Antifungal activity of chitosan oligomers-amino acid conjugate complexes against Fusarium culmorum in spelt (Triticum spelta L.)

Fusarium head blight (FHB) is a complex disease of cereals caused by Fusarium species, which causes severe damages in terms of yield quality and quantity worldwide, and which produces mycotoxin contamination, posing a serious threat to public health. In the study presented herein, the antifungal activity against Fusarium culmorum of chitosan oligomers (COS)–amino acid conjugate complexes was investigated both in vitro and in vivo. The amino acids assayed were cysteine, glycine, proline and tyrosine. In vitro tests showed an enhancement of mycelial growth inhibition, with EC50 and EC90 effective concentration values ranging from 320 to 948 µg·mL−1 and from 1107 to 1407 µg·mL−1 respectively, for the conjugate complexes, as a result of the synergistic behavior between COS and the amino acids, tentatively ascribed to enhanced cell membrane damage originating from lipid peroxidation. Tests on colonies showed a maximum percentage reduction in the number of colonies at 1500 µg·mL−1 concentration, while grain tests were found to inhibit fungal growth, reducing deoxynivalenol content by 89%. The formulation that showed the best performance, i.e., the conjugate complex based on COS and tyrosine, was further investigated in a small-scale field trial with artificially inoculated spelt (Triticum spelta L.), and as a seed treatment to inhibit fungal growth in spelt seedlings. The field experiment showed that the chosen formulation induced a decrease in disease severity, with a control efficacy of 83.5%, while the seed tests showed that the treatment did not affect the percentage of germination and resulted in a lower incidence of root rot caused by the pathogen, albeit with a lower control efficacy (50%). Consequently, the reported conjugate complexes hold enough promise for crop protection applications to deserve further examination in larger field trials, with other Fusarium spp. pathogens and/or Triticum species

Antifungal activity against Fusarium culmorum of stevioside, Silybum marianum seed extracts, and their conjugate complexes

Fusarium head blight (FHB) is a disease that poses a major challenge in cereal production that has important food and feed safety implications due to trichothecene contamination. In this study, the effect of stevioside—a glycoside found in the leaves of candyleaf (Stevia rebaudiana Bertoni)—was evaluated in vitro against Fusarium culmorum (W.G. Smith) Sacc., alone and in combination (in a 1:1 molar ratio) with polyphenols obtained from milk thistle seeds (Silybum marianum (L.) Gaertn). Different concentrations, ranging from 32 to 512 µg·mL−1, were assayed, finding EC50 and EC90 inhibitory concentrations of 156 and 221 µg·mL−1, respectively, for the treatment based only on stevioside, and EC50 and EC90 values of 123 and 160 µg·mL−1, respectively, for the treatment based on the stevioside–polyphenol conjugate complexes. Colony formation inhibition results were consistent, reaching full inhibition at 256 µg·mL−1. Given that synergistic behavior was observed for this latter formulation (SF = 1.43, according to Wadley’s method), it was further assessed for grain protection at storage, mostly directed against mycotoxin contamination caused by the aforementioned phytopathogen, confirming that it could inhibit fungal growth and avoid trichothecene contamination. Moreover, seed tests showed that the treatment did not affect the percentage of germination, and it resulted in a lower incidence of root rot caused by the pathogen in Kamut and winter wheat seedlings. Hence, the application of these stevioside–S. marianum seed extract conjugate complexes may be put forward as a promising and environmentally friendly treatment for the protection of cereal crops and stored grain against FHB

Reactivity and Applications of Singlet Oxygen Molecule

Reactive oxygen species (ROS) are molecules produced in living organisms, in the environment, and in various chemical reactions. The main species include, among others, singlet oxygen (1O2), the superoxide anion radical (•O2−), the hydroxyl radical (HO•), and the hydroperoxyl radical (HOO•). In general, the reactivity of 1O2 is lower than that of HO• but even higher than that of •O2−. Singlet oxygen is the lowest energy excited state of molecular oxygen, but it is also a highly reactive species, which can initiate oxidation reactions of biomolecules such as amino acids, proteins, nucleic acids, and lipids, either by a direct reaction or by the induction of ROS. Singlet oxygen is a highly reactive electrophilic species that reacts with electron-rich molecules and is related to several types of pathologies. To inhibit the oxidation of biomolecules with this species, some substances act as antioxidants by performing a quenching effect. In this chapter, aspects such as its physicochemical properties, methods of generation and detection, as well as the reactivity of this molecule are detailed

In vitro antifungal activity of chitosan-polyphenol conjugates against Phytophthora cinnamomi

Producción CientíficaPhytophthora cinnamomi is responsible for radical rot in a wide range of hosts, resulting in large economic and ecological losses worldwide. In Spain, it is responsible for diseases such as the oak decline or the chestnut blight. In this study, different polyphenol-stevioside inclusion compounds dispersed in a hydroalcoholic solution of chitosan oligomers have been investigated, with a view to their application as natural bioactive complexes to replace conventional systemic fungicides against this fungus. The polyphenols tested in vitro were curcumin, ferulic acid, gallic acid and silymarin. Three concentrations (125, 250 and 500 µg·mL−1) were assayed, with and without silver nanoparticles (AgNPs), and notable differences were found in the inhibition of mycelium growth, with EC50 and EC90 values ranging from 171 to 373.6 µg·mL−1, and from 446.2 to 963.7 µg·mL−1, respectively. The results obtained showed that the addition of AgNPs, despite their antimicrobial activity, did not always lead to synergies. In the case of P. cinnamomi, an unexpected antagonistic behavior was found for two of the polyphenols (curcumin and silymarin), while an additive behavior for ferulic acid and a synergistic behavior for gallic acid were attained. In view of their inhibitory power, the preparations based on ferulic acid with AgNPs and on silymarin without AgNPs are proposed for applications in crop and forests protection against P. cinnamomi.Junta de Castilla y León - Fondo Europeo de Desarrollo Regional (project VA258P18)Universidad de Zaragoza (project UZ2019-TEC-07

Efficient microwave-assisted acid hydrolysis of lignocellulosic materials into total reducing sugars in ionic liquids

Different types of lignocellulosic materials (carnauba leaves, macauba shell and pine nut shell) and native cellulose have been studied for the production of total reducing sugars (TRS) through microwave-assisted acid-catalyzed hydrolysis in ionic liquids (ILs). Four reaction media have been assessed: two deep eutectic solvents (DES), choline chloride-oxalic acid (ChCl/ox) and choline chloride-urea (ChCl/urea), and two conventional ionic liquids, tetraethylammonium chloride (TEAC) and tetraethylammonium bromide (TEAB). Five acids (H2SO4, HCl, HNO3, H3PO4andp-toluensulfonic acid) have been evaluated in varying concentrations (5-30%) and time intervals (0-60 min), at different temperatures (100-140°C). Significant TRS production yields (as high as 83.7% in ChCl/ox for carnauba leaves) have been attained for both DES in combination with HNO3 10%, at 120°C for 30 min, with the additional advantage of low furfural and HMF by-products generation

Estudio de la actividad antifúngica in vitro de materiales compuestos basados en complejos de inclusión de polifenoles contra Phytophthora cinnamomi

El hongo Phytophthora cinnamomi es responsable de la podredumbre radical en una amplia gama de hospedantes, produciendo grandes pérdidas económicas y ecológicas a nivel mundial. En el caso de España, es responsable de enfermedades como la ‘seca’ de la encina y el alcornoque, o la ‘tinta’ del castaño. En este estudio se han investigado diferentes compuestos de inclusión de polifenoles-esteviósido dispersados en una solución hidroalcohólica de oligómeros de quitosano, con aplicación como complejos naturales bioactivos para reemplazar a los fungicidas sistémicos convencionales. Los polifenoles ensayados in vitro han sido curcumina, ácido ferúlico, ácido gálico y silimarina. Se han ensayado tres concentraciones (125, 250 y 500 µg·mL−1), con y sin nanopartículas de plata (AgNPs), y se han encontrado diferencias notables en la inhibición del crecimiento del micelio, con valores de CE50 y CE90 que han oscilado entre 171 y 373,6 µg·mL−1, y entre 446,2 y 963,7 µg·mL−1, respectivamente, observándose un rendimiento superior de los preparados a base de ácido ferúlico con AgNPs y de silimarina sin nAg. Los resultados obtenidos ponen en evidencia que la adición de AgNPs, pese a su actividad antimicrobiana, no siempre da lugar a sinergismos. En el caso de P. cinnamomi, se ha encontrado un comportamiento antagonístico inesperado para dos de los polifenoles (curcumina y silimarina), un comportamiento aditivo para el ácido ferúlico y un comportamiento sinergístico para el ácido gálico. Los preparados referidos pueden ser prometedores para aplicaciones de protección de cultivos y masas forestales contra P. cinnamomi

Actividad antifúngica contra Fusarium culmorum de los extractos de hojas de Stevia rebaudiana y sus combinaciones con extractos de semillas de Silybum marianum y Lycium barbarum

Fusarium culmorum es un hongo que produce la fusariosis (scab o FHB), una enfermedad que plantea un importante reto en la producción de cereales y conlleva notables implicaciones de seguridad alimentaria por la contaminación con micotoxinas (tricotecenos). Ocasionalmente, se desarrolla en almacén cuando las condiciones le son favorables (a bajas temperaturas y alta humedad) o si el grano ha sido secado insuficientemente y de forma rápida. En este estudio se ha evaluado in vitro el efecto antifúngico de los extractos de hojas de estevia, solos o en combinación con polifenoles (procedentes de semillas de cardo asnal, de semillas de goji o ácido gálico puro), contra este fitopatógeno. Se han ensayado distintas concentraciones, desde 31.25 µg·mL-1 a 1 mg·mL-1 y se han obtenido, para el tratamiento basado sólo en las hojas de estevia, concentraciones inhibitorias CE50 y CE90 de 112 y 283 µg·mL-1, respectivamente; y para los tratamientos basados en hojas de estevia y polifenoles, valores de CE50 y CE90 de 160 y 500 µg·mL-1, respectivamente. Estos resultados llevan a concluir que, aunque el comportamiento para las mezclas es no-sinérgico para este patógeno (la respuesta a la presencia de polifenoles es impredecible), la actividad antifúngica de las hojas de estevia (referida, principalmente a esteviósido como principio activo) es incuestionable. En consecuencia, se propone la aplicación de extractos de hojas de estevia para la protección contra la fusariosis de cosechas y del grano en almacenes

Inhibition of Yeast Hexokinase by Acyl Glucosides of Phloretin and its Implication in the Warburg Effect

Contrary to differentiated cells, cancer cells predominantly convert glucose to lactate even under conditions of adequate oxygen supply (“Warburg effect”). The initial enzyme implicated in this route is hexokinase, which transforms D-glucose into D-glucose-6-phosphate. We proposed the use of different polyphenols (resveratrol, epigallocatechin gallate, pterostilbene, phloretin) and their derivatives (α-glucosides and acylated α-glucosides) to inhibit this enzyme. For this study, we used Saccharomyces cerevisiae hexokinase, whose two isoforms show high resemblance at the active site with human hexokinase HK2. To monitor the reactions, a method of anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) was developed. Remarkably, most of the assayed compounds inhibited the enzyme more than 50 % in the standard assay. Among them, phloretin 4’-O-(6’’-O-octanoyl)-α-D-glucopyranoside showed the highest inhibition and was studied in depth to determine the inhibition pattern and inhibition constant. The Ki for glucose was calculated to be 22.1±0.4 μM. Computational models of inhibition were carried out with the three molecules displaying the highest inhibition, and correlated adequately with the observed inhibitory effects on the enzyme. The inhibitory effect of several of the assayed polyphenols on hexokinase and their lack of toxicity renders them promising candidates as adjuvant drugs for cancer therapy.This work was supported by: (1) Grant PDC2022-133134-C21 “ACYLGLUFLAV_APP” funded by MCIN/AEI/10.13039/501100011033 by the “European Union Next Generation EU/PRTR”; (2) Grant PID2019-105838RB−C31 “GLYCOENZ-PHARMA” funded by MCIN/AEI/10.13039/501100011033; (3) Grant PID2022-136367OB−C31 “GLYCOENZ-GREEN” funded by MCIN/AEI/10.13039/501100011033/ and through FEDER, a Way of Making Europe (4) Grant CM_5779 “Programa Investigo” (Madrid Region, Call 2022, European Union Next Generation EU). We thank Carlos Uceda for technical help.Peer reviewe

Theoretical Three-Dimensional Zinc Complexes with Glutathione, Amino Acids and Flavonoids

Zinc plays an important role in the regulation of many cellular functions; it is a signaling molecule involved in the transduction of several cascades in response to intra and extracellular stimuli. Labile zinc is a small fraction of total intracellular zinc, that is loosely bound to proteins and is easily interchangeable. At the cellular level, several molecules can bind labile zinc and promote its passage across lipophilic membranes. Such molecules are known as ionophores. Several of these compounds are known in the scientific literature, but most of them can be harmful to human health and are therefore not allowed for medical use. We here performed a theoretical three-dimensional study of known zinc ionophores, together with a computational energetic study and propose that some dietary flavonoids, glutathione and amino acids could form zinc complexes and facilitate the transport of zinc, with the possible biological implications and potential health benefits of these natural compounds. The study is based on obtaining a molecular conformational structure of the zinc complexes with the lowest possible energy content. The discovery of novel substances that act as zinc ionophores is an attractive research topic that offers exciting opportunities in medicinal chemistry. We propose that these novel complexes could be promising candidates for drug design to provide new solutions for conditions and diseases related to zinc deficiency or impairment derived from the dysregulation of this important metal.This research was funded by “Junta de Castilla y León”, projects FEDER-VA115P17 and VA149G18); by projects APOGEO (Cooperation Program INTERREG-MAC 2014–2020, with European Funds for Regional Development-FEDER, “Agencia Canaria de Investigación, Innovación y Sociedad de la Información (ACIISI) del Gobierno de Canarias” (project ProID2020010134), CajaCanarias (project 2019SP43); and Spanish Ministry of Economy and Competitiveness (Grant PID2019-105838RB-C31).Peer reviewe

Impact of Zinc, Glutathione, and Polyphenols as Antioxidants in the Immune Response against SARS-CoV-2

SARS-CoV-2, the coronavirus triggering the disease COVID-19, has a catastrophic health and socioeconomic impact at a global scale. Three key factors contribute to the pathogenesis of COVID-19: excessive inflammation, immune system depression/inhibition, and a set of proinflammatory cytokines. Common to these factors, a central function of oxidative stress has been highlighted. A diversity of clinical trials focused predominantly on antioxidants are being implemented as potential therapies for COVID-19. In this study, we look at the role of zinc, glutathione, and polyphenols, as key antioxidants of possible medicinal or nutritional significance, and examine their role in the antiviral immune response induced by SARS-Cov-2. An unresolved question is why some people experience chronic COVID and others do not. Understanding the relationship between SARS-CoV-2 and the immune system, as well as the role of defective immune responses to disease development, would be essential to recognize the pathogenesis of COVID-19, the risk factors that affect the harmful consequences of the disease, and the rational design of successful therapies and vaccinations. We expect that our research will provide a novel perspective that contributes to the design of clinical or nutritional targets for the prevention of this pandemic.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (Grant PID2019-105838RB-C31).Peer reviewe