ABSTRACT
Botrytis cinerea (B. cinerea) and Colletotrichum gloeosporioides (C. gloeosporioides) were isolated from the decaying strawberry tissue. The antifungal properties of Monarda didyma essential oil (MEO) and its nanoemulsion were confirmed, demonstrating complete inhibition of the pathogens at concentrations of 0.45 µL/mL (0.37 mg/mL) and 10 µL/mL, respectively. Thymol, a primary component of MEO, was determined as an antimicrobial agent with IC50 values of 34.51 (B. cinerea) and 53.40 (C. gloeosporioides) µg/mL. Hippophae rhamnoides oil (HEO) was confirmed as a potent antioxidant, leading to the development of a thymol-HEO-chitosan film designed to act as an antistaling agent. The disease index and weight loss rate can be reduced by 90 and 60%, respectively, with nutrients also being well-preserved, offering an innovative approach to preservative development. Studies on the antifungal mechanism revealed that thymol could bind to FKS1 to disrupt the cell wall, causing the collapse of mitochondrial membrane potential and a burst of reactive oxygen species.
Subject(s)
Botrytis , Colletotrichum , Food Preservation , Fragaria , Fragaria/chemistry , Fragaria/microbiology , Botrytis/drug effects , Botrytis/growth & development , Colletotrichum/drug effects , Food Preservation/methods , Apoptosis/drug effects , Plant Diseases/microbiology , Food Preservatives/pharmacology , Food Preservatives/chemistry , Oils, Volatile/pharmacology , Oils, Volatile/chemistry , Antifungal Agents/pharmacology , Antifungal Agents/chemistry , Fungicides, Industrial/pharmacology , Fungicides, Industrial/chemistry , Fruit/chemistry , Fruit/microbiology , Thymol/pharmacology , Thymol/chemistry , Surface Properties , Chitosan/chemistry , Chitosan/pharmacologyABSTRACT
Mycotoxins are representative contaminants causing food losses and food safety problems worldwide. Thymol can effectively inhibit pathogen infestation and aflatoxin accumulation during grain storage, but high volatility limits its application. Here, a thymol-betaine co-crystal system was synthesized through grinding-induced self-assembly. The THY-TMG co-crystal exhibited excellent thermal stability with melting point of 91.2 °C owing to abundant intermolecular interactions. Remarkably, after 15 days at 30 °C, the release rate of thymol from co-crystal was only 55%, far surpassing that of pure thymol. Notably, the co-crystal demonstrated the ability to bind H2O in the environment while controlling the release of thymol, essentially acting as a desiccant. Moreover, the co-crystals effectively inhibited the growth of Aspergillus flavus and the biosynthesis of aflatoxin B1. In practical terms, the THY-TMG co-crystal was successful in preventing AFB1 contamination and nutrients loss in peanuts, thereby prolonging their shelf-life under conditions of 28 °C and 70% RH.
Subject(s)
Aspergillus flavus , Betaine , Thymol , Thymol/chemistry , Thymol/pharmacology , Aspergillus flavus/growth & development , Aspergillus flavus/drug effects , Aspergillus flavus/chemistry , Betaine/chemistry , Betaine/pharmacology , Food Preservatives/pharmacology , Food Preservatives/chemistry , Food Contamination/prevention & control , Food Contamination/analysis , Delayed-Action Preparations/chemistry , Arachis/chemistry , Arachis/microbiology , Crystallization , Aflatoxins/chemistry , Aflatoxin B1/chemistryABSTRACT
Phytopathogen, pest, weed, and nutrient deficiency cause severe losses to global crop yields every year. As the core engine, agrochemicals drive the continuous development of modern agriculture to meet the demand for agricultural productivity and increase the environmental burden due to inefficient use. With new advances in nanotechnology, introducing nanomaterials into agriculture to realize agrochemical accurate and targeted delivery has brought new opportunities to support the sustainable development of green agriculture. Metal-Organic frameworks (MOFs), which weave metal ions/clusters and organic ligands into porous frameworks, have exhibited significant advantages in constructing biotic/abiotic stimuli-responsive nanoplatforms for controlled agrochemical delivery. This review emphasizes the recent developments of MOF-based nanoplatforms for crop protection, including phytopathogen, pest, and weed control, and crop growth promotion, including fertilizer/plant hormone delivery. Finally, forward-looking perspectives and challenges on MOF-based nanoplatforms for future applications in crop protection and growth promotion are also discussed.
ABSTRACT
The reduction in blueberry harvest due to pathogen infection was reported to reach 80%. Essential oil (EO) can provide a new way to preserve blueberry. Here, in search for plants volatiles with preservation ability, a novel device was designed for the screening of aromatic plants led to the discovery of hit plant Monarda didyma L. Consequently, antifungi activity of M. didyma EO (MEO) and its nano-emulsion (MNE) were tested. 2 species of pathogenic fungi were isolated from blueberries, namely Alternaria sp. and Colletotrichum sp. were used as the target strains. In the in vitro activity test, the pathogenic were completely inhibited when the EO was 4 µL or 1.0 µL/mL. Compared with EO, MNE exhibited superior antimicrobial activity. Moreover, MNE can cause serious morphological changes and result in a decrease in the rot and weightlessness rate of blueberry. Hence, NME represents a promising agent for the preservation of postharvest blueberry.
Subject(s)
Blueberry Plants , Monarda , Oils, Volatile , Oils, Volatile/pharmacology , AlternariaABSTRACT
Anthocyanin is a natural antioxidant agent extracted from the fruits of Sambucus canadensis, which has been considered to have potential anti-aging effects. Cell senescence is the primary cause of aging and related diseases. Recently, research on the development of compounds for eliminating senescent cells or damaged organs have shown prospects. The compounds which promote the clearing of senescent cells are called "senolytics". Though anthocyanin is considered to have potential anti-aging effects owing to its anti-inflammatory and antioxidant activities, the mechanism of the elimination of senescent cells remains unclear. In this study, we prepared anthocyanins extracted from the fruits of Sambucus canadensis and evaluated their anti-aging effects in vivo and in vitro. We found that anthocyanin could significantly reduce cell senescence and aging of the lens by inhibiting the activity of the PI3K/AKT/mTOR signaling pathway, consequently promoting the apoptosis of senescent cells, increasing the autophagic and mitophagic flux, and enhancing the renewal of mitochondria and the cell to maintain cellular homeostasis, leading to attenuating aging. Therefore, our study provided a basis for anthocyanin to be used as new "senolytics" in anti-aging.
Subject(s)
Anthocyanins , Sambucus , Anthocyanins/pharmacology , Antioxidants/pharmacology , Phosphatidylinositol 3-Kinases , Cellular Senescence , Oxidative StressABSTRACT
Hyperosmolality-gated calcium-permeable channels (OSCA) are characterized as an osmosensor in plants; they are able to recognize and respond to exogenous and endogenous osmotic changes, and play a vital role in plant growth and adaptability to environmental stress. To explore the potential biological functions of OSCAs in maize, we performed a bioinformatics and expression analysis of the ZmOSCA gene family. Using bioinformatics methods, we identified twelve OSCA genes from the genome database of maize. According to their sequence composition and phylogenetic relationship, the maize OSCA family was classified into four groups (â , â ¡, â ¢, and â £). Multiple sequence alignment analysis revealed a conserved DUF221 domain in these members. We modeled the calcium binding sites of four OSCA families using the autodocking technique. The expression profiles of ZmOSCA genes were analyzed in different tissues and under diverse abiotic stresses such as drought, salt, high temperature, and chilling using quantitative real-time PCR (qRT-PCR). We found that the expression of twelve ZmOSCA genes is variant in different tissues of maize. Furthermore, abiotic stresses such as drought, salt, high temperature, and chilling differentially induced the expression of twelve ZmOSCA genes. We chose ZmOSCA2.2 and ZmOSCA2.3, which responded most strongly to temperature stress, for prediction of protein interactions. We modeled the calcium binding sites of four OSCA families using autodocking tools, obtaining a number of new results. These results are helpful in understanding the function of the plant OSCA gene family for study of the molecular mechanism of plant osmotic stress and response, as well as exploration of the interaction between osmotic stress, high-temperature stress, and low-temperature stress signal transduction mechanisms. As such, they can provide a theoretical basis for crop breeding.
Subject(s)
Gene Expression Regulation, Plant , Zea mays , Zea mays/metabolism , Phylogeny , Plant Proteins/metabolism , Temperature , Calcium/metabolism , Gene Expression Profiling , Droughts , Stress, Physiological/genetics , Sodium Chloride/pharmacologyABSTRACT
Aflatoxins, including aflatoxin B1, B2, G1, G2, M1, and M2, are one of the major types of mycotoxins that endangers food safety, human health, and contribute to the immeasurable loss of food and agricultural production in the world yearly. In addition, aflatoxin B1 (AFB1) mainly produced by Aspergilus sp. is the most potent of these compounds and has been well documented to cause the development of hepatocellular carcinoma in humans and animals. This paper reviewed the detoxification and degradation of AFB1, including analysis and summary of the major technologies in physics, chemistry, and biology in recent years. The chemical structure and toxicity of the transformed products, and the degradation mechanisms of AFB1 are overviewed and discussed in this presented review. In addition to the traditional techniques, we also provide a prospective study on the use of emerging detoxification methods such as natural products and photocatalysis. The purpose of this work is to provide reference for AFB1 control and detoxification, and to promote the development of follow-up research.
ABSTRACT
The calcium-sensing receptor (CAS), as a chloroplast thylakoid membrane protein, is involved in the process of external Ca2+-induced cytosolic Ca2+ increase in plants. However, the underlying mechanism regulating this process is lacking. Furthermore, recent evidence suggests that CAS may perform additional roles in plants. Here, we provided an update covering the multiple roles of CAS in stomatal movement regulation and Ca2+ signaling in plants. We also analyzed the possible phosphorylation mechanism of CAS by light and discuss the role of CAS in abiotic stress (drought, salt stress) and biotic stresses (plant immune signaling). Finally, we proposed a perspective for future experiments that are required to fill gaps in our understanding of the biological function of CAS in plants.
ABSTRACT
Infundibulicybe trachyspora is described as a new species from northeastern China. The species is characterized by clitocyboid to omphalioid habit, carneous, greyish-yellow to brownish pileus, brown to dark reddish-brown, longitudinally fibrillose-striate stipe, non-amyloid, non-smooth spores, the absence of cystidia and the presence of clamp connections. A comprehensive description of the species is provided together with photo-illustrations and comparisons with phenotypically similar and phylogenetically related species. The nuclear ribosomal internal transcribed spacer (ITS) region and the nuclear, large subunit rDNA (nrLSU) region of the new species was sequenced and analyzed. The phylogenetic analysis supported the novelty of the species and its placement within the genus. Furthermore, a discussion on the proposal to establish a new section is made, and a key is provided for the Infundibulicybe species reported from China.
Subject(s)
Agaricales , DNA, Fungal/genetics , DNA, Ribosomal Spacer/genetics , Phylogeny , Sequence Analysis, DNAABSTRACT
Two new polyketides named rufoolivacin E and viocristin B, a new natural product named 1-hydroxy-3,6,8-trimethoxyanthraquinone, and 13 known compounds were isolated from edible mushroom Cortinarius purpurascens in this work. Their structures were assigned on the basis of high-resolution electrospray ionization mass spectrometry, 1D and 2D nuclear magnetic resonance, and electronic circular dichroism data. Notably, the enzyme activity test on glutamate dehydrogenase indicated that 1, 3, 4, 5, 6, 10, 11, and 15 displayed an excellent inhibition effect. Further kinetic studies showed that the most potent compounds, 4 and 10, possess great potential as competitive inhibitors of glutamate dehydrogenase. Molecular docking and computational chemistry were applied to illustrate the binding mechanism in detail. 2,2-Diphenyl-1-picrylhydrazyl and reactive oxygen species assay results showed that compounds 1, 2, 3, and 8 exhibited significant antioxidant activities with IC50 values of 7.0 ± 0.3, 8.6 ± 0.1, 7.5 ± 0.1, and 2.8 ± 0.2 µg mL-1, respectively. Thus, Cortinarius caerulescens represents a novel resource of these polyketides to be utilized in food selection and drug discovery.
Subject(s)
Agaricales , Polyketides , Antioxidants/pharmacology , Cortinarius , Glutamate Dehydrogenase , Kinetics , Molecular Docking Simulation , Molecular Structure , Polyketides/pharmacology , PolymersABSTRACT
Controlled fungicide delivery in response to the specific microenvironment produced by fungal pathogens is an advisable strategy to improve the efficacy of fungicides. Herein, the authors construct a smart fungicide nanoplatform, using mesoporous silica nanoparticles (MSNs) as nanocarriers loaded with eugenol (EU) and Ag+ coordinated polydopamine (Ag+ -PDA) as a coating to form Ag+ -PDA@MSNs-EU NPs for Botrytis cinerea (B. cinerea) control. As a botanical fungicide, EU offers an eco-friendly alternative to synthetic fungicides and can upregulate several defense-related genes in the tomato plant. The Ag+ -PDA coating can lock the EU inside the nanocarriers and respond to the oxalic acid produced by B. cinerea to corelease the loaded EU and Ag+ . The results demonstrate that Ag+ -PDA@MSNs-EU NPs can effectively inhibit the mycelial growth of B. cinerea on detached and potted tomato leaves. The construction of such a smart fungicide nanoplatform provides new guidance to design controlled fungicides release systems, which can respond to the microenvironment associated with plant pathogen to realize fungus control.
Subject(s)
Fungicides, Industrial , Nanoparticles , Botrytis/genetics , Fungicides, Industrial/pharmacology , Silicon Dioxide/pharmacologyABSTRACT
Analysis, purification, and characterization of AFB1 degraded products are vital steps for elucidation of the photocatalytic mechanism. In this report, the UPLC-Q-TOF-MS/MS technique was first coupled with purification and NMR spectral approaches to analyze and characterize degraded products of AFB1 photocatalyzed under UV irradiation. A total of seventeen degraded products were characterized based on the UPLC-Q-TOF-MS/MS analysis, in which seven ones (1-7) including four (stereo) isomers (1,2, 5, and 6) were purified and elucidated by NMR experiments. According to the structural features of AFB1 and degraded products (1-7), the possible photocatalytic mechanisms were suggested. Furthermore, AFB1 and degraded products (1-7) were evaluated against different cell lines. The results indicated that the UPLC-Q-TOF-MS/MS technique combined with purification, NMR spectral experiments, and biological tests was an applicable integrated approach for analysis, characterization, and toxic evaluation of degraded products of AFB1, which could be used to evaluate other mycotoxin degradation processes.
ABSTRACT
Synthetic fungicides have been widely used to protect crops from fungal diseases. However, excessive use of synthetic fungicides leads to the generation of fungicide resistance in fungal pathogens. Recently, smart cargo delivery systems have been introduced for the construction of a pesticide delivery nanoplatform, benefiting from their controlled release performance. Herein, a fungal pathogen microenvironment-responsive supramolecular fungicide nanoplatform has been designed and constructed, using quaternary ammonium salt (Q)-modified mesoporous silica nanoparticles (MSN-Q NPs) as nanocarriers loaded with berberine hydrochloride (BH) and carboxylatopillar[5]arene (CP[5]A) as nanogates to form BH-loaded CP[5]A@MSN-Q NPs for effective inhibition of Botrytis cinerea. CP[5]A as nanogates can endow the fungicide nanoplatform with pH stimuli-responsive release features for the control of fungicide release. The loaded BH, as a natural plant fungicide, provides an ecofriendly alternative to synthetic fungicides for controlling B. cinerea. Interestingly, we use oxalic acid (OA) secreted by B. cinerea as a trigger so that BH can be released from the fungicide nanoplatform on demand under pathogen microenvironments for controlling B. cinerea. The experimental results indicate that the fabricated fungicide nanoplatform could effectively inhibit the mycelial growth and spore germination, providing a new way for the management of B. cinerea in actual application.
Subject(s)
Drug Carriers/chemistry , Fungicides, Industrial/chemistry , Fungicides, Industrial/pharmacology , Nanoparticles/chemistry , Silicon Dioxide/chemistry , Berberine/chemistry , Berberine/pharmacology , Botrytis/drug effects , Delayed-Action Preparations , Drug Liberation , Drug Resistance, Fungal/drug effects , Porosity , Quaternary Ammonium Compounds/chemistryABSTRACT
Controllable and on-demand delivery of agrochemicals such as plant hormones is conducive to improving agrochemicals utilization, tackling water and environmental pollution, reducing soil acidification, and realizing the goals of precision agriculture. Herein, a smart plant hormone delivery system based on metal-organic frameworks (MOFs) and supramolecular nanovalves, namely gibberellin (GA)-loaded CLT6@PCN-Q, is constructed through supramolecular host-guest interaction to regulate the growth of dicotyledonous Chinese cabbage and monocotyledonous wheat. The porous nanoscale MOF (NMOF) with a uniform diameter of 97 nm modified by quaternary ammonium (Q) stalks is served as a cargo reservoir, followed by the decoration of carboxylated leaning tower[6]arene (CLT6) based nanovalves on NMOF surfaces through host-guest interactions to fabricate CLT6@PCN-Q with a diameter of â¼101 nm and a zeta potential value of -13.2 mV. Interestingly, the as-fabricated supramolecular nanoplatform exhibits efficient cargo loading and multi-stimuli-responsive release under various external stimuli including pH, temperature, and competitive agent spermine (SPM), which can realize the on-demand release of cargo. In addition, GA-loaded CLT6@PCN-Q is capable of effectively promoting the seeds germination of wheat and stem growth of dicotyledonous Chinese cabbage and monocotyledonous wheat (1.86 and 1.30 times of control groups, respectively). The smart supramolecular nanoplatform based on MOFs and supramolecular nanovalves paves a way for the controlled delivery of plant hormones and other agrochemicals for promoting plant growth, offering new insights and methods to realize precision agriculture. STATEMENT OF SIGNIFICANCE: To achieve controllable and sustainable release of cargos such as agrochemicals, a smart MOF-based multi-stimuli-responsive supramolecular nanoplatform equipped with supramolecular nanovalves was fabricated via the host-guest interaction between quaternary ammonium stalks-functionalized nanoMOFs and water-soluble leaning tower[6]arene. The as-prepared supramolecular nanoplatform with uniform diameter distribution demonstrated good cargo release in response to various external stimuli. The installation of synthetic macrocycles could effectively reduce cargo loss in the pre-treatment process. This type of supramolecular nanoplatform exhibited good promoting effect on seed germination and plant growth dicotyledonous Chinese cabbage and monocotyledonous wheat. As an eco-friendly, controlled, and efficient cargo delivery system, this supramolecular nanoplatform will be a promising candidate in precision agriculture and controlled drug release to attract the broad readership.
Subject(s)
Water , Drug Liberation , PorosityABSTRACT
Agricultural chemicals have been widely utilized to manage pests, weeds, and plant pathogens for maximizing crop yields. However, the excessive use of these organic substances to compensate their instability in the environment has caused severe environmental consequences, threatened human health, and consumed enormous economic costs. In order to improve the utilization efficiency of these agricultural chemicals, one strategy that attracted researchers is to design novel eco-friendly nanoplatforms. To date, numerous advanced nanoplatforms with functional components have been applied in the agricultural field, such as silica-based materials for pesticides delivery, metal/metal oxide nanoparticles for pesticides/mycotoxins detection, and carbon nanoparticles for fertilizers delivery. In this review, the synthesis, applications, and mechanisms of recent eco-friendly nanoplatforms in the agricultural field, including pesticides and mycotoxins on-site detection, phytopathogen inactivation, pest control, and crops growth regulation for guaranteeing food security, enhancing the utilization efficiency of agricultural chemicals and increasing crop yields are highlighted. The review also stimulates new thinking for improving the existing agricultural technologies, protecting crops from biotic and abiotic stress, alleviating the global food crisis, and ensuring food security. In addition, the challenges to overcome the constrained applications of functional nanoplatforms in the agricultural field are also discussed.
Subject(s)
Crop Protection/methods , Crops, Agricultural/growth & development , Green Chemistry Technology/methods , Nanotechnology/methods , Pest Control, Biological/methods , Quality Control , Biosensing Techniques , Fertilizers , Humans , Nanoparticles , PesticidesABSTRACT
Eight natural biphenyl-type phytoalexins exhibiting antifungal effect were isolated from the leaves of Sorbus pohuashanensis, which invaded by Alternaria tenuissi, and their growth inhibition rate towards A. tenuissi were 50.3 %, 54.0 %, 66.4 %, 58.8 %, 48.5 %, 51.0 %, 33.3 %, and 37.0 %, respectively. In vivo activity assay verified the protective effect of these natural biphenyls on tobacco leaves. The observation of mycelial morphology revealed that these compounds possessed adverse effects on mycelial growth of A. tenuissi. Subsequently, the most potent active compounds, 3',4',5'-trimethoxy[1,1'-biphenyl]-4-ol (3) and 3,4,4',5-tetramethoxy-1,1'-biphenyl (4), were conducted to the further antifungal evaluation and showed significant activity against the other four crop pathogens, Fusarium graminearum, Helminthosporium maydis, Sclerotinia sclerotiorum, and Exserohilum turcicum. Further, the structure-activity relationships and biosynthesis of these compounds were speculated in this work.
Subject(s)
Alternaria/drug effects , Antifungal Agents/pharmacology , Biphenyl Compounds/pharmacology , Sorbus/chemistry , Alternaria/growth & development , Alternaria/pathogenicity , Antifungal Agents/chemistry , Antifungal Agents/isolation & purification , Ascomycota/drug effects , Ascomycota/pathogenicity , Biphenyl Compounds/chemistry , Biphenyl Compounds/isolation & purification , Bipolaris/drug effects , Bipolaris/pathogenicity , Fusarium/drug effects , Fusarium/pathogenicity , Microbial Sensitivity Tests , Molecular Structure , Plant Extracts , Plant Leaves/chemistryABSTRACT
Due to the unique environmental conditions and vast territory, marine habitat breeds more abundant biological resources than terrestrial environment. Massive marine biological species provide valuable resources for obtaining a large number of natural products with diverse structure and excellent activity. In recent years, new breakthroughs have been made in the application of marine natural products in drug development. In addition, the use of marine natural products to develop insecticides and other pesticide products has also been widely concerned. Targeting marine plants, animals, and microorganisms, we have collected information on marine natural products with insecticidal activity for nearly decade, including alkaloids, terpenes, flavonoids and phenols fatty acids, peptides, and proteins, etâ al. In addition, some active crude extracts are also included. This review describes the insecticidal activities of marine natural products and their broad applications for future research in agriculture and health.
Subject(s)
Biological Products/chemistry , Insecticides/chemistry , Animals , Biological Products/chemical synthesis , Insecticides/chemical synthesis , Molecular StructureABSTRACT
Globosumin, an undescribed chromene-4,7(4aH)-dione-tetramic acid PKS-PKS-NRPS hybrid, and globosumone, an undescribed azaphilone, together with ten known metabolites, were isolated from the desert plant-associated endophytic fungus Chaetomium globosum (Chaetomiaceae). The planar structures and relative configurations of globosumin and globosumone were determined by high-resolution ESI-MS and NMR data, and the absolute configurations of these two metabolites were determined by electronic circular dichroism (ECD) and circular dichroism (CD) combined with time-dependent density functional theory (TDDFT)-based quantum-chemical calculations. Chaetoglobosin A displayed biological effects against the seedling growth of Arabidopsis thaliana (Brassicaceae) in a dose-dependent manner, and this compound also exhibited biological activity against two cancer cell lines, A549 and HepG2, with IC50 values of 6.82 ± 2.34 and 38.62 ± 7.44 µM, respectively.
Subject(s)
Chaetomium , Circular Dichroism , Magnetic Resonance Spectroscopy , PlantsABSTRACT
The phenomenon of the long leaching time and low leaching rate is presented in the acid leaching process under the conventional conditions of low reaction temperature and acid concentration. In order to promote leaching rates of indium and tin in waste liquid crystal display, an optimized process combining rapid milling and acid leaching has been proposed, which is more time and energy-efficient, environmentally sound compared with the traditional acid leaching process. Leaching mechanism analysis was conducted to uncover the different leaching behavior of indium and tin. And the external factors affecting the leaching rates of indium and tin were studied to optimize. In this process, the fine powder with a weight ratio of 97.6%, which particle size less than 0.075 mm, was obtained with the optimal milling time of 30 min by rapid grinding in the planetary high energy ball milling. About -0.003 l/s of grinding rate constant was performed in the grinding size fraction from 3 mm to 0.075 mm. The research results indicated that the particle size less than 0.035 mm was agglomerated, and the addition of H2O2 reduced the leaching rate for the particle size less than 0.075 mm. Moreover, 86.3% and 76.1% of indium and tin were leached in a short leaching time of 10 min by using 3 M H2SO4 at 85 °C for particle size range from 0.075 to 0.035 mm, while 96.9% and 85.6%, respectively in 90 min.