EFFECT OF SAR INDUCERS ON GRAPE SECONDARY METABOLITES

Fungicide application is now the most efficacious method for controlling plant diseases caused by oomycetes and fungi. As legislation is limiting and reducing their use, it is strongly stimulating studies for the identification of additional and environmentally friendly approaches in the control of their associated diseases. Among these, systemic acquired resistance (SAR) offers the prospect of long-lasting, broad-spectrum disease control through activation of the resistance defence machinery of the plant itself. Plant activators are products employed in crop protection able to elicit SAR. Therefore, they may trigger the plant own defence response against pathogen attacks, mainly stimulating mechanisms such as the biosynthesis of phytoalexins, plant secondary metabolites with a broad spectrum biological activity. In this study, two plant defence inducers, benzothiadiazole (BTH) and chitosan (CHT), deserving particular attention because of their efficacy and low toxicity, have been used. CHT is a natural and low-cost polymer (from the waste products of the crustacean carapace), obtained by chitin deacetylation. Its effectiveness is higher when molecular weight is between 10 and 100 kD and the deacetylation degree range is from 80 to 90 percent. BTH [benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester] is a synthetic compound and a functional analogue of salicylic acid, a plant hormone-like compound deeply involved in resistance against pathogens. Two different phytoiatric campaigns were planned, in 2009 and 2010, on two red grapevine (Vitis vinifera L.) varieties cultivated in experimental vineyards located at distinct sites: Groppello, an autochthonous cultivar of Lombardia, at Raffa di Puegnago (Brescia), and Merlot at Conegliano Veneto (Treviso). Open field treatments carried out on Groppello grapevines were: i) 0.03% (w/v) CHT (76 kDa molecular weight and 85% deacetylation degree), ii) 0.03% CHT in combination with 150 g hL-1 copper hydroxide (CHT/Cu) and iii) 0.3 mM BTH; whereas, on Merlot, besides CHT and CHT/Cu, BTH were replaced by 300 g hL-1 potassium phosphites. Untreated vines were used as negative control, while plants treated with conventional fungicides (penconazole and methyldinocap) were the positive control. In both field surveys, the trial was set up as a complete randomized block design in 4 replications, with 10 vines (a parcel) per treatment in each block. Plants were sprayed approximately every 10 days, according to the meteorological conditions, from the beginning of grape susceptibility to fungal diseases until the complete v\ue9raison. The phytosanitary status of vineyards was assessed weekly on leaves and bunches, by visual inspections, though both sites were not particularly predisposed to severe fungal or other epidemics. The epidemiological evaluations were performed on bunches alone and infection indexes were calculated. All the treatments were effective in controlling fungal infections (downy mildew, Plasmopara viticola and powdery mildew, Erysiphe necator), in terms of disease incidence (I %), disease severity (S %) and infection degree (ID %), though these indexes were low in untreated control grapevines, particularly in 2010. Sampling was scheduled at two phenological phases: pre-v\ue9raison and 100% v\ue9raison; bunches were randomly collected from plants during the morning and stored at -20 \ub0C until analyses. Berry tissues were separated into skin, flesh and seeds, powdered and extracted. Then, extracts were tested to evaluate their melatonin levels [by ultra performance liquid chromatography (UPLC)-MS/MS], total polyphenols (TP, by Folin-Ciocalteau assay) and antiradical activity [by DPPH, 2,2-diphenyl-1-pycryl hydrazyl and ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay]. Groppello and Merlot experimental wines were produced, by standard microvinification techniques, in the Centro Vitivinicolo Provinciale of Brescia and Centro di Ricerca per la Viticoltura of Conegliano (TV), respectively, and stored at 4 \ub0C in the dark until analyses. Microvinificates were produced from grapes treated with both elicitors and conventional fungicides (controls), and the following analyses, besides those also conducted for all berry tissues, were carried out: tryptophan, serotonin and melatonin detection (by UPLC-MS/MS); content of cis and trans resveratrol (by UPLC-MS/MS); levels of mycotoxins (manly ochratoxin A, OTA, by UPLC-MS/MS). In general, berry tissues treated with elicitors and the corresponding experimental wines showed higher levels of melatonin and polyphenols, as well as a higher antiradical activity than samples treated with conventional fungicides. High concentrations of tryptophan were detected in all samples, contrary to serotonin, which was not detected. The most effective elictors were CHT/Cu and CHT. In all wines, the level of OTA was below the allowable threshold of 2 ng/mL. To the best of our knowledge, these results represent the first data on the effects of agrochemicals on the melatonin content of red wine, and the presence of melatonin was reported, for the first time, in berry seeds and flash, after its previously detection in skin. Furthermore, the level of the indolamine in berry tissues varies according to the phenological stage, resulting more abundant in seed at pre-v\ue9raison and in skin at v\ue9raison. The good agreement between the data obtained in 2009 and 2010 for Groppello cultivar, and, in 2009, between Groppello and Merlot varieties cultivated in different geographical areas, suggests that, in general, plant activators may improve some qualitative/healthy treats ascribed to red wine, though their efficacy in controlling grapevine fungal diseases should be better ascertained. Finally, the role of melatonin, a powerful antioxidant, in grapevine physiology is still somewhat obscure. It is possible that, at pre-v\ue9raison, when skin anthocyanic pigmentation is still lacking, this compound may defend berry from damage due to photooxidation or UV radiation, whereas, at v\ue9raison, the indolamine may protect the germ tissues, particularly rich in storage lipids and membranes and vulnerable to oxidative damage. In conclusion, the possibility of enhancing the pharmaconutritional potential of grape/wine with phytosanitary treatments should be further tajen into account

Impact of Cooking on Bioactive Compounds and Antioxidant Activity of Pigmented Rice Cultivars

Pigmented rice cultivars, namely Venere and Artemide, are a source of bioactive molecules, in particular phenolics, including anthocyanins, exerting a positive effect on cardiovascular systems thanks also to their antioxidant capacity. This study aimed to determine the total phenol index (TPI), total flavonoids (TF), total anthocyanins (TA) and in vitro antioxidant capacity in 12 batches of Venere cultivar and two batches of Artemide cultivar. The rice was cooked using different methods (boiling, microwave, pressure cooker, water bath, rice cooker) with the purpose to individuate the procedure limiting the loss of bioactive compounds. TPI, TF and TA were spectrophotometrically determined in both raw and cooked rice samples. Rice samples of Artemide cultivars were richer in TPI (17.7-18.8 vs. 8.2-11.9 g gallic acid/kg in Venere rice), TF (13.1 vs. 5.0-7.1 g catechin/kg rice for Venere rice) and TA (3.2-3.4 vs. 1.8-2.9 g Cy-3glc/kg for Venere rice) in comparison to those of Venere cultivar; as well, they showed higher antioxidant capacity (46.6-47.8 vs. 14.4-31.9 mM Trolox/kg for Venere rice). Among the investigated cooking methods, the rice cooker and the water bath led to lower and comparable losses of phenolics. Interestingly, the cooking water remaining after cooking with the rice cooker was rich in phenolics. The consumption of a portion of rice (100 g) cooked with the rice cooker with its own cooking water can supply 240 mg catechin and 711 mg cyanidin 3-O-glucoside for Venere rice and 545 mg catechin and 614 mg cyanidin 3-O-glucoside for Artemide rice, with a potential positive effect on health

Validation of a method for diosgenin extraction from fenugreek (Trigonella foenum-graecum L.)

Background. Diosgenin is a very important plant secondary metabolite and raw material for the drug industry. Plant sources rich in diosgenin include yam (Dioscorea spp.) and fenugreek (Trigonella foenum-graecum L.). A method for diosgenin extraction from yam extracts has previously been validated, but its extraction from fenugreek plants still requires validation. In addition, all available methods require time-consuming additional purification steps. The present study was aimed at developing a low cost, less time-consuming single-step method for diosgenin extraction from fenugreek. Material and methods. This study represents a method developed for diosgenin extraction from fenugreek plants without any additional/supportive purification methods such as chromatography or thin-layer chromatography. Diosgenin yield estimation and purity analysis by HPLC method, along with accuracy and precision analysis, is presented. Results. Five different fenugreek varieties were subjected to a newly developed diosgenin extraction method, and an HPLC chromatogram showed a single peak corresponding to diosgenin. Yield was determined by the standard curve method. Limit of detection (LOD) and limit of quantification (LOQ) for the assay were found to be 0.0312 and 0.102 \u3bcg, respectively; t calculated for slope and other statistical parameters were found to be significant (P value < 0.001) for this method. Conclusion. We have developed a fast, accurate and low cost method for diosgenin extraction from fenugreek. Although the authors have studied this method only in fenugreek plants, it could be applied to the extraction of a few other plant secondary metabolites, which will help researchers to save time and effort

LC-MS/MS-Based Profiling of Tryptophan-Related Metabolites in Healthy Plant Foods

Food plants contain hundreds of bioactive phytochemicals arising from different secondary metabolic pathways. Among these, the metabolic route of the amino acid Tryptophan yields a large number of plant natural products with chemically and pharmacologically diverse properties. We propose the identifier "indolome" to collect all metabolites in the Tryptophan pathway. In addition, Tryptophan-rich plant sources can be used as substrates for the fermentation by yeast strains to produce pharmacologically active metabolites, such as Melatonin. To pursue this technological development, we have developed a UHPLC-MS/MS method to monitor 14 Tryptophan, Tryptamine, amino-benzoic, and pyridine metabolites. In addition, different extraction procedures to improve the recovery of Tryptophan and its derivatives from the vegetal matrix were tested. We investigated soybeans, pumpkin seeds, sesame seeds, and spirulina because of their botanical diversity and documented healthy effects. Four different extractions with different solvents and temperatures were tested, and water extraction at room temperature was chosen as the most suitable procedure to extract the whole Tryptophan metabolites pattern (called by us "indolome") in terms of ease, high efficiency, short time, low cost, and sustainability. In all plant matrices, Tryptophan was the most abundant indole compound, while the pattern of its metabolites was different in the diverse plants extracts. Overall, 5-OH Tryptamine and Kynurenine were the most abundant compounds, despite being 100-1000-fold lower than Tryptophan. Melatonin was undetected in all extracts, but sesame showed the presence of a Melatonin isomer. The results of this study highlight the variability in the occurrence of indole compounds among diverse food plants. The knowledge of Tryptophan metabolism in plants represents a relevant issue for human health and nutrition

Synthesis of imine congeners of resveratrol and evaluation of their anti-platelet activity

Resveratrol (3,5,4\u2032-trihydroxy-trans-stilbene) is a cardioprotective phytochemical occurring in many plant products. In this study, a new series of imine congeners of resveratrol has been synthesized in which the imine moiety replaced the double bond in the structure of resveratrol. In addition, the in vitro antiplatelet activity of these resveratrol derivatives has been evaluated against adenosine diphosphate (ADP), arachidonic acid (AA), and collagen as platelet aggregation inducers. In general, the synthesized compounds were active as antiplatelet agents, and, therefore, the imine functional group may be considered as an effective replacement for a double bond in resveratrol for developing new and promising antiplatelet drugs

Characterization of the biogenic volatile organic compounds (BVOCs) and analysis of the PR1 molecular marker in Vitis vinifera L. inoculated with the nematode Xiphinema index

Upon pathogen attack, plants very quickly undergo rather complex physico-chemical changes, such as the production of new chemicals or alterations in membrane and cell wall properties, to reduce disease damages. An underestimated threat is represented by root parasitic nematodes. In Vitis vinifera L., the nematode Xiphinema index is the unique vector of Grapevine fanleaf virus, responsible for fanleaf degeneration, one of the most widespread and economically damaging diseases worldwide. The aim of this study was to investigate changes in the emission of biogenic volatile organic compounds (BVOCs) in grapevines attacked by X. index. BVOCs play a role in plant defensive mechanisms and are synthetized in response to biotic damages. In our study, the BVOC profile was altered by the nematode feeding process. We found a decrease in \u3b2-ocimene and limonene monoterpene emissions, as well as an increase in \u3b1-farnesene and \u3b1-bergamotene sesquiterpene emissions in nematode-treated plants. Moreover, we evaluated the PR1 gene expression. The transcript level of PR1 gene was higher in the nematode-wounded roots, while in the leaf tissues it showed a lower expression compared to control grapevines

Surface functionalization of bioactive glasses with polyphenols from Padina pavonica algae and in situ reduction of silver ions : physico-chemical characterization and biological response

Bioactive glasses (BGs) are attractive materials for bone replacement due to their tailorable chemical composition that is able to promote bone healing and repair. Accordingly, many attempts have been introduced to further improve BGs' biological behavior and to protect them from bacterial infection, which is nowadays the primary reason for implant failure. Polyphenols from natural products have been proposed as a novel source of antibacterial agents, whereas silver is a well-known antibacterial agent largely employed due to its broad-ranged activity. Based on these premises, the surface of a bioactive glass (CEL2) was functionalized with polyphenols extracted fromthe Egyptian algae Padina pavonica and enriched with silver nanoparticles (AgNPs) using an in situ reduction technique only using algae extract. We analyzed the composite's morphological and physical-chemical characteristics using FE-SEM, EDS, XPS and Folin-Ciocalteau; all analyses confirmed that both algae polyphenols and AgNPs were successfully loaded together onto the CEL2 surface. Antibacterial analysis revealed that the presence of polyphenols and AgNPs significantly reduced the metabolic activity (> 50%) of Staphylococcus aureus biofilm in comparison with bare CEL2 controls. Finally, we verified the composite's cytocompatibility with human osteoblasts progenitors that were selected as representative cells for bone healing advancement

Rice allelopathy in weed management : an integrated approach

The intensive use of pesticides with low biodegradability and high persistence in soil, surface and ground waters, represents a considerable environmental risk, especially under high weed pressure conditions. Furthermore, the number of herbicide-resistant weeds is increasing. Against this background, the investigation of alternative weed control strategies has taken on considerable importance. Among these, allelopathy as a negative effect of one plant on another due to the direct or indirect (including microorganisms) release of chemicals in the environment can be a useful tool for the integrated management of weeds in agroecosystems. In particular, the paddies have been considered in this work by reviewing the data both on rice allelopathy and rice weed agronomic control methods developed to improve the crop yield

Phenolic compounds from Achillea millefolium L. and their bioactivity

Since antiquity, Achillea millefolium L. (Asteraceae) has been used in traditional medicine of several cultures, from Europe to Asia. Its richness in bioactive compounds contributes to a wide range of medicinal properties. In this study, we assessed A. millefolium methanolic extract and its isolated components for free radical scavenging activity against 2,2-diphenyl-pycrilhydrazyl, total antioxidant capacity (based on the reduction of Cu ++ to Cu +), and ability to inhibit lipid peroxidation. The activity against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum was also tested. Chlorogenic acid, its derivatives and some flavonoids isolated by semipreparative HPLC and identified by NMR and spectrometric techniques were the major bioactive constituents of the methanolic extract. The latter exhibited significant antioxidant properties, as well as its flavonol glycosides and chlorogenic acids. With regard to the antiplasmodial activity, apigenin 7-glucoside was the most effective compound, followed by luteolin 7-glucoside, whereas chlorogenic acids were completely inactive. On the whole, our results confirmed A. millefolium as an important source of bioactive metabolites, justifying its pharmaceutical and ethnobotanical use

Anticancer Effects of Wild Mountain Mentha longifolia Extract in Adrenocortical Tumor Cell Models

Mint [Mentha longifolia (L.) Hudson] is an aromatic plant that belongs to Lamiaceae family. It is traditionally used as herbal tea in Europe, Australia and North Africa and shows numerous pharmacological effects, such as spasmolytic, antioxidant, antimicrobial and anti-hemolytic. Recently, its antiproliferative role has been suggested in a small number of tumor cell models, but no data are available on adrenocortical carcinoma, a malignancy with a survival rate at 5 years of 20%\u201330% which frequently metastasize. This work aimed to study the effects of Mentha longifolia L. crude extract (ME) on two adrenocortical tumor cell models (H295R and SW13 cells). Chemical composition of ME was assessed by gas-chromatography/mass spectrometry and NMR spectroscopy analysis. Brine shrimp lethality assay showed ME effects at >0.5 \ub5g/\ub5l (p 0.5 \ub5g/\ub5l, p 0.5 \ub5g/\ub5l, p < 0.05), while Wright staining demonstrated the presence of both necrotic and apoptotic cells. Cell cycle analysis showed a strong increase in subG0/G1 phase, related to cell death. Furthermore, MAPK and PI3k/Akt pathways were modulated by Western blot analysis when treating cells with ME alone or combined with mitotane. The crude methanolic extract of wild mountain mint can decrease cell viability, vitality and survival of adrenocortical tumor cell models, in particular of SW13 cells. These data show the potential anticancer effects of ME, still more work is needed to corroborate these findings