Everaldo Attard

Cucurbitacin E has neuroprotective properties and autophagic modulating activities on dopaminergic neurons

Natural molecules are under intensive study for their potential as preventive and/or adjuvant therapies for neurodegenerative disorders such as Parkinsons disease (PD). We evaluated the neuroprotective potential of cucurbitacin E (CuE), a tetracyclic triterpenoid phytosterol extracted from the Ecballium elaterium (Cucurbitaceae), using a known cellular model of PD, NGF-differentiated PC12. In our postmitotic experimental paradigm, neuronal cells were treated with the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP+) to provoke significant cellular damage and apoptosis or with the potent N,N-diethyldithiocarbamate (DDC) to induce superoxide (O2 •−) production, and CuE was administered prior to and during the neurotoxic treatment. We measured cellular death and reactive oxygen species to evaluate the antioxidant and antiapoptotic properties of CuE. In addition, we analyzed cellular macroautophagy, a bulk degradation process involving the lysosomal pathway. CuE showed neuroprotective effects on MPP+-induced cell death. However, CuE failed to rescue neuronal cells from oxidative stress induced by MPP+ or DDC. Microscopy and western blot data show an intriguing involvement of CuE in maintaining lysosomal distribution and decreasing autophagy flux. Altogether, these data indicate that CuE decreases neuronal death and autophagic flux in a postmitotic cellular model of PD.

A rapid microtitre plate Folin-Ciocalteu method for the assessment of polyphenols

Several methods have been described for the determination of phenolic compounds in animal and plant products using the Folin-Ciocalteu (FC) assay. Most of these methods describe the use of this reagent and sodium carbonate in spectrophotometric methods. The macro FC assay was compared with two micro FC assays carried out on a microplate reader. Excellent correlation was obtained among the three assays with a molar extinction coefficient of 5.228±0.187x103 M−1 cm−1. The micro assay may serve as a high throughput method for the rapid determination of polyphenols in various samples.

The Effects of Cucurbitacin E on Two Lymphocyte Models

Cucurbitacin E (CuE), like other cucurbitacins, is a potential anti-neoplastic agent. The aim of the present study was to investigate the in vitro effects of CuE on phytohaemagglutinin (PHA)-activated and unstimulated lymphocytes. CuE did not produce any significant cytotoxic effects on the two models. On the contrary, it had a stimulatory effect, and was capable of inducing and maintaining high proliferation rates in lymphocytes. The stimulatory effect of CuE was concentration-dependent with a median stimulatory concentration (SC50) of 1.166 µM and reaching maximal effect at a concentration of 10–20 µM. The stimulatory effect of CuE was about 25% lower than that of PHA, but a combination of CuE and PHA had an additive effect producing a greater response than that induced by the two substances used separately. Agarose gel electrophoresis for DNA fragmentation failed to show any significant apoptotic activity in the cells after 48 h exposure to CuE.

Oleuropein Prevents Neuronal Death, Mitigates Mitochondrial Superoxide Production and Modulates Autophagy in a Dopaminergic Cellular Model.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, primarily affecting dopaminergic neurons in the substantia nigra. There is currently no cure for PD and present medications aim to alleviate clinical symptoms, thus prevention remains the ideal strategy to reduce the prevalence of this disease. The goal of this study was to investigate whether oleuropein (OLE), the major phenolic compound in olive derivatives, may prevent neuronal degeneration in a cellular dopaminergic model of PD, differentiated PC12 cells exposed to the potent parkinsonian toxin 6-hydroxydopamine (6-OHDA). We also investigated OLE’s ability to mitigate mitochondrial oxidative stress and modulate the autophagic flux. Our results obtained by measuring cytotoxicity and apoptotic events demonstrate that OLE significantly decreases neuronal death. OLE could also reduce mitochondrial production of reactive oxygen species resulting from blocking superoxide dismutase activity. Moreover, quantification of autophagic and acidic vesicles in the cytoplasm alongside expression of specific autophagic markers uncovered a regulatory role for OLE against autophagic flux impairment induced by bafilomycin A1. Altogether, our results define OLE as a neuroprotective, anti-oxidative and autophagy-regulating molecule, in a neuronal dopaminergic cellular model.

Antitrypsin activity of extracts from Ecballium elaterium seeds.

The antitrypsin activity of extracts obtained from two different seed types of Ecballium elaterium, was tested in vitro. The presence of trypsin inhibitors in three chromatographed samples for each extract, showed a strong and specific antitrypsin activity.

Investigative study on the angiotensin converting enzyme (ACE) inhibiting properties of the terpenoid extract of Crataegus monogyna using in silico models

Crataegus monogyna is mainly used in the treatment of cardiac and circulatory system disorders. In vitro and clinical studies are indicative of the fact that the hydroethanolic extract of C. monogyna has angiotensin converting enzyme (ACE) inhibitory activity. This study sought to support these claims through the use of in silico modelling techniques. Possible binding conformations for β-amyrin, oleanolic acid and ursolic acid were generated using captopril, as well as enalaprilat and lisinopril, as template ligands. The ligand binding affinity (LBA) of each was calculated and the best binding conformation of each triterpene was established. Results indicate that these naturally occuring terpenes possess in silico predicted ligand binding affinities that are superior to both the small molecule captopril and the larger molecules enalaprilat and lisinopril.

The Phytochemical and In Vitro Pharmacological Testing of Maltese Medicinal Plants

The Maltese archipelago is composed of a small number of islands with a total surface area of approximately 457 km2. Albeit this small size the Maltese islands host a vast number of plant and animal species. Plant biodiversity, with its 1264 vascular species, is mainly attributed to the strategic position of Malta within the Mediterranean, in which throughout the years several conquerors and civilisations sought to possess Malta particularly for military purposes. In part, the plant diversity of Malta is attributed to introductions brought about by various military forces, as an aid during injury and sickness. Naturally, the phytodiversity has an inclination towards the Mediterranean type of flora with an approximately 66% of the Maltese flora pertaining to this region (E. Attard, 2004). Typical Mediterranean medicinal plants include conifers (Pinus halepensis and Cupressus sempervirens), broad-leaved trees (Laurus nobilis, Morus nigra and Tamarix gallica), fruit trees (Ceratonia siliqua, Citrus trees, Nerium oleander, Olea europaea and Punica granatum), and others (Allium sativum, Aloe ferox, Capparis spinosa, Opuntia ficus-indica, Origanum vulgare, Papaver somniferum, Phytolacca decandra and Pistacia lentiscus). The other portion (34%) is attributed to plants originating from the warm North African (Cynomorium coccineum, Ficus carica and Myrtus communis) and the colder South Europaean regions (Crataegus monogyna, Populus alba and Salix species).

In vitro fermentation of feed ingredients by fresh or frozen pig fecal inocula

In vitro techniques can offer a rapid, repeatable and cost-effective alternative to in vivo experiments. We investigated the gas production (GP) of nine different feeds commonly used in Italian and Maltese pig farms in order to assess the use of the Ankom system in this species and to compare the in vitro microbial activity fresh or frozen inocula derived from piglet feces. Proximate and polyphenolic analyses were determined for all substrates. Fermentation profiles were measured according to the cumulative GP technique. Analysis of GP values revealed significant differences in the fermentation patterns mainly due to substrate and inoculum effects. Fermentation parameters varied significantly according to the substrate ingredients. The frozen inoculum generally led to more total GP than for fresh, with the exception of hard wheat bran pellets (HWBP), pelleted pulp and mature carob. Conversely the fresh fecal inoculum had an earlier maximum time to reach maximum GP than the frozen, showing a higher maximum rate of GP in all substrates excepting for HWBP. A significant difference in frozen and fresh inocula was found for the complete formulation. Further studies are required to confirm the variation between fresh and frozen fecal inocula to a wider range of feed, including those that could have negative biological effects.

Could EU herbal monographs contribute to Malta's treatment armamentarium?

Ten years have passed since Directive 2004/24/EC regulating herbal medicinal products across the EU were published. The directive created the Committee on Herbal Medicinal Products within the European Medicines Agency whose remit includes the creation and publishing of official EU monographs on herbal medicinal products. These monographs include the official uses of the products and their evidence for efficacy and safety. To this effect, we are interested in analysing the potential impact herbal product EU monographs could have on the therapeutic treatment options available for prescribers in Malta. Therefore our aim was two-fold. First, to rationalise the spread of indications of the herbal substances listed in the community herbal monograph inventory and subsequently determine if these herbal substances could potentially contribute to the treatment options available in our local scenario (Malta). 128 EU monographs were analysed resulting in a total of 230 indications which subsequently codified into 42 unique ATC codes. The Malta Medicines List contains 1456 unique ATC codes. Comparative analysis of the Malta Medicines List revealed that the 21 therapeutic areas had 4 or less pharmaceutically used substances (5th level ATC codes) registered and therefore in our opinion are areas with limited therapeutic choice. The following 4 therapeutic areas, A05 bile and liver therapy, A13 tonics, A15 appetite stimulants and D03 preparations for treatment of wounds and ulcers, could potentially benefit from the registration of herbal medicinal products according to the EU herbal monographs. If such registration is effected the aforementioned areas would no longer be considered limited because more than 4 therapeutic choices would be available to prescribers. This study is the first study across the EU to analyse the potential impact of published EU herbal monographs on therapeutic coverage in an EU member state and confirms the notion that herbal products could potentially increase the treatment options available in areas where few medical products have been registered due to Maltas small market size.

Egg proteome: the protein and carotenoids characteristics deriving from laying hens reared in different housing systems and under different environmental conditions

The rearing and breeding of poultry both for meat and egg production has long been an important part of the Maltese agricultural activity. In fact, it can safely be said that historical evidence affirm the highly importance of poultry in the hands of Maltese farmers. This can nowadays be proofed by the presence of a local rustic dual purpose poultry breed known as the ‘Black Maltese’. Nowadays local egg production has been entirely replaced by intensively reared hybrid strains of poultry, particularly those deriving from the White Leghorn and Rhode Island Red, both of which are more productive.