial effect of the fruit rind of Punica granatum for the treatment of malarial disease may be attributed to the anti-parasitic activity and the inhibition of the pro-inflammatory mechanisms involved in the onset of cerebral malaria.

Inhibition of platelet aggregation by olive oil phenols via cAMP-phosphodiesterase

The aim of the present study was to confirm that olive oil phenols reduce human platelet aggregability and to verify the hypothesis that cAMP- and cGMP- phosphodiesterases (PDE) could be one of the targets of the biological effect. Four extracts from oils characterized by a high phenol content (HPE), and low phenol levels (LPE) were prepared and analyzed qualitatively and quantitatively by HPLC-UV and electrospray ionization-MS/MS. Human washed platelets stimulated with thrombin were used for the aggregation assay. Human platelet cAMP-PDE and recombinant PDE5A1 were used as enzyme source. Platelet aggregation and enzyme activity were assayed in the presence of HPE, LPE and individual phenols. The phenol content of HPE ranged between 250 and 500 mg/kg, whereas the LPE content was 46 mg/kg. The compounds identified were hydroxytyrosol (HT), tyrosol (TY), oleuropein aglycone (OleA) and the flavonoids quercetin (QU), luteolin (LU) and apigenin (AP). OleA was the most abundant phenol (range 23.3 to 37.7 %) and LU was the most abundant flavonoid in the extracts. Oil extracts inhibited platelet aggregation with an 50% inhibitory concentration interval of 1.23-11.2 microg/ml. The inhibitory effect of individual compounds (10 microm) including homovanillyl alcohol (HVA) followed this order: OleA>LU>HT = TY = QU = HVA, while AP was inactive. All the extracts inhibited cAMP-PDE, while no significant inhibition of PDE5A1 (50 microg/ml) was observed. All the flavonoids and OleA inhibited cAMP-PDE, whereas HT, TY, HVA (100 microm) were inactive. Olive oil extracts and part of its phenolic constituents inhibit platelet aggregation; cAMP-PDE inhibition is one mechanism through which olive oil phenols inhibit platelet aggregation.

Olive Oil Phenols Modulate the Expression of Metalloproteinase 9 in THP-1 Cells by Acting on Nuclear Factor-κB Signaling

In vivo studies suggest that the phenolic component contributes to the anti-inflammatory and antiatherosclerotic actions of olive oil; however, the effects in circulating cells are not fully characterized. Monocytes play a key role in inflammation-based diseases by expressing several molecules, including metalloproteinases (MMPs). In the present study, we investigated the effects of olive oil phenolic extract and individual compounds on MMP-9 in THP-1 cells, a human monocyte-like cell line. Olive oil extract prevented the stimulation of MMP-9 expression and secretion in tumor necrosis factor alpha-treated THP-1 cells. Oleuropein aglycone, a typical olive oil phenol, was active at concentrations found in the extract, although other compounds probably contribute to the biological activity. We also found that the effect of the extract and individual compounds on MMP-9 is due to impaired nuclear factor-kappaB signaling. Our findings provide further evidence on the mechanisms by which olive oil reduces the inflammatory burden associated with disorders, such as atherosclerosis.

Inhibition of NF-kB and metalloproteinase-9 expression and secretion by parthenolide derivatives

Semisynthetic derivatives of parthenolide (1) were tested on NF-kB driven transcription and metalloproteinase-9 (MMP-9) expression and secretion. The four membered ring compounds 5 and 6, obtained by acidic treatment of 1, exhibited a higher activity with respect to 1 in all the biological assays. Then an increased ability of the 5 and 6 to inhibit NF-kB driven transcription may lead to a down-regulation of MMP-9 expression and secretion. This work provides new details about the structural requisites for NF-kB inhibition.

Dietary polyphenols and regulation of gelatinase expression and activity

The interaction of cells with the extracellular matrix (ECM) is critical for the normal development and function of organisms. The matrix metalloproteinases (MMPs) are a family of Zn(++) and Ca(++) dependent endopeptidases, which are key mediators of ECM remodelling. The turnover and remodelling of ECM must be tightly regulated, since uncontrolled proteolysis would contribute to abnormal development and to the generation of many pathological conditions characterized by either excessive degradation, or lack of degradation of ECM components. In particular, the gelatinases (MMP-2 and -9) are abundantly expressed in various malignant tumors, play an active role in angiogenesis, and may also influence the process of atherosclerotic lesion formation. In recent years, much consideration has been given to the role of diet in preventing degenerative diseases, such as cancer and cardiovascular diseases. Polyphenols are abundant components/micronutrients of the human diet that have been shown in vitro to profoundly affect ECM turnover by regulating gelatinases expression and activity, acting at both the pre- and post-transcriptional level. Therefore, they could have a beneficial effect in many pathological conditions implicated in connective tissue destruction and remodelling associated with degenerative diseases.

A structure-activity study for the inhibition of metalloproteinase-9 activity and gene expression by analogues of gallocatechin-3-gallate

Abstract.Catechins are able to modulate the gelatinolytic activity of matrix metalloproteinase-9 (MMP-9) by reducing its release from macrophages. Gallocatechins decrease MMP-9 secretion by lowering MMP-9 promoter activity and mRNA levels. The effect appears to be dependent on some structural and stereochemical requirements. In this study, the relationship between chemical structure and activity was studied by testing the effect of analogues of (±)-gallocatechin-3-gallate (±)-GCG, selectively deprived of hydroxyl groups, on MMP-9 activity, transcription, and secretion. Our results indicate that (±)-GCG and (±)-catechin-3-gallate are characterized by a substitution pattern compatible with direct inhibition of MMP-9 activity. Conversely, when transcription was the target, (±)-trans-3-flavanol-3-benzoate, lacking all the hydroxyl groups, was the most effective both in lowering MMP-9 promoter activity and consequently protein secretion, and in inhibiting nuclear-factor-κB-driven transcription. Our results suggest that the structural requirements for enzyme inhibition are different from those necessary for targeting gene expression.

Atovaquone-statine "double-drugs" with high antiplasmodial activity.

Malaria is one of the most widespread parasitic infections in the world; the unavailability of a vaccine and the spread and intensification of drug resistance over the past 15–20 years have led to a dramatic decline in the efficacy of the most affordable antimalarial drugs. The reported presence of sporadic cases of artemether resistance indicates the need for increased vigilance and a coordinated and rapid deployment of drug combinations. Plasmepsins are a family of proteases comprising aspartic proteases Plasmepsins I, II, and IV (PLM I, PLM II, and PLM IV) and an histoaspartic protease (HAP) localized in the digestive vacuole of Plasmodium falciparum (Pf) together with other proteases (that is, falcipains). These proteases are involved in the degradation of haemoglobin during the intraerythrocytic cycle of Pf. Haemoglobin digestion is a crucial process for the survival of the parasite in the red blood cell (RBC) both for providing amino acids for protein biosynthesis and for reducing the osmotic pressure in the RBC. The inhibition of these enzymes has been proposed as a promising target for the development of a new antimalarial therapeutic approach. The protease machinery developed by the parasite to accomplish this task is quite redundant and the role of each enzyme needs further investigation. The developed PLM inhibitors, including molecules with a statinebased core and peptidomimetic structures, showed a limited effectiveness in inhibiting the intraerythrocytic parasite growth (IC50 range 1–20 mm), although their Ki values against PLMs were in the nanomolar range. Their low potency against cultured parasites may be explained by the redundancy of proteases which implies that the blockade of one or two PLMs may not be lethal. The “double-drug” approach combines two different inhibitors into a single chemical entity by a linker, with the aim of improving the physicochemical characteristics of the individual compounds. We have previously reported the high antiplasmodial activity of double-drug 1, designed by linking a statinebased inhibitor of PLMs, with primaquine, a known antimalarial drug active against the hepatic stage of Plasmodium. 10] We describe herein the synthesis and biological evaluation of a series of ATQ–statine double-drugs. ATQ is a very potent anti-

Anti-plasmodial activity of Ailanthus excelsa

The anti-plasmodial activity of Ailanthus excelsa stem bark was investigated. The methanolic extract inhibited in vitro growth of chloroquine-sensitive (D10) and resistant strains (W2) of Plasmodium falciparum (IC50 4.6 and 2.8 microg/ml, respectively). The effect was retained in the chloroform fraction (3.1 and 2.1 microg/ml, respectively). The anti-plasmodial activity could be ascribed to the impairment of haemoglobin degradation through the inhibition of plasmepsin II activity (IC50 of 13.43+/-1.74 microg/ml) and of the haem detoxification to haemozoin.

Th-W57:3 A structure-activity study for the inhibition of metalloproteinase-9 activity and gene expression by analogues of gallocatechin-3-gallate

Catechins are able to modulate the gelatino- lytic activity of matrix metalloproteinase-9 (MMP-9) by reducing its release from macrophages. Gallocatechins decrease MMP-9 secretion by lowering MMP-9 promot- er activity and mRNA levels. The effect appears to be de- pendent on some structural and stereochemical require- ments. In this study, the relationship between chemical structure and activity was studied by testing the effect of analogues of (±)-gallocatechin-3-gallate (±)-GCG, selec- tively deprived of hydroxyl groups, on MMP-9 activity, transcription, and secretion. Our results indicate that (±)- GCG and (±)-catechin-3-gallate are characterized by a substitution pattern compatible with direct inhibition of MMP-9 activity. Conversely, when transcription was the target, (±)-trans-3-fl avanol-3-benzoate, lacking all the hydroxyl groups, was the most effective both in lower- ing MMP-9 promoter activity and consequently protein secretion, and in inhibiting nuclear-factor-kB-driven transcription. Our results suggest that the structural requirements for enzyme inhibition are different from those necessary for targeting gene expression.