In Vitro Activity of Propolis on Oral Microorganisms and Biofilms.

Natural products are being discussed as alternatives to commonly used chemicals in antimicrobial therapy. The study aimed to investigate the antimicrobial activity of propolis against microbial species associated with caries, periodontal disease, and Candida infections. Two commercially available ethanolic extracts of Brazilian and one of European propolis (EEP) were used. The minimal inhibitory concentrations (MIC) of propolis and controls against eight microbial strains were determined. Scanning and transmission electron microscopy (SEM and TEM) images visualized the effect of propolis on microorganisms. Subsequently, the activity on three different multi-species biofilms (both formation and existing biofilms) was assessed. All MIC values of the Brazilian EEPs were low against the tested oral species (≤0.1 mg/mL-3.13 mg/mL propolis (Candida albicans)). The European EEP had slightly higher MICs than the Brazilian EEPs. The SEM and TEM images suggest an interaction of propolis with the microbial cell wall. The European EEP exhibited the strongest effect on retarding biofilm formation, whereas the Brazilian EEPs were highly active against preformed biofilms (100 mg/mL propolis of both EEPs reduced colony forming unit counts always by more than 6 log10). The antimicrobial and anti-biofilm activities point to the potential of propolis as an adjunct in oral health care products.

Leaves and Fruits Preparations of Pistacia lentiscus L.: A Review on the Ethnopharmacological Uses and Implications in Inflammation and Infection.

There is an increasing interest in revisiting plants for drug discovery, proving scientifically their role as remedies. The aim of this review was to give an overview of the ethnopharmacological uses of Pistacia lentiscus L. (PlL) leaves and fruits, expanding the search for the scientific discovery of their chemistry, anti-inflammatory, antioxidative and antimicrobial activities. PlL is a wild-growing shrub rich in terpenoids and polyphenols, the oil and extracts of which have been widely used against inflammation and infections, and as wound healing agents. The more recurrent components in PlL essential oil (EO) are represented by α-pinene, terpinene, caryophyllene, limonene and myrcene, with high variability in concentration depending on the Mediterranean country. The anti-inflammatory activity of the oil mainly occurs due to the inhibition of pro-inflammatory cytokines and the arachidonic acid cascade. Interestingly, the capacity against COX-2 and LOX indicates PlL EO as a dual inhibitory compound. The high content of polyphenols enriching the extracts provide explanations for the known biological properties of the plant. The protective effect against reactive oxygen species is of wide interest. In particular, their anthocyanins content greatly clarifies their antioxidative capacity. Further, the antimicrobial activity of PlL oil and extracts includes the inhibition of Staphylococcus aureus, Escherichia coli, periodontal bacteria and Candida spp. In conclusion, the relevant scientific properties indicate PlL as a nutraceutical and also as a therapeutic agent against a wide range of diseases based on inflammation and infections.

Antioxidant and Biological Activities of Hydroxytyrosol and Homovanillic Alcohol Obtained from Olive Mill Wastewaters of Extra-Virgin Olive Oil Production.

Some constituents of the Mediterranean diet, such as extra-virgin olive oil (EVOO) contain substances such as hydroxytyrosol (HT) and its metabolite homovanillic alcohol (HA). HT has aroused much interest due to its antioxidant activity as a radical scavenger, whereas only a few studies have been made on the HA molecule. Both chemical synthesis and extraction techniques have been developed to obtain these molecules, with each method having its advantages and drawbacks. In this study, we report the use of tyrosol from olive mill wastewaters as a starting molecule to synthesize HT and HA, using a sustainable procedure characterized by high efficiency and low cost. The effects of HT and HA were evaluated on two cell lines, THP-1 human leukemic monocytes and L-6 myoblasts from rat skeletal muscle, after treating the cells with a radical generator. Both HT and HA efficiently inhibited ROS production. In particular, HT inhibited the proliferation of the THP-1 leukemic monocytes, while HA protected L-6 myoblasts from cytotoxicity.

Resin foraging dynamics in Varroa destructor-infested hives: a case of medication of kin?

Social insects have evolved colony behavioral, physiological, and organizational adaptations (social immunity) to reduce the risks of parasitization and/or disease transmission. The collection of resin from various plants and its use in the hive as propolis is a clear example of behavioral defense. For Apis mellifera, an increased propolis content in the hive may correspond to variations in the microbial load of the colony and to a downregulation of an individual bee's immune response. However, many aspects of such antimicrobial mechanism still need to be clarified. Assuming that bacterial and fungal infection mechanisms differ from the action of a parasite, we studied the resin collection dynamics in Varroa destructor-infested honeybee colonies. Comparative experiments involving hives with different mite infestation levels were conducted in order to assess the amount of resin collected and propolis quality within the hive, over a 2-year period (2014 and 2015). Our study demonstrates that when A. mellifera colonies are under stress because of Varroa infestation, an increase in the number of resin foragers is recorded, even if a general intensification of the foraging activity is not observed. A reduction in the total polyphenolic content in propolis produced in infested versus uninfested hives was also noticed. Considering that different propolis types show varying levels of inhibition against a variety of honey bee pathogens in vitro, it would be very important to study the effects against Varroa of two diverse types of propolis: from Varroa-free and from Varroa-infested hives.

The effects of raw propolis on Varroa-infested honey bee (Apis mellifera) workers.

Self-medication plays a major role in the behavioral defense against pathogens and parasites that animals have developed during evolution. The conditions defining this adaptive behavior are: (1) contact with the substance in question must be deliberate; (2) the substance must be detrimental to one or more parasites; (3) the detrimental effect on parasites must lead to increased host fitness. Recent studies have shown that A. mellifera colonies are able to increase resin foraging rates when infested by V. destructor, whereas further investigations are needed for evidence of parasite and host fitness. In order to understand whether Varroa-infested colonies could benefit from increasing levels of resin, we carried out laboratory bioassays to investigate the effects of propolis on the fitness of infested workers. The longevity and energetic stress of adult bees kept in experimental cages and artificially infested with the mite were thus monitored over time. At the same time, in vitro experiments were performed to study the contact effects of crude propolis on Varroa mites. Our results clearly demonstrate the positive effects of raw propolis on the lifespan of Varroa-infested adult bees. A low narcoleptic effect (19-22%) of raw propolis on phoretic mites after 5 h was also observed. In terms of energetic stress, we found no differences between Varroa-free and Varroa-infested bees in terms of the daily sucrose solution demand. Our findings seem to confirm the hypothesis that resin collection and propolis use in the hive represent an example of self-medication behavior in social insects.

Traditional knowledge about plant, animal, and mineral-based remedies to treat cattle, pigs, horses, and other domestic animals in the Mediterranean island of Sardinia.

BACKGROUND: Mediterranean farmers traditionally utilized plants, animals, and minerals sourced locally to treat their animals. Research is needed to understand at what extent such knowledge of domestic animal care still survives and to document such traditions for further developments. METHODS: We carried out our field study to recover ancient ethno-veterinary practices by means of questionnaires and interviews to farmers in rural areas of the Mediterranean island of Sardinia (Italy). Quantitative indices were used to evaluate the distribution and diversity of the acquired information. RESULTS: We report here 98 sources (42 plant taxa, 14 animal-based substances, 15 minerals, and 27 other materials of various origin) emerged from the survey for the care of 41 ailments of cattle, pigs, and horses. Ethno-veterinary treatments, detailed in their formulations and applications, were used against ecto- and endo-parasites, gastrointestinal diseases, heart diseases, viral and bacterial diseases, wounds, sprains, and bruises. CONCLUSION: Our survey can be useful to implement the use of phyto-therapeutics and other remedies of non-herbal origin for diseased animals, and, as elderly farmers held most of the knowledge, it can contribute to the conservation of Mediterranean ethno-veterinary knowledge.

Analysis of chromosome damage by sister chromatid exchange (SCE) and redox homeostasis characterization on sheep flocks from Sardinian pasturelands.

Over the last decades, an increase of pollutants of diverse origin (industrial, military, mining, etc.) was recorded in several areas of Sardinia Island. We report the results of a multidisciplinary and complementary study based on cytogenetic and physiological analyses. The data obtained show the effects of the environmental impact on six sheep flocks (Sardinian breed) grazing on natural pasturelands next to possible polluted areas and compared to three herds grazing in different areas far from those potentially contaminated and used as control. Sister chromatid exchange (SCE) test was used as cytogenetic test to analyze chromosomal damages and it was performed on peripheral blood samples collected from 129 adult sheep (age > 4 years) randomly selected from polluted (92 animals) and control (37 animals) areas. Two types of cell cultures were performed: without (normal cultures) and with the addition of 5-BrdU. SCE-mean values estimated over 35 cells counted for each animal were 8.65 ± 3.40, 8.10 ± 3.50, 8.05 ± 3.08, 7.42 ± 3.34, 9.28 ± 3.56 and 8.38 ± 3.29 in the exposed areas, whereas the average values were 7.86 ± 3.31 in the control group. Significant increases (P < 0.01) of SCEs were found in three investigated areas of Southern Sardinia. Furthermore, sheep of the same flocks were characterized for blood redox homeostasis in order to define the potential targets of oxidative damage and to identify biomarkers of the extent of animal exposure to environmental contaminants. The plasma levels of Asc, Toc and Ret were found to be significantly lower (P < 0.001) in exposed sheep (I, II, IV and V) than in the control group. TAC as well as GPx and SOD activities were higher in control than in the exposed groups (P < 0.001). Finally, plasma levels of N-Tyr, PC, and LPO were significantly lower (P < 0.001) in the control group than in the exposed groups.

Condensed tannin accumulation and nitrogen fixation potential of Onobrychis viciifolia Scop. grown in a Mediterranean environment.

BACKGROUND: Sainfoin (Onobrychis viciifolia Scop.) is a forage legume found in temperate areas but is less widespread in Mediterranean environments. Compared with other perennial legumes, it has the advantage of containing condensed tannins (CT) that can be important for their implications on ruminant nutrition and health. Data on nitrogen (N) fixation by sainfoin in the literature originate from very different environments and only a few field data are available, so it is important to improve knowledge on the N fixation potential of this species, particularly under a Mediterranean climate. Here the accumulation pattern of polyphenolic compounds (total, non-tannic polyphenols and CT) and the N fixation potential of sainfoin were studied in order to contribute to its valorisation for sustainable farming management in Mediterranean environments. RESULTS: CT concentrations were always in the range considered beneficial for animals, not exceeding 50 g delphinidin equivalent kg⁻¹ dry matter (DM). The regression of aerial fixed N on aerial DM showed a relationship of 22 kg fixed N t⁻¹ aerial DM in a Mediterranean environment. CONCLUSION: A wider exploitation of sainfoin is suggested for production under rain-fed conditions, thus enlarging the limited set of available perennial legumes suitable for Mediterranean environments.

Correlations between phenolic content and antioxidant properties in twenty-four plant species of traditional ethnoveterinary use in the Mediterranean area.

CONTEXT: Scientific information on antioxidant properties and phenolic content of less widely used plants can be useful. Therefore, the assessment of such properties remains an interesting and useful task, particularly for finding new sources for natural antioxidants, functional foods, and nutraceuticals. OBJECTIVE: As knowledge about antioxidant properties and phenolic content of many plant species used as traditional plant remedies is limited, we determined in vitro the total antioxidant activity and the phenolic content of several plant species traditionally used for ethnoveterinary practices. MATERIALS AND METHODS: For 24 extracts (70% acetone) from wild and cultivated plant species traditionally used for health care of animals we determined the Trolox equivalent antioxidant capacity (TEAC) by the two assays 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the 2,2'-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS). The phenolic content was determined by the Folin Ciocalteu method. RESULTS: Total phenolics, calculated as gallic acid equivalent (GAE), showed variation ranging from 3.18 (Allium sativum L. (Liliaceae)) to 147.68 (Pistacia lentiscus L. (Anacardiaceae)) mgGAE/g dry weight (DW). High TEAC values corresponded to high phenolic content, while plants with low antioxidant activity exhibited low total phenolic content. The TEAC determined through each assay and total phenolic content were positively correlated, R² = 0.9152 and R² = 0.8896, respectively, for DPPH and ABTS assay. DISCUSSION AND CONCLUSION: These findings suggest that phenolic content could be used as an indicator of antioxidant properties. The results of this study encourage investigations on Mediterranean plant species as sources of antioxidants.

The dynamics of phenolic concentration in some pasture species and implications for animal husbandry.

BACKGROUND: Although secondary metabolites have often been studied considering their primary role as plant-defensive and their effect on consumers detrimental, their effects are usually dose-dependent, so it is important to consider their content at all phenological phases and in different plant organs. In this study the content of total phenolics (TotP), non-tannic phenolics (NTP) and proanthocyanidins (PA) was determined in natural populations of pasture species at defined phenological phases and in different plant organs and also in pathogen-infected plants. RESULTS: Variation in TotP, NTP and PA content was observed in all species, phenological phases and plant organs. Leaf blades were the organs with the highest content in all species. The content of all examined compounds increased in infected leaves, reaching the maximum when 20% of plant tissues were attacked and decreasing at higher infection levels. CONCLUSION: Delphinidin was found as an appropriate standard related to the PA structure of each plant species. PA concentrations were always in the range considered beneficial for animals, not exceeding 60 g delphinidin equivalent kg(-1) dry matter. The Folin-Ciocalteu and butanol/HCl/Fe(3+) assays remain the most practical methods for screening large numbers of different plant species for TotP and PA.

Presence of a novel 16S-23S rRNA gene intergenic spacer insert in Bradyrhizobium canariense strains.

Seven slow-growing bacterial strains isolated from root nodules of yellow serradella (Ornithopus compressus) that originated from Asinara Island on North Western Sardinia in Italy were characterized by partial 16S rRNA gene and intergenic spacer (ITS) sequencing as well as amplified fragment length polymorphism (AFLP) genomic fingerprinting. The results indicated that the O. compressus isolates belong to the Bradyrhizobium canariense species. The analysis of ITS sequences divided the branch of B. canariense strains into two statistically separated groups (ITS clusters I and II). All the strains in ITS cluster I showed the presence of unique oligonucleotide insert TTAGAGACTTAGGTTTCTK. This insert was neither found in other described species of the family Rhizobiaceae nor in any other bacterial families and can be used as a natural and high selective genetic marker for ITS cluster I of B. canariense strains. ITS grouping of O. compressus isolates was supported by the unweighted pair group method with arithmetic averages cluster analysis of their AFLP patterns, suggesting that the strains of ITS cluster II were genetically closer to each other than to isolates from the ITS cluster I. A taxonomic importance is supposed of the revealed 19 bp ITS insert for an intraspecific division within high heterogeneous B. canariense species.