Enrica Bosisio

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 microg/mL) and its active principle icariin (1) (IC50 5.9 microM) 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.

Antiplasmodial activity of Punica granatum L. fruit rind.

AIM OF THE STUDY Sun-dried rind of the immature fruit of Punica granatum L. (Punicaceae) (Pg) is presently used as a herbal formulation (OMARIA) in Orissa, India, for the therapy and prophylaxis of malaria. The aims of this study were (i) to assess in vitro the antiplasmodial activity of the methanolic extract, of a tannin enriched fraction and of compounds/metabolites of the antimalarial plant, (ii) to estimate the curative efficacy of the Pg extracts and (iii) to explore the mechanism of action of the antiplasmodial compounds. Urolithins, the ellagitannin metabolites, were also investigated for antiplasmodial activity. MATERIALS AND METHODS Chloroquine-susceptible (D10) and -resistant (W2) strains of Pf were used for in vitro studies and the rodent malaria model Plasmodium berghei-BALB/c mice was used for in vivo assessments. Recombinant plasmepsins 2 and 4 were used to investigate the interference of Pg compounds with the metabolism of haemoglobin by malaria parasites. RESULTS The Pg methanolic extract (Pg-MeOH) inhibited parasite growth in vitro with a IC(50) of 4.5 and 2.8 microg/ml, for D10 and W2 strain, respectively. The activity was found to be associated to the fraction enriched with tannins (Pg-FET, IC(50) 2.9 and 1.5 microg/ml) in which punicalagins (29.1%), punicalins, ellagic acid (13.4%) and its glycoside could be identified. Plasmepsin 2 was inhibited by Pg-MeOH extract and by Pg-FET (IC(50) 7.3 and 3.0 microg/ml), which could partly explain the antiparasitic effect. On the contrary, urolithins were inactive. Both Pg-MeOH extract and Pg-FET did not show any in vivo efficacy in the murine model. CONCLUSIONS The in vitro studies support the use of Pg as antimalarial remedy. Possible explanations for the negative in vivo results are discussed.

Inhibition of Human cAMP-Phosphodiesterase as a Mechanism of the Spasmolytic Effect of Matricaria recutita L.

Mechanisms underlying the spasmolytic activity of chamomile still remain unclear. Inhibition of cAMP- and cGMP-phosphodiesterases (PDE) is one of the mechanisms operated by spasmolytic drugs. In this study, the effect of chamomile on PDE was investigated. Human platelet cAMP-PDE and recombinant PDE5A1 were assayed in the presence of infusions prepared from sifted flowers and capitula. LC-ESI-MS/MS analysis showed different compositions in infusions made with sifted flowers and capitula. Chamomile inhibited cAMP-PDE activity (IC50 = 17.9-40.5 microg/mL), while cGMP-PDE5 was less affected (-15% at 50 microg/mL). Among the individual compounds tested, only flavonoids showed an inhibitory effect (IC50 = 1.3-14.9 microM), contributing to around 39% of the infusion inhibition; other compounds responsible for cAMP-PDE inhibition still remain unknown. Although experimental evidence supporting the use of chamomile for gastrointestinal minor spasms dates back to the fifties, cAMP-PDE inhibition as a likely mechanism underlying the spasmolytic activity is reported for the first time.

A Bio-Guided Fractionation to Assess the Inhibitory Activity of Calendula officinalis L. on the NF-κB Driven Transcription in Human Gastric Epithelial Cells.

Calendula officinalis L. has been largely known for its topical anti-inflammatory properties; however, there are no experimental evidences about its antiphlogistic effect at the gastric level. To investigate whether marigold might exert an activity against gastric inflammation, a CH2Cl2 extract obtained from C. officinalis flowers was evaluated in vitro on the NF-κB pathway. The lipophilic extract demonstrated a significant inhibitory effect on the NF-κB driven transcription. The identification of active compounds was conducted by a bio-guided fractionation of the extract that afforded 16 fractions. Fraction J exhibited a concentration-dependent inhibitory activity on the NF-κB driven transcription and significantly contributed to the antiphlogistic effect showed by CH2Cl2 extract. The main components of fraction J were loliolide and the fucoside acetates of β-eudesmol and viridiflorol. HPLC analysis of fractions D and E led to the identification and isolation of triterpene esters that showed a concentration-dependent inhibition of the NF-κB driven transcription, with faradiol-3-myristate and the corresponding aglycone being the most active compounds. The present study provides some experimental evidences that Calendula officinalis L. may exert an anti-inflammatory activity on the gastric district by the inhibition of the NF-κB system, identifying the compounds responsible, at least in part, for the observed effect.