Development and Validation of QuEChERS Followed by UHPLC-ToF-MS Method for Determination of Multi-Mycotoxins in Pistachio Nuts.

Pistachios are one of the types of tree nut fruits with the highest mycotoxin contamination, especially of aflatoxins, worldwide. This study developed a Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method that was followed by Ultra-High Performance Liquid Chromatography combined with Time-of-Flight Mass Spectrometry (UHPLC-ToF-MS) for the determination of mycotoxins in pistachios. Different approaches to dispersive solid phase extraction as a clean-up method for high lipid matrices were evaluated. For this, classic sorbents such as C18 (octadecyl-modified silica) and PSA (primary secondary amine), and new classes of sorbents, namely EMR-Lipid (enhanced matrix removal-lipid) and Z-Sep (modified silica gel with zirconium oxide), were used. The QuEChERS method, followed by Z-Sep d-SPE clean-up, provided the best analytical performance for aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), zearalenone (ZEA), toxin T2 (T2) and toxin HT-2 (HT2) in pistachios. The method was validated in terms of linearity, sensitivity, repeatability, interday precision and recovery; it achieved good results according to criteria imposed by Commission Regulation (EC) no. 401/2006. The method was applied to real samples and the results show that pistachios that are available in Portuguese markets are safe from mycotoxins that are of concern to human health.

Dehiscence and prolonged storage of 'Kerman' Pistachios: Effects on morphometry and nutraceutical value.

'Kerman' pistachios (KP; Pistacia vera L.) are an important crop for several countries but their commercial value is diminished by their shell dehiscence status and prolonged storage in popular marketplaces. The aim was to evaluate the independent/synergistic effect of prolonged storage (1-4 year) and dehiscence status (split/unsplit) on KP's morphometry and chemical composition. Whole nut's and kernel's length, width, thickness, surface area, and volume were more affected by dehiscence (split > unsplit; p ≤ 0.01) than storage time; Kernel's mass, macronutrient composition and tocopherols (T)/tocotrienols (T3) were not much affected by dehiscence but time-trend correlations were observed with macronutrient composition (split/unsplit; ρ = - 0.57-0.42) and T + T3 (unsplit; ρ = 0.81). Specific/total fatty acids were affected by a complex dehiscence × storage time interaction, and they linearly correlated with certain morphometric characteristics (r ≥ 0.6). Shell dehiscence status more than prolonged storage substantially modifies KP's quality.

Determination of the polyphenolic fraction of Pistacia vera L. kernel extracts by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry detection.

Pistachio (Pistacia vera L.) belongs to the Anacardiaceae family and it is a small tree species. It is native of the Middle East and Central Asia, but currently, it is cultivated also in California and in some Mediterranean countries, such as Greece and Italy. The most important pistachio producers are Iran, the USA, and Turkey. Besides being a delicious nut, pistachio, due to its wholesome nutritional properties, could be considered as a functional food. According to the results of several studies, pistachios have been proven to have various groups of valuable phytochemicals such as anthocyanins, flavan-3-ols, proanthocyanidins, flavonols, isoflavones, flavanones, stilbenes, and phenolic acids, possessing excellent biological activities. The most common analytical technique employed for their analysis is represented by liquid chromatography coupled to photodiode array and mass spectrometry detection. However, conventional LC can present some limits especially in terms of resolving power. In this contribution, as a powerful alternative, comprehensive two-dimensional liquid chromatography (LC×LC) was applied to the determination of the polyphenolic fraction of pistachio kernels from different geographical origins. A 150-mm micro-bore cyano column (2.7 µm dp) and 50-mm superficially porous C18 silica column (2.7 µm dp) in the first (1D) and second (2D) dimensions were employed, respectively. For boosting orthogonality, a shift 2D gradient was investigated leading to an increase in the overall peak capacity. The newly developed LC×LC method showed satisfactory linearity, sensitivity, precision and accuracy, which was then applied to sample quantitative analysis. A total of 51 different polyphenolic compounds were determined in the four samples investigated and 18 out of them are hereby reported for the first time.

Characterization and Phytotoxicity Assessment of Essential Oils from Plant Byproducts.

The present work describes the chemical characterization and the phytotoxicity assessment of essential oils (EOs) obtained from spent materials or pruning waste of four plant species: Zingiber officinale Roscoe used in the juicing industry, Pistacia vera L. var. Bronte used in the food industry, discarded material of industrial hemp (Cannabis sativa L. var. Futura 75), and pruning waste from Cupressus sempervirens L. The phytochemical profile of the EOs was evaluated by gas chromatographic flame ionization detection (GC-FID) and GC-MS analyses, which highlighted the presence of several compounds with a wide range of biological activities. Among them, application possibilities in agriculture were evaluated by studying the phytotoxic activity in vitro against germination and initial radical growth of several seeds such as Raphanus sativus L., Lepidium sativum L., Lactuca sativa L., Solanum lycopersicum L., Lolium multiflorum Lam., and Portulaca oleracea L.

Functional and sensory properties of pistachio nuts as affected by cultivar.

BACKGROUND: Modern agriculture allows farmers to choose among different cultivars of the same fruit to fulfill their agronomic needs and consumers' demands; however, there are only a few studies that describe and compare key functional and sensory properties of different pistachio cultivars. The main objective of this study was to compare eight pistachio cultivars by analyzing key functional properties (phenolic compounds, polymeric procyanidins, antioxidant activity, and inhibition of α-amylase and ß-glucosidase), aromatic compounds with gas chromatography-mass spectrometry (GC-MS), and by examining their sensory properties with a trained panel. RESULTS: A combination of LC-PDA-MS-QTof (liquid chromatography photodiode array quadrupole time-off flight mass spectrometry) and electrospray ionization was used to determine two phenolic acids, nine flavonols, one anthocyanin, and three flavan-3-ols in pistachio cultivars, with a total concentration ranging from 500 to 6065 mg 100 g-1 dry weight (dw). The total polymeric procyanidins concentrations oscillated between 348 and 5919 mg 100 g-1 dw, (-)-epicatechin being the major monomer contributor. Pinene was the most abundant volatile compound (∼200 mg kg-1 dw), and, in the sensory analysis of samples, 23 sensory attributes were found to differ significantly among the cultivars. CONCLUSION: The cultivar 'Larnaka' stood out as having the best functional profile (high polyphenolic content, high antioxidant activity, and high values of α-amylase and ß-glucosidase inhibition), and the cultivars 'Kastel' and 'Kerman' showed the most attractive sensory properties, mainly the most intense flavor. © 2019 Society of Chemical Industry.

Conserved DNA-derived polymorphism, new markers for genetic diversity analysis of Tunisian Pistacia vera L.

Pistachio trees (Pistacia vera L.) have been cultivated in Tunisia for decades and the plantation was extended mostly in the center of the country contributing to the economic growth of marginalized areas. Herein we used conserved DNA derived polymorphism (CDDP) technique, which target specifically conserved sequences of plant functional genes, to assess the genetic diversity and construct genetic relationships among 65 Tunisian pistachio trees. A set of nine primers were used and 157 CDDP markers were revealed with an average of 17.44 showing a high degree of polymorphism (99.37%). The average of polymorphism information content of CDDP markers was of 0.86, which indicates the efficiency of CDDP primers in the estimation of genetic diversity between pistachios. UPGMA dendrogram and the principal component analysis showed four clusters of analyzed pistachios trees. Our results showed that the genetic structure depends on: (1) the gene exchanges between groups, (2) the geographical origin and (3) the sex of the tree. The same result was revealed by the Bayesian analysis implemented in STRUCTURE at K = 4, in which the pistachio genotypes of El Guettar, Kasserine and Sfax were assigned with more than 80% of probability. Our results prove polymorphism and efficiency of CDDP markers in the characterization and genetic diversity analysis of P. vera L. genotypes to define conservation strategy.

In Vitro Evaluation of the Antioxidant, Cytoprotective, and Antimicrobial Properties of Essential Oil from Pistacia vera L. Variety Bronte Hull.

Although the chemical composition and biological properties of some species of the genus Pistacia has been investigated, studies on hull essential oil of Pistacia vera L. variety Bronte (HEO) are currently lacking. In this work, we have carried out an in-depth phytochemical profile elucidation by Gas Chromatography-Mass Spectrometry (GC-MS) analysis, and an evaluation of antioxidant scavenging properties of HEO, using several different in vitro methods, checking also its cytoprotective potential on lymphocytes treated with tert-butyl hydroperoxide. Moreover, the antimicrobial activity against Gram-positive and Gram-negative strains, both American Type Culture Collection (ATCC) and clinical isolates, was also investigated. GC-MS analysis highlighted the richness of this complex matrix, with the identification of 40 derivatives. The major components identified were 4-Carene (31.743%), α-Pinene (23.584%), d-Limonene (8.002%), and 3-Carene (7.731%). The HEO showed a strong iron chelating activity and was found to be markedly active against hydroxyl radical, while scarce effects were found against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Moreover, pre-treatment with HEO was observed to significantly increase the cell viability, decreasing the lactate dehydrogenase (LDH) release. HEO was bactericidal against all the tested strains at the concentration of 7.11 mg/mL, with the exception of Pseudomonas aeruginosa ATCC 9027. The obtained results demonstrate the strong free-radical scavenging activity of HEO along with remarkable cytoprotective and antimicrobial properties.

Pistachio (Pistacia vera L.) consumption improves cognitive performance and mood in overweight young adults: A pilot study.

This pilot study evaluated the impact of pistachio consumption on cognitive performance and mood in overweight young adults. Pistachios were characterized (chemical and nutraceutical), and a baseline-final, uncontrolled nutritional intervention was performed (28 g of pistachio/28 days). Psychometric tests were applied to estimate cognitive performance and mood; anthropometric evaluation, biochemical analysis, and plasma antioxidant activity were included. The main component of nuts was lipids (48.1%). Pistachios consumption significantly (p ≤ 0.05) reduced waist circumference (-1.47 cm), total cholesterol (-10.21 mg/dL), LDL (-6.57 mg/dL), and triglycerides (-21.07 mg/dL), and increased plasma antioxidant activity. Pistachio supplementation improved risk tolerance (p ≤ 0.006) and decision-making strategy (p ≤ 0.002; BART-task), executive functions (BCST-task; p ≤ 0.006), and selective and sustained attention (Go/No-Go-test; p ≤ 0.016). The mood state was positively modulated (p ≤ 0.05) for anxiety, anger-hostility, and sadness-depression. These results show for the first time the benefits of pistachio consumption on cognitive performance and mood in overweight young adults.

Evaluation of bioactivities of Pistacia vera L. hull extracts as a potential antimicrobial and antioxidant natural source.

Pistacia vera L. hull, a the major byproduct of pistachio processing, is a source of functional compounds with antioxidant and antimicrobial activities. The extraction of these natural compounds from pistachio hulls and their use instead of synthetic chemicals has gained great attention. In this work, the phytochemical contents and antioxidant and antimicrobial activities of pistachio hull ethanolic (PVE) and aqueous (PVD) extracts obtained by microwave-assisted extraction (MAE) were investigated. Gallic acid (1.9 and 1.5 mg g-1 dw), quercetin (0.025 and 0.009 mg g-1 dw), total phenolic (23.3 and 14.7 mg GAE g-1 dw) and flavonoid (5.0 and 2.9 mg QE g-1 dw) contents and antioxidant activities (SC50 0.63 and 0.56 mg mL-1) of PVE and PVD extracts were determined, respectively. The extracts exhibited antimicrobial effects against Enterococcus faecalis, Staphylococcus aureus, Streptococcus uberis, Bacillus cereus, and Bacillus subtilis. Minimal inhibitory concentrations (MICs, 0.8-49.0 and 9.6-82.5 mg mL-1) and the minimal bactericidal concentrations (MBCs, 1.3-99.1 and 15.5-150.0 mg mL-1) of PVE and PVD extracts were determined, respectively. Kill curves revealed that PVE and PVD extracts could inhibit the growth of bacteria. It was shown that PVE and PVD extracts could represent a good economical source of functional and bioactive compounds.

Evaluation of Phenolic Profile and Antioxidant Activity of Eleven Pistachio Cultivars (Pistacia vera L.) Cultivated in Andalusia.

Pistachio (Pistacia vera L.) is a nut with a good adaptability to the Mediterranean conditions of cultivation, specifically in the Andalusian region, becoming an emerging crop. Moreover, it has been getting attention in the past years for the great content of bioactive compounds such as polyphenols. Although some studies have reported the polyphenolic profile of pistachios, most of them have analyzed the hull part, considered as a residue, and not the kernel which is the edible part. Therefore, characterization of eleven varieties of pistachios kernels cultivated in Andalusia and harvested in 2019 and 2020 was carried out by UHPLC-MS (ultra-high-performance liquid chromatography high-resolution mass spectrometry). The identification and quantification of 56 polyphenolic compounds was performed, being the hydroxybenzoic acids group the most abundant with a 71−86% of the total amount followed by flavan-3-ols group that accounted for 8−24%. Moreover, 3,4-dihydroxybenzoic acid was the main compound in most of the varieties, followed by vanillic acid hexoside. Larnaka, Avdat, Aegina, and Mateur presented the highest amount of total polyphenols, while Kalehghouchi, Joley, Lost Hills, Kerman, and Golden Hills were the varieties with the lowest content. Regarding the harvest season, no significant differences (p < 0.01) were found in the total amount of polyphenols between 2019 and 2020. In addition, the antioxidant activity was measured by DPPH (1,1-diphenyl-2-picryl-hydrazyl), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), and ORAC (oxygen radical absorbance capacity) assays, showing a similar trend as that of the polyphenols.

In vivo wound healing effect of Italian and Algerian Pistacia vera L. resins.

Pistacia vera oleoresin is one of the natural products used traditionally for the management of wounds. However, there were no scientific reports documented so far on the wound healing activities to substantiate the claim. This study assesses the potential of the oleoresin of P. vera collected in Italy and Algeria for wound healing efficacy via in vivo circular wound excision model. Italian and Algerian oleoresins were subjected to purification and successive fractionation to obtain three matrices. The fractions have been characterized using GC-FID and GC-MS analyses. Oleoresins mixed with vaseline (5% w/w) were topically applied on wound excision induced on the dorsum of rabbits. Wound healing effects were evaluated by percent of wound contraction. Biopsies performed after healing were histologically assessed. Phytochemical results showed a high content of terpenoids components inducing an efficient wound healing effect determined by an in vivo study. Italian and Algerian oleoresins ointments showed significant wound contraction from day 8 to day 16 as compared to the negative control. The two ointments have not showed statistically difference as compared to Cicatryl, reference drug. These results have also been confirmed by the histological evaluation of the tissues involved. The absence of signs of toxicity on the skin of rabbits indicated the safety of the ointments. The study showed that both oleoresins have a very high effectiveness as wound healing agents and appear to justify their traditional use in wound healing in several countries and offer a scientific support to the treatment of traditional healers.

Synthesis and biological impacts of pollen shells/Fe3O4 nanoparticles composites on human MG-63 osteosarcoma cells.

INTRODUCTION: Cell-adhesive surfaces play a pivotal role in biomedical engineering, as most biological reactions take place on surfaces. Pollen shell (PSh) ofPistacia vera L., as a new medical device, has previously been reported to cause cytotoxicity and apoptosis in MG-63 bone cancer cells. METHODS: Iron oxide nanoparticles (Fe3O4NPs) were synthesized and their reaction to PShs was gauged at different concentrations, and then characterized using field emission scanning electron microscopy (FESEM), Fourier-transform infrared spectroscopy, energy dispersion X-ray spectrometer, X-ray diffraction spectra, dynamic light scattering, and vibrating sample magnetometer. Then, the biological impacts of PShs/Fe3O4NPs composites on MG-63 cells were investigated in-vitro using MTT assay, quantitative polymerase chain reaction (qPCR), Annexin V/propidium iodide, FESEM, and DAPI staining. RESULTS: Fe3O4NPs with a size range of 24-40 nm and a zeta potential value of -37.4 mV were successfully assembled on the PShs. The viability of MG-63 cells was significantly decreased when cultured on the magnetic PShs as compared to non-magnetic PShs, in Fe3O4 concentration and time-dependent manner. In contrast, magnetic PShs had a positive effect on the viability of normal human bone marrow-derived mesenchymal stem cells (hBM-MSCs). The analysis of apoptosis-related genes in cancer cells revealed that loading Fe3O4NPs on PShs increased expression of BAX/BCL2 and caspase-3 genes. The increased apoptotic activity of combined PShs/Fe3O4NPs was further confirmed by flow cytometric measurement, morphological analysis, and DAPI staining. CONCLUSION: The incorporation of Fe3O4NPs into PShs could effectively increase anticancer effects on MG-63 cells via the mitochondria-mediated apoptosis pathway, evident by upregulation of BAX/BCL2 ratio and caspase-3.

Identification of an anti-herpetic compound isolated from Pistacia vera L. male floral buds.

Due to the numerous side effects of conventional drugs against herpetic infections and the growing phenomenon of resistance, the researchers turned to natural compounds as a source of new drugs because they are less toxic than the synthetic molecules. This study aimed to analyse the activity of Pistacia vera L. male floral bud extracts, against the replication of herpes simplex virus type 2, as well as to investigate their mode of action, isolate, and identify the active compound. Cell viability and anti-herpes virus activity were performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the plaque reduction assay, respectively. Three extracts (ethanolic, aqueous and polysaccharide extracts) were tested, only aqueous and polysaccharide extracts had anti-herpetic activity with a selectivity index of 29.12 and 20.25, respectively. Investigation about the mechanism of action indicated that the two active extracts inhibited the virus replication by direct contact with virucidal selectivity indexes of 39.15 and 32.09, respectively. An active compound was isolated from the aqueous extract using TLC bio-guided assay: it was identified as gallic acid by high-performance liquid chromatography-diode array detection coupled with electrospray ionization mass spectrometry (HPLC-DAD-ESI-MSn). The antiviral activity of Pistacia vera L. has been previously shown. The selectivity index of gallic acid is much lower than that of the active extract from which it has been isolated. Therefore, we can consider the aqueous extract prepared from Pistacia vera L. male floral buds as a promising natural product for treating herpetic diseases.

Hollow pollen grains as scaffolding building blocks in bone tissue engineering.

Introduction: The current study, for the first time, suggests nature-made pollen grains (PGs) of Pistacia vera L. as a potential candidate for using as scaffolding building blocks with encapsulation capability of bioactive compounds, such as bone morphogenetic protein 4 (BMP4). Methods: A modified method using KOH (5%, 25ºC) was developed to produce nonallergic hollow pollen grains (HPGs), confirmed by energy dispersive X-ray (EDX) analysis, field emission scanning electron microscopy (FESEM), and DNA and protein staining techniques. The in-vitro study was conducted on human adipose-derived mesenchymal stem cells (hAD-MSCs) to investigate the applicability of HPGs as bone scaffolding building blocks. Cytocompability was evaluated by FESEM, MTT assay, and gene expression analysis of apoptotic markers (BAX and BCL2). The osteoconductive potential of HPGs was assessed by alkaline phosphatase (ALP) activity measurement and gene expression analysis of osteogenic markers (RUNX2 and osteocalcin). Results: Findings demonstrated that HPGs can be considered as biocompatible compounds increasing the metabolic activities of the cells. Further, the bioactive nature of HPGs resulted in suitable cellular adhesion properties, required for a potent scaffold. The investigation of apoptotic gene expression indicated a reduced BAX/BCL2 ratio reflecting the protective effect of HPGs on hAD-MSCs. The increased ALP activity and expression of osteogenic genes displayed the osteoconductive property of HPGs. Moreover, the incorporation of BMP4 in HPGs initiated a synergistic effect on osteoblast maturation. Conclusion: Owing to the unique compositional and surface nanotopographical features of the Pistacia vera L. HPG, this microscale architecture provides a favorable microenvironment for the bottom-up remodeling of bone.

Mycotoxins in Pistachios (Pistacia vera L.): Methods for Determination, Occurrence, Decontamination.

The consumption of pistachios (Pistacia vera L.) has been increasing, given their important benefit to human health. In addition to being an excellent nutritional source, they have been associated with chemical hazards, such as mycotoxins, resulting in fungal contamination and its secondary metabolism. Aflatoxins (AFs) are the most common mycotoxins in pistachio and the most toxic to humans, with hepatotoxic effects. More mycotoxins such as ochratoxin A (OTA), fumonisins (FBs), zearalenone (ZEA) and trichothecenes (T2, HT2 and DON) and emerging mycotoxins have been involved in nuts. Because of the low levels of concentration and the complexity of the matrix, the determination techniques must be very sensitive. The present paper carries out an extensive review of the state of the art of the determination of mycotoxins in pistachios, concerning the trends in analytical methodologies for their determination and the levels detected as a result of its contamination. Screening methods based on immunoassays are useful due to their simplicity and rapid response. Liquid chromatography (LC) is the gold standard with new improvements to enhance accuracy, precision and sensitivity and a lower detection limit. The reduction of Aspergillus' and aflatoxins' contamination is important to minimize the public health risks. While prevention, mostly in pre-harvest, is the most effective and preferable measure to avoid mycotoxin contamination, there is an increased number of decontamination processes which will also be addressed in this review.

Characterization of the complete chloroplast genome of the Pistacia vera L.

The complete chloroplast genome sequence of Pistacia vera was mapped and determined based on Illumina sequencing data. The complete chloroplast genome is 160,654 bp and contains a pair of inverted repeat regions of 26,596 bp each, a large single-copy region of 88,376 bp, and a small single-copy region of 19,086 bp. It harbors 113 genes, including 79 protein-coding genes,4 ribosomal RNA genes, and 30 transfer RNA genes. Phylogenetic analysis based on chloroplast genomes indicates that Pistacia vera is closely related to that of Pistacia weinmanniifolia.

Cumulative risk assessment of pesticide residues in different Iranian pistachio cultivars: Applying the source specific HQS and adversity specific HIA approaches in Real Life Risk Simulations (RLRS).

Iran is a major supplier of the world pistachio market. In this study, we collected five pistachio cultivars from four main pistachio-producing zones in August and September 2016, and determined the residues of 18 organophosphorus, carbamate, pyrethroid and nicotinoid pesticide in these samples using Gas Chromatography-Mass Spectrometry, as an efficient method for determination of pesticides' residues. Next, single-chemical and chronic cumulative risk assessment was done based on the new approaches of the food specific Hazard Quotient and adversity specific Hazard Index. Fifteen from eighteen food-specific Hazard Quotients were above 1 even in cases when the respective contamination was bellow MRLs. The adversity specific Hazard Indexes values were above 1 for five from six adversities indicating various risks in the resulted levels of pistachios' contamination from the pesticides' mixture. However, no risk for carcinogenicity was found. Our results indicate the necessity of taking appropriate measures to control/standardize pesticides practice in pistachio cultivation in Iran and the need to re-establish the MRLs based on cumulative exposure.

Effect of roasting conditions on pigment composition and some quality parameters of pistachio oil.

Pistachio roasting before oil extraction increases consumer preference but may cause changes in the oil composition. In this work, the effect of different roasting conditions on the physical parameters, oxidative stability, and pigment composition of pistachio oil extracted by pressure was studied. Density value of pistachio oil was reduced with severe roasting conditions (125 °C), while viscosity increased slightly. This adverse effect was compensated by a significant increase in both oxidative stability and, especially, in the content of chlorophyll and carotenoid pigments. Pistachio roasting temperature had a clear impact on the color of the pistachio oils, changing from yellow in oils from raw or minimally roasted pistachios (50-75 °C) to brilliant green in oils from pistachios subjected to higher temperature treatments (100-125 °C). An increase in temperature favored the pigment transfer to the oil. The green oils had a total pigment content between 2.3 and 4 times higher than the yellow oils.

Chemical composition of Pistacia vera L. oleoresin and its antibacterial, anti-virulence and anti-biofilm activities against oral streptococci, including Streptococcus mutans.

OBJECTIVE: The aim of this study was to characterize the chemical composition of oleoresin of Pistacia vera L. and to determine its antimicrobial and anti-virulence activity versus selected oral streptococci. DESIGN: A gaschromatografic analysis of the oleoresin was performed. The antimicrobial and anti-virulence activity of the oleoresin and its fractions was evaluated by the Minimum Inhibitory Concentration (MIC) and/or Minimum Bactericidal Concentration (MBC), biofilm production and haemolytic activity inhibition experiments. RESULTS: The oleoresin MBCs were ≥1024 µg/mL for all tested strains; the neutral and acidic fraction MBCs ranged from 128 to 2048 µg/mL. Essential oil's MBCs (from 256 to 2048 µg/mL) were almost identical to MICs, suggesting a bactericidal effect. P. vera oleoresin at sub-lethal concentrations significantly reduced biofilm production by Streptococcus mutans (up to 49.4%) and by Streptococcus sanguinis (up to 71.2%). In addition, the acidic fraction showed a specific anti-biofilm activity against S. mutans (up to 41.3% reduction). A significant dose-dependent reduction in the haemolytic activity of S. mutans (up to 65.9%) and of S. anginosus (up to 78.3%) was observed after growth in the presence of oleoresin at sub-lethal concentrations. The acidic fraction reduced haemolytic activity (up to 54.3% at 64 µg/mL) of S. mutans only. CONCLUSIONS: Given the anti-virulence activity of the P. vera oleoresin and its acidic fraction against S. mutans, our findings suggest their potential use in oral hygiene. These data represent the first step in the exploitation of P. vera L. oleoresin.