Eugenol oil nanoemulsion antifungal activity agai

Authors: Kamel A AbdElsalam Alexei R Khokhlov

Publish Date: 2015/01/10

Volume: 5, Issue: 2, Pages: 255-265

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Abstract

The current research deals with the formulation and characterization of biobased oilinwater nanoemulsion The formulated eugenol oil nanoemulsion was characterized by dynamic light scattering stability test transmission electron microscopy and thin layer chromatography The nanoemulsion droplets were found to have a Zaverage diameter of 80 nm and TEM study reveals the spherical shape of eugenol oil nanoemulsion EON The size of the nanoemulsion was found to be physically stable up to more than 1month when it was kept at room temperature 25 °C The TEM micrograph showed that the EON was spherical in shape and moderately mono or didispersed and was in the range of 50–110 nm Three concentrations of the nanoformulation were used to evalute the antifusarium activity both in vitro and in vivo experiments SDSPAGE results of total protein from the Fusarium oxysporum f sp vasinfectum FOV isolate before and after treatment with eugenol oil nanoemulsion indicate that the content of extra cellular soluble small molecular proteins decreased significantly in EONtreated fungus Light micrographs of mycelia and spores treated with EON showed the disruption of the fungal structures The analysis of variance ANOVA for Fusarium wilt incidence indicated highly significant p = 0000 effects of concentration genotype and their interaction The difference in wilt incidence between concentrations and control was not the same for each genotype that is the genotypes responded differently to concentrations Effects of three EON concentration on germination percentage and radicle length were determined in the laboratory One very interesting finding in the current study is that cotton genotypes was the most important factors in determining wilt incidence as it accounted for 9318  of the explained model variation In vitro experiments were conducted to evaluate the potential phytotoxic effect of three EON concentrations Concentration genotype and concentration x genotype interaction were all highly significant sources of variation in seed germination however interaction was the first in importance as a source of variation followed by the concentration while genotype was the least important source of variation These results suggest the potential use of eugenol oil nanoemulsion for protecting seedcotton from Fusarium wilt infectionFusarium oxysporum f sp vasinfectum causes important crop losses in the most cottonproducing countries Fusarium wilt is a destructive disease of cotton Gossypium spp in several countries of the globe including Australia USA Egypt Tanzania and China Watkins 1981 Hillocks 1992 AbdElsalam et al 2014 The first recorded of this disease outside the USA came from Egypt where it was quickly disseminated after the release of the highly susceptible cultivar ‘Sakal’ during the 1920s Fahmy 1927 Currently selection and breeding of new genotypes with higher levels of resistance to fusarium wilt are the main approaches to control this disease AbdElsalam et al 2014 The improvement of cotton cultivar resistance through conventional plant breeding methods is timeconsuming and laborious Tohidfar et al 2012Longterm extensive fungicides in open field have led to the appearance of fungicideresistant populations of fungal pathogens Brent and Hollomon 1998 Overall intensive fungicide application results in several negative effects in the environment that cannot be ignored Feng and Zheng 2007 Therefore development of alternative ecosafe antifungal agents like biobased nanomaterials is urgently needed A number of nanotechnologies can improve the current crop management protocols within a short term PérezdeLuque and Rubiales 2009 AbdElsalam 2012 Kah et al 2013 Recently nanomaterials particle size 100 nm are being developed and offer the opportunity to more efficiently and safely administration of pesticides fungicides herbicides and fertilizers by better delivery of active ingredients and less environmental drift Li et al 2007 Gogoi et al 2009 Antimicrobial nanoemulsions are highly stable oilinwater emulsions composed of nanometersized positively charged droplets that have broadspectrum activity against enveloped viruses fungi and bacteria Hamouda et al 2001 Myc et al 2002 Wang et al 2007 Nanobiocide a product prepared by mixing several biobased chemicals was reported to eliminate fungus Magnaporthe grisea the causal agent of rice blast disease Gogoi et al 2009 Clove and eugenol oil showed good potential to inhibit growth of A niger Penicillium sp and Rhizopus sp on media Eugenol a main component of clove oil was proposed as the agent responsible for clove oil’s antifungal activity against a wide range of plant pathogens Pinto et al 2009 Wang et al 2010 Matan et al 2011 Ghosh et al 2014 Therefore the present study was aimed to formulate and characterize the eugenol oil nanoemulsion using nonionic surfactant Tween 20 with water as a continuous aqueous phase The antifungal activities of the synthesized EONs was evaluated in vitro and in vivo SDS–PAGE analysis of FOV treated by EON was studied to determine the mode of action of EON One of the most important issues to be addressed before the extensive utilization of nanomaterials is the evolution of their possible toxicity Thus the phytotoxicity effects of EON on cotton genotypes were assessedMean droplet size of each prepared composition was completed to verify nanoemulsion region using pseudoternary phase diagram Every corner correspond to 100  of water surfactants Tween and oil Phase diagrams were created using TRIANGULAR softwareCommercial products of eugenol oil were used in the present study Eugenol C10H12O2 a phenylpropanoid is an allyl chainsubstituted guaiacol The eugenol oil 99  MW16421 Fluka code 46100 and nonionic surfactant Tween 20 were added slowly under gentle stirring until a homogeneous mixture formed Then water was added drop wise at 37 °C and stirred at 43 g This mixture was sonicated using an Ultrasonicator Bandelin SONOPULS HD 2200 Germany for 10 min at 700 W The particle size and shape of 10  eugenol nanoemulsion were detected by stability measurement particle size analyzer and transmission electron microscopeThe stability analysis was performed by centrifuging the 10  eugenol nanoemulsion at 3500 rpm for 30 min Shafiq and Shakeel 2010 The stability was further checked at both refrigerator temperature 4 °C and room temperature 25 °C Three freeze–thaw cycles between −21 °C and +25 °C were also checked Stability assay was also performed by storage for 3 months Ten percent of eugenol nanoemulsion was diluted to 1 2 and 5  eugenol nanoemulsion solution by distilled water and store at RT until usage The purpose of these tests was to select a stable lowsurfactant formulation with a nanoemulsionsize droplet and stable physicochemical propertiesMeasurement of droplet size of nanoemulsion was performed by a dynamic light scattering method using Zetasizer Nano ZS Malvern Instruments UK at room temperature Prior to measurement 30 µl of the nanoemulsion was diluted with 3 ml of water at 25 °C Particle size data were expressed as the mean of the Zaverage of 3 independent batches of the nanoemulsionsTwenty microliters of diluted samples was placed on a filmcoated 200mesh copper specimen grid for 10 min and the fluid excess was eliminated using filter paper The grid was then stained with one drop of 3  phosphotungstic acid and allowed to dry for 3 min The coated grid was dried and examined under the TEM microscope Philips CM 12 The samples were observed by operating at 120 kV

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