Anna Baldisserotto

Activity and stability studies of verbascoside, a novel antioxidant, in dermo-cosmetic and pharmaceutical topical formulations.

We here report the results of our investigations carried out on verbascoside, a phenylpropanoid glycoside known for its antioxidant, anti-inflammatory and photoprotective actions. Verbascoside was obtained from Buddleia davidii meristematic cells, obtained in turn using a sustainable biotechnology platform which employs an in vitro plant cell culture technology. Verbascoside was first investigated to assess the behaviour of the active ingredient in solution or in finished preparations, in view of its potential topical use, especially in skin protection. Stability studies were performed by HPLC, and a PCL assay was adopted to determine the radical scavenging activity toward superoxide anion. The high hydrophilic character of verbascoside, suggested in a somewhat limited range of possible applications, leading us to explore its derivatization to obtain the semi-synthetic derivative VPP, an acyl derivative of verbascoside, with an improved range of applications due to its lower hydrophilic profile. Alone, VPP revealed increased antioxidant activity, both as an active ingredient and in dermocosmetic preparations. Stability studies showed a greater stability of VPP in lipophilic vehicles, whereas the parent verbascoside proved more stable in an O/W emulsions. Verbascoside was also stable in suppositories, an interesting pharmaceutical form for possible applications in treatment of inflammation of the intestinal mucosa.

In vitro and in vivo studies on UFP-112, a novel potent and long lasting agonist selective for the nociceptin/orphanin FQ receptor

[(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112) has been designed as a novel ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) by combining into the same peptide different chemical modifications reported to increase N/OFQ potency. In vitro data obtained in the electrically stimulated mouse vas deferens demonstrated that UFP-112 behaved as a high potency (pEC(50) 9.43) full agonist at the NOP receptor. UFP-112 effects were sensitive to the NOP antagonist UFP-101 but not to naloxone and no longer evident in tissues taken from NOP(-/-) mice. In vitro half life of UFP-112 in mouse plasma and brain homogenate was 2.6- and 3.5-fold higher than that of N/OFQ. In vivo, in the mouse tail withdrawal assay, UFP-112 (1-100pmol, i.c.v.) mimicked the actions of N/OFQ producing pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t.; in both cases, UFP-112 was approximately 100-fold more potent than the natural peptide and produced longer lasting effects. UFP-112 also mimicked the hyperphagic effect of N/OFQ producing a bell shaped dose response curve with the maximum reached at 10pmol. The hyperphagic effects of N/OFQ and UFP-112 were absent in NOP(-/-) mice. Equi-effective high doses of UFP-112 (0.1nmol) and N/OFQ (10nmol) were injected i.c.v. in mice and spontaneous locomotor activity recorded for 16h. N/OFQ produced a clear inhibitory effect which lasted for 60min while UFP-112 elicited longer lasting effects (>6h). In conscious rats, UFP-112 (0.1 and 10nmol/kg, i.v.) produced a marked and sustained decrease in heart rate, blood pressure, and urinary sodium excretion and a profound increase in urine flow. Collectively, these findings demonstrate that UFP-112 behaves in vitro and in vivo as a highly potent and selective ligand able to produce full and long lasting activation of NOP receptors.

Synthesis, antioxidant and antimicrobial activity of a new phloridzin derivative for dermo-cosmetic applications.

The phenolic compound phloridzin (phloretin 2′-O-glucoside, variously named phlorizin, phlorrhizin, phlorhizin or phlorizoside) is a prominent member of the chemical class of dihydrochalcones, which are phenylpropanoids. Phloridzin is specifically found in apple and apple juice and known for its biological properties. In particular we were attracted by potential dermo-cosmetic applications. Here we report the synthesis, stability studies and antimicrobial activity of compound F2, a new semi-synthetic derivative of phloridzin. The new derivative was also included in finished formulations to evaluate its stability with a view to a potential topical use. Stability studies were performed by HPLC; PCL assay and ORAC tests were used to determine the antioxidant activity. F2 presented an antioxidant activity very close to that of the parent phloridzin, but, unlike the latter, was more stable in formulations. To further explore potential health claims, antifungal activity of phloridzin and its derivative F2 were determined; the results, however, were rather low; the highest value was 31,6% of inhibition reached by F2 on Microsporum canis at the highest dose.

Further Studies on Lead Compounds Containing the Opioid Pharmacophore Dmt-Tic

Some reference opioids containing the Dmt-Tic pharmacophore, especially the delta agonists H-Dmt-Tic-Gly-NH-Ph (1) and H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid (4) (UFP-512) were evaluated for the influence of the substitution of Gly with aspartic acid, its chirality, and the importance of the -NH-Ph and N(1)H-Bid hydrogens in the inductions of delta agonism. The results provide the following conclusions: (i) Asp increases delta selectivity by lowering the mu affinity; (ii) -NH-Ph and N(1)H-Bid nitrogens methylation transforms the delta agonists into delta antagonists; (iii) the substitution of Gly with L-Asp/D-Asp in the delta agonist H-Dmt-Tic-Gly-NH-Ph gave delta antagonists; the same substitution in the delta agonist H-Dmt-Tic-NH-CH2-Bid yielded more selective agonists, H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid and H-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid; (iv) L-Asp seems important only in functional bioactivity, not in receptor affinity; (v) H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N(1)-Me) (10) evidenced analgesia similar to 4, which was reversed by naltrindole only in the tail flick. 4 and 10 had opposite behaviours in mice; 4 caused agitation, 10 gave sedation and convulsions.

Essential Oil Extraction, Chemical Analysis and Anti-Candida Activity of Calamintha nepeta (L.) Savi subsp. glandulosa (Req.) Ball-New Approaches.

A comprehensive study on essential oils extracted from different Calamintha nepeta (L.) Savi subsp. glandulosa (Req.) Ball samples from Tarquinia (Italy) is reported. In this study, the 24-h steam distillation procedure for essential oil preparation, in terms of different harvesting and extraction times, was applied. The Gas chromatography–mass spectrometry (GC/MS) analysis showed that C. nepeta (L.) Savi subsp. glandulosa (Req.) Ball essential oils from Tarquinia belong to the pulegone-rich chemotype. The analysis of 44 samples revealed that along with pulegone, some other chemicals may participate in exerting the related antifungal activity. The results indicated that for higher activity, the essential oils should be produced with at least a 6-h steam distillation process. Even though it is not so dependent on the period of harvesting, it could be recommended not to harvest the plant in the fruiting stage, since no significant antifungal effect was shown. The maximum essential oil yield was obtained in August, with the highest pulegone percentage. To obtain the oil with a higher content of menthone, September and October should be considered as the optimal periods. Regarding the extraction duration, vegetative stage material gives the oil in the first 3 h, while material from the reproductive phase should be extracted at least at 6 or even 12 h.

Proteasome inhibitors induce the presentation of an Epstein–Barr virus nuclear antigen 1‐derived cytotoxic T lymphocyte epitope in Burkitt’s lymphoma cells

The Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is generally expressed in all EBV‐associated tumours and is therefore an interesting target for immunotherapy. However, evidence for the recognition and elimination of EBV‐transformed and Burkitt’s lymphoma (BL) cells by cytotoxic T lymphocytes (CTLs) specific for endogenously presented EBNA1‐derived epitopes remains elusive. We confirm here that CTLs specific for the HLA‐B35/B53‐presented EBNA1‐derived HPVGEADYFEY (HPV) epitope are detectable in the majority of HLA‐B35 individuals, and recognize EBV‐transformed B lymphocytes, thereby demonstrating that the GAr domain does not fully inhibit the class I presentation of the HPV epitope. In contrast, BL cells are not recognized by HPV‐specific CTLs, suggesting that other mechanisms contribute to providing a full protection from EBNA1‐specific CTL‐mediated lysis. One of the major differences between BL cells and lymphoplastoid cell lines (LCLs) is the proteasome; indeed, proteasomes from BL cells demonstrate far lower chymotryptic and tryptic‐like activities compared with proteasomes from LCLs. Hence, inefficient proteasomal processing is likely to be the main reason for the poor presentation of this epitope in BL cells. Interestingly, we show that treatments with proteasome inhibitors partially restore the capacity of BL cells to present the HPV epitope. This indicates that proteasomes from BL cells, although less efficient in degrading reference substrates than proteasomes from LCLs, are able to destroy the HPV epitope, which can, however, be generated and presented after partial inhibition of the proteasome. These findings suggest the use of proteasome inhibitors, alone or in combination with other drugs, as a strategy for the treatment of EBNA1‐carrying tumours.

Design, synthesis and biological activity of a novel Rutin analogue with improved lipid soluble properties.

Recent interest in flavonoids has increased greatly due to their biological and pharmacological activities. Flavonoids, consist of a large group of low molecular weight polyphenolic substances, naturally occurring in fruits, vegetables, tea, and wine, and are an integral part of the human diet. Rutin is a common dietary flavonoid that is widely consumed worldwide from plant-derived beverages and foods as traditional and folk medicine remedy as well. Rutin exhibit important pharmacological activities, including anti-oxidation, anti-inflammation, anti-diabetic, anti-adipogenic, neuroprotective and hormone therapy. Here, we present the synthesis, antimicrobial, antiproliferative and pro-apoptotic effect on human leukemic K562 cells of compound R2, a new semi-synthetic derivative of Rutin as compared to Rutin itself. The new derivative was also included in finished topical formulations to evaluate a potential application to the dermatology field in view of the antioxidant/antimicrobial/antiinflammatory properties. Stability studies were performed by HPLC; PCL assay and ORAC tests were used to determine the antioxidant activity. R2 presented an antioxidant activity very close to that of the parent Rutin while bearing much better lipophilic character. Regarding antiproliferative effects on the human K562 cell line, R2 was found to be more effective than parent Rutin. Preliminary experiments demonstrated that R2 inhibits NF-kB activity and promotes cellular apoptosis.

Essential oil extraction, chemical analysis and anti-Candida activity of Foeniculum vulgare Miller – new approaches

Abstract A comprehensive study on essential oil samples of Foeniculum vulgare Miller from Tarquinia (Italy) is reported. A 24-h systematic steam distillation was performed on different harvested samples applying different extraction times. The GC-MS analysis of the residue outcome showed o-cymene, α-phellandrene, α-pinene and estragole as the major constituents. The predominance and continued presence of o-cymene makes this fennel oil a rather unique chemotype. An evident correlation between the antifungal activity and phenological stage is demonstrated. The most active fractions were particularly rich in estragole, as well as a significant amount of fenchone that possibly exerts some additive effect in the expression of overall antifungal potency. Pre-fruiting material produced oil particularly rich in o-cymene. With reference to the duration of the extraction, the maximum amount of oil was released within the first 3 h, whereas the reproductive phase material needed at least 6 h for the extraction.

Trypanocidal activity of peptidyl vinyl ester derivatives selective for inhibition of mammalian proteasome trypsin-like activity

Nine vinyl ester tripeptides selective for inhibition of mammalian proteasome trypsin-like activity were tested for in vitro activity against Trypanosoma brucei. Interestingly, two compounds showed trypanocidal activity in the low micromolar range without displaying cytotoxicity against human cells. However, the compounds did not inhibit the trypsin-like activity of the trypanosome proteasome although their effect correlates with inactivation of the chymotrypsin-like activity. This finding shows that the inhibitor sensitivities between mammalian and trypanosome proteasome are distinct. This difference may be exploited for rational anti-trypanosomal drug development.

New cyclic peptide proteasome inhibitors

Here we report the study of a new series of vinyl ester cyclopeptide analogues synthesized on the basis of our previous development of a class of cyclopeptides derived from our linear prototype inhibitors. In these compounds, the exocyclic pharmacophoric unit Leu-VE was linked to the gamma-carboxyl group of the glutamic acid residue at the C-terminal. The best analogues of the series have been shown to inhibit the caspase-like activity of the proteasome at nanomolar concentrations and have also demonstrated good resistance to proteolysis and a capacity to permeate the cell membrane.