Peptides Derived From the α-Core and γ-Core Regions of a Putative Silybum marianum Flower Defensin Show Antifungal Activity Against Fusarium graminearum.

Fusarium graminearum is the etiological agent of Fusarium head blight (FHB), a disease that produces a significant decrease in wheat crop yield and it is further aggravated by the presence of mycotoxins in the affected grains that may cause health problems to humans and animals. Plant defensins and defensin-like proteins are antimicrobial peptides (AMPs); they are small basic, cysteine-rich peptides (CRPs) ubiquitously expressed in the plant kingdom and mostly involved in host defence. They present a highly variable sequence but a conserved structure. The γ-core located in the C-terminal region of plant defensins has a conserved ß-hairpin structure and is a well-known determinant of the antimicrobial activity among disulphide-containing AMPs. Another conserved motif of plant defensins is the α-core located in the N-terminal region, not conserved among the disulphide-containing AMPs, it has not been yet extensively studied. In this report, we have cloned the putative antimicrobial protein DefSm2, expressed in flowers of the wild plant Silybum marianum. The cDNA encodes a protein with two fused basic domains of an N-terminal defensin domain (DefSm2-D) and a C-terminal Arg-rich and Lys-rich domain. To further characterize the DefSm2-D domain, we built a 3D template-based model that will serve to support the design of novel antifungal peptides. We have designed four potential antifungal peptides: two from the DefSm2-D α-core region (SmAPα1-21 and SmAPα10-21) and two from the γ-core region (SmAPγ27-44 and SmAPγ29-35). We have chemically synthesized and purified the peptides and further characterized them by electrospray ionization mass spectrometry (ESI-MS) and Circular dichroism (CD) spectroscopy. SmAPα1-21, SmAPα10-21, and SmAPγ27-44 inhibited the growth of the phytopathogen F. graminearum at low micromolar concentrations. Conidia exposure to the fungicidal concentration of the peptides caused membrane permeabilization to the fluorescent probe propidium iodide (PI), suggesting that this is one of the main contributing factors in fungal cell killing. Furthermore, conidia treated for 0.5h showed cytoplasmic disorganization as observed by transmission electron microscopy (TEM). Remarkably, the peptides derived from the α-core induced morphological changes on the conidia cell wall, which is a promising target since its distinctive biochemical and structural organization is absent in plant and mammalian cells.

A Case Report of Accidental Intoxication following Ingestion of Foxglove Confused with Borage: High Digoxinemia without Major Complications.

Foxglove (Digitalis purpurea L.) leaves are frequently confused with borage (Borago officinalis L.), which is traditionally used as a food ingredient. Due to the presence of the cardiac glycosides, mostly digitoxin, foxglove leaves are poisonous to human and may be fatal if ingested. A 55-year-old Caucasian woman complaining weakness, fatigue, nausea, and vomiting was admitted to the Emergency Department. Her symptoms started following consumption of a home-made savory pie with 5 leaves from a plant bought in a garden nursery as borage. Digoxinemia was high (10.4 µg/L). The patient was admitted to the cardiac intensive care unit for electrocardiographic monitoring. Two days after admission, a single episode of advanced atrioventricular (AV) block was recorded by telemetry, followed by a second-degree AV block episode. Plasma samples at day 11 were analysed by LC-MS spectrometry, and gitoxin was identified suggesting that this compound may be responsible for the clinical toxicity rather than digoxin. In the case of Digitalis spp. poisoning, laboratory data should be interpreted according to the clinical picture and method of analysis used since a variety of glycosides, which are chemically similar to the cardioactive glycosides but without or with fewer cardiac effects, may be incorrectly recognized as digoxin by the test, giving misleading results.

ß-carbonic anhydrases play a role in salicylic acid perception in Arabidopsis.

The plant hormone salicylic acid (SA) is required for defense responses. NON EXPRESSER OF PATHOGENESIS RELATED 1 (NPR1) and NON RECOGNITION OF BTH-4 (NRB4) are required for the response to SA in Arabidopsis (Arabidopsis thaliana). Here, we isolated several interactors of NRB4 using yeast two-hybrid assays. Two of these interactors, ßCA1 and ßCA2, are ß-carbonic anhydrase family proteins. Since double mutant ßca1 ßca2 plants did not show any obvious phenotype, we investigated other ßCAs and found that NRB4 also interacts with ßCA3 and ßCA4. Moreover, several ßCAs interacted with NPR1 in yeast, including one that interacted in a SA-dependent manner. This interaction was abolished in loss-of-function alleles of NPR1. Interactions between ßCAs and both NRB4 and NPR1 were also detected in planta, with evidence for a triple interaction, NRB4-ßCA1-NPR1. The quintuple mutant ßca1 ßca2 ßca3 ßca4 ßca6 showed partial insensitivity to SA. These findings suggest that one of the functions of carbonic anhydrases is to modulate the perception of SA in plants.

Artemisia umbelliformis Lam. and Génépi Liqueur: Volatile Profile as Diagnostic Marker for Geographic Origin and To Predict Liqueur Safety.

Artemisia umbelliformis, commonly known as "white génépi", is characterized by a volatile fraction rich in α- and ß-thujones, two monoterpenoids; under European Union (EU) regulations these are limited to 35 mg/L in Artemisia-based beverages because of their recognized activity on the human central nervous system. This study reports the results of an investigation to define the geographical origin and thujone content of individual plants of A. umbelliformis from different geographical sites, cultivated experimentally at a single site, and to predict the thujone content in the resulting liqueurs through their volatile fraction. Headspace solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) and non-separative HS-SPME-MS were used as analytical platforms to create a database suitable for chemometric description and prediction through linear discriminant analysis (LDA). HS-SPME-MS was applied to shorten analysis time. With both approaches, a diagnostic prediction of (i) plant geographical origin and (ii) thujone content of plant-related liqueurs could be made.

Food safety and nutritional quality for the prevention of non communicable diseases: the Nutrient, hazard Analysis and Critical Control Point process (NACCP).

BACKGROUND: The important role of food and nutrition in public health is being increasingly recognized as crucial for its potential impact on health-related quality of life and the economy, both at the societal and individual levels. The prevalence of non-communicable diseases calls for a reformulation of our view of food. The Hazard Analysis and Critical Control Point (HACCP) system, first implemented in the EU with the Directive 43/93/CEE, later replaced by Regulation CE 178/2002 and Regulation CE 852/2004, is the internationally agreed approach for food safety control. Our aim is to develop a new procedure for the assessment of the Nutrient, hazard Analysis and Critical Control Point (NACCP) process, for total quality management (TMQ), and optimize nutritional levels. METHODS: NACCP was based on four general principles: i) guarantee of health maintenance; ii) evaluate and assure the nutritional quality of food and TMQ; iii) give correct information to the consumers; iv) ensure an ethical profit. There are three stages for the application of the NACCP process: 1) application of NACCP for quality principles; 2) application of NACCP for health principals; 3) implementation of the NACCP process. The actions are: 1) identification of nutritional markers, which must remain intact throughout the food supply chain; 2) identification of critical control points which must monitored in order to minimize the likelihood of a reduction in quality; 3) establishment of critical limits to maintain adequate levels of nutrient; 4) establishment, and implementation of effective monitoring procedures of critical control points; 5) establishment of corrective actions; 6) identification of metabolic biomarkers; 7) evaluation of the effects of food intake, through the application of specific clinical trials; 8) establishment of procedures for consumer information; 9) implementation of the Health claim Regulation EU 1924/2006; 10) starting a training program. RESULTS AND DISCUSSION: We calculate the risk assessment as follows: Risk (R) = probability (P) × damage (D). The NACCP process considers the entire food supply chain "from farm to consumer"; in each point of the chain it is necessary implement a tight monitoring in order to guarantee optimal nutritional quality.

Determination of major, minor and trace elements in Glyceric Macerates and Mother Tinctures and in the starting plant materials.

Glyceric Macerates (GMs) and Mother Tinctures (MTs) are liquid preparations obtained from plant buds (for GMs) and flowers, leaves or roots (for MT) by extraction with a mixture of solvents. Their quality depends on the quality of the plant materials and on the preparation procedures. In this work we determined the concentrations of major, minor and trace elements in buds, flowers and other plant components and in the GMs and MTs obtained from them by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) after microwave mineralization. To the best of our knowledge, this procedure has been applied for the first time here to the analysis of buds. We have taken into account spectral interferences and other causes of errors. Analogies and differences with regard to the method reported by European Pharmacopoeia for heavy metal determination in herbal drugs have been highlighted. The experimental results have been interpreted with chemometric techniques. No significant contamination was detected during the manufacturing step. Element concentrations in GMs and MTs, taking into account their daily dosages, are lower than acceptable intake levels.

Partial Molecular Characterization of Arctium minus Aspartylendopeptidase and Preparation of Bioactive Peptides by Whey Protein Hydrolysis.

In this article, we report the cloning of an aspartic protease (AP) from flowers of Arctium minus (Hill) Bernh. (Asteraceae) along with the use of depigmented aqueous flower extracts, as a source of APs, for the hydrolysis of whey proteins. The isolated cDNA encoded a protein product with 509 amino acids called arctiumisin, with the characteristic primary structure organization of typical plant APs. Bovine whey protein hydrolysates, obtained employing the enzyme extracts of A. minus flowers, displayed inhibitory angiotensin-converting enzyme (ACE) and antioxidant activities. Hydrolysates after 3 and 5 h of reaction (degree of hydrolysis 2.4 and 5.6, respectively) and the associated peptide fraction with molecular weight below 3 kDa were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization/time of flight mass spectrometry, and reverse phase-high-performance liquid chromatography. The results obtained in this study demonstrate the viability of using proteases from A. minus to increase the antioxidant and inhibitory ACE capacity of whey proteins.

Properties and applications of phytepsins from thistle flowers.

Aqueous extracts of thistle flowers from the genus Cynara-Cardueae tribe Cass. (Cynareae Less.), Asteraceae Dumortier-are traditionally used in the Mediterranean region for production of artisanal cheeses. This is because of the presence of aspartic proteases (APs) with the ability to coagulate milk. Plant APs, collectively known as phytepsins (EC 3.4.23.40), are bilobed endopeptidases present in an ample variety of plant species with activity mainly at acidic pHs, and have two aspartic residues located on each side of a catalytic cleft that are responsible for catalysis. The cleavage of the scissile peptide-bond occurs primarily between residues with large hydrophobic side-chains. Even when aspartylendopeptidase activity in plants is normally present at relatively low levels overall, the flowers of several species of the Cardueae tribe possess APs with extremely high specific activities in certain tissues. For this reason, in the last two decades, APs present in thistle flowers have been the subject of intensive study. Present here is a compilation of work that summarizes the known chemical and biological properties of these proteases, as well as their biomedical and biotechnological applications.

Analysis of fluid extracts obtained from Papaver rhoeas petals contaminated with Papaver bracteatum petals.

In this paper, we report a case of misidentification of medicinal plants involving dried petals of Papaver rhoeas (red poppy) contaminated with Papaver bracteatum (scarlet poppy) petals. Preliminary TLC analysis indicated the presence of thebaine either in the fluid extracts or in the petals. It was therefore necessary to carry out an accurate botanic examination of the plant material, which revealed contamination of the red poppy petals with scarlet poppy petals. Moreover, to confirm the adulteration, we developed and validated an efficient, reversed-phase ion pair HPLC method for determination of the alkaloids specific for the Papaver species. Six petal batches and five commercial fluid extracts were analyzed. Only one petal batch from Iran contained thebaine and its analogue oripavine while the alkaloids typical for the Papaver bracteatum species were identified in all fluid extracts, meaning that they were all prepared with contaminated petals.

Determination of the active principles of Catha Edulis: quali-quantitative analysis of cathinone, cathine, and phenylpropanolamine.

In the last years, all the vegetable material supposed to belong to the Catha Edulis species, seized at the Malpensa and Orio al Serio airports, were analyzed in our laboratory on behalf of the Tribunals of Busto Arsizio and Bergamo, respectively. After a preliminary botanic examination, the quali-quantitative determination of the active principles cathinone, cathine and phenylpropanolamine (PPA) was carried out by means of GC/MS and GC/FID techniques, which meet the requirements of the forensic analyses. We developed a fast, effective and reliable derivatization procedure which allowed to simultaneously detect cathine and PPA, whose discrimination is mandatory since PPA is not a psychoactive agent. Cathine was distributed in the various parts of the plant (leaves and stems) and its quantity ranged from 0.03% to 0.17% of the weight of the vegetable material; PPA was not detected in the twigs and its quantity in the leaves ranged from 0.07% to 0.16%. The quantitative determination of cathinone was carried out directly on the methanol solution after maceration of the vegetable material, its quantity ranging from 0.02% to 0.10%. No significant difference in the content of the two active principles was found between the fresh and the dried material.

Hypovolemic shock due to severe gastrointestinal bleeding in a child taking an herbal syrup.

We report the case of a 4-years-old boy who was admitted with hypovolemic shock due to a severe gastrointestinal bleeding. The esophagogastroduodenoscopy (EGDS) showed hiatus hernia, erosions and ulcerations of the lower esophagus, possibly due to a gastroesophageal reflux, and a small duodenal erosion. The child was previously healthy and he had never shown any symptoms related to this condition. The only product taken by the child in the previous days was a syrup containing several herbs, among which Filipendula ulmaria (L.) Maxim. and Salix spp. (known to contain salicylates), marketed as food and prescribed by his paediatrician to treat a mild cold accompanied by fever. Quali-quantitative analysis confirmed the presence of salicylates in the syrup. Naranjo algorithm showed a probable correlation between the onset of symptoms and the consumption of the herbal remedy. The child recovered after receiving intensive care. The product was withdrawn from Italian market.

An update on vitamin E, tocopherol and tocotrienol-perspectives.

Vitamin E, like tocotrienols and tocopherols, is constituted of compounds essential for animal cells. Vitamin E is exclusively synthesized by photosynthetic eukaryotes and other oxygenic photosynthetic organisms such as cyanobacteria. In order to prevent lipid oxidation, the plants mainly accumulate tocochromanols in oily seeds and fruits or in young tissues undergoing active cell divisions. From a health point of view, at the moment there is a great interest in the natural forms of tocochromanols, because they are considered promising compounds able to maintain a healthy cardiovascular system and satisfactory blood cholesterol levels. Some evidence suggests that the potency of the antioxidant effects may differ between natural or synthetic source of tocochromanols (vitamin E).

Analytical aspects of diterpene alkaloid poisoning with monkshood.

A sensitive and specific method for aconitine extraction from biological samples was developed. Aconitine, the main toxic alkaloid from plants belonging to Aconitum species (family Ranunculaceae), was determined in plant material by an external standard method, and by a standard addition calibration method in biological fluids. Described here is one fatal case and five intoxications of accidental aconitine poisoning following the ingestion of aconite mistaken for an edible grass, Aruncus dioicus (Walt.) Fernald, "mountain asparagus", and Cicerbita alpina (L.) Wallroth. The aconitine content in urine was in the range 2.94 microg/mL (dead patient)-0.20 microg/mL (surviving patients), which was almost two to four times higher than that in plasma.

Digitalis must be banished from the table: a rare case of acute accidental Digitalis intoxication of a whole family.

Advanced Digitalis intoxication is a rare event, mainly associated with overdose in patients with Digitalis therapy. We report an unusual case of acute 'familiar' digitalis poisoning in three patients who had eaten potato dumplings flavoured with leaves of Borago officinalis L. unconsciously mixed with leaves of Digitalis purpurea L. A complicated clinical course with marked bradyarrhythmias was presented, with good evolution thanks to the use of digoxin-specific antibody Fab fragments. The theme of the domestic use of plants with medicinal effects has been treated and discussed.

Analysis of food supplements containing iodine: a survey of Italian market.

Iodine is an essential nutrient for humans as a critical constituent of thyroid hormones; both iodine deficiency and excess have adverse consequences for the thyroid gland. The aims of this research were to compare claimed concentrations of iodine with measured ones in various iodine-supplemented products, estimate the amount of iodine ingested by Italian consumers who use these products, and compare the calculated intakes to the Recommended Daily Allowance and tolerable Upper Level. A convenience sample of 43 food supplements was analyzed for iodine concentration. Analytical values resembled those declared in the label in fewer than half of the examples; in four cases, the maximal daily dose was higher than the tolerable upper level for iodine of 600 microg/day. Labeling of iodine-rich food supplements appears to be unreliable and caution should be exercised in the consumption of food supplements rich in iodine as there is a risk of exceeding the established safe upper level of daily intake.