Enhancement in Antibacterial Activities of Eugenol-Entrapped

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Agricultural and Environmental Chemistry

Enhancement in antibacterial activities of eugenol-entrapped ethosome nanoparticles via strengthening its permeability and sustained-release Peng Jin, Rui Yao, Dingkui Qin, Qing Chen, and Qizhen Du J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06278 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 11, 2019

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Journal of Agricultural and Food Chemistry

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Enhancement in antibacterial activities of eugenol-entrapped

2

ethosome nanoparticles via strengthening its permeability and

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sustained-release

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Peng Jin, Rui Yao, Dingkui Qin, Qing Chen, Qizhen Du*

8

The Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang Province, The

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College of Agricultural and Food Sciences, Zhejiang A & F University, Linan, 311300, China

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

*Corresponding Author

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The College of Agricultural and Food Sciences, Zhejiang A & F University, Linan

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311300, China. Tel.: +86-571-15958126861; Fax: +86-571-88218710; Email:

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[email protected] (Q. Du)

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ACS Paragon Plus Environment


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ABSTRACT

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The antibacterial efficiency and synergistic mechanisms of novel formulated eugenol

36

entrapped ethosome nanoparticles (ELG-NPs) against fruit anthracnose were

37

investigated. The results showed that concentrations of eugenol and ethanol

38

significantly influenced the particle size and entrapment efficiency of nanoethosome,

39

and the particle size significantly influenced the antibacterial effect. A superior

40

ELG-NPs with optimized process (0.5% eugenol, 2% lecithin and 30% ethanol) was

41

obtained with size of 44.21 nm and entrapment efficiency of 82%. ELG-NPs

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exhibited greater antibacterial activity (>93%) against fruit pathogens than that of

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free-eugenol, showed 100% inhibition of the anthracnose incidence in postharvest

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loquat after 6-d. The permeability study firstly visualized in banana cortex with

45

fluorescent indicators, demonstrated that eugenol delivered to the interior with

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ELG-NPs was 6-fold higher than that of free-eugenol. ELG-NPs showed a

47

satisfactory slow-release and prolonged antibacterial action. This work provides a

48

promising strategy for disease controls in agricultural, food, cosmetic, and medical

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areas.

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KEY WORDS: Eugenol; Nanoethosomes; Anthracnose; Antibacterial activity;

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Permeability; Postharvest diseases

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INTRODUCTION

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Fresh fruits and vegetables are susceptible to anthracnose caused by pathogens

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contaminated at any step from pre-harvest treatment to post-harvest storage and

59

transportation. The development of postharvest pathogens quickly results in the

60

quality deterioration and even serious decay of fruits and vegetables, which are the

61

major cause of economic losses through the supply chain. Therefore, many

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decay-controlling measures have been taken in recent decades to provide protection

63

against postharvest diseases and to reduce the postharvest losses. To date, application

64

of synthetic chemicals (i.e., fungicides, pesticides, inorganic salts) is the major

65

approach for disease control.1,2 However, with growing concern over the food safety

66

and human health, the development of alternative eco-safe strategies against

67

postharvest diseases is urgently needed.

68

Recently, a number of natural plant extracts have been applied as effective

69

alternatives to conventional antibacterial or antifungal agents because of their special

70

characteristics–low toxicity, comparable efficacy, and customer approval. For

71

example, polysaccharides,3 organic acids,2,4 essential oils,5 terpenoids, and

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phytosterols6 are reported to have significant antimicrobial, antiviral, and (or)

73

antioxidant potentials due to the presence of saponins, flavonoids, ketones, and

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aldehydes.

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(4-allyl-2-methoxyphenol) has been found to be able to fight against various

76

pathogens owing to its effective antimicrobial and antioxidant properties. In addition,

77

eugenol is classified as ''generally regarded as safe'' (GRAS),7 and has been

As

a

major

constituent

in

clove

3


essential

oil,

eugenol


78

considered as an ideal antibacterial agents in the food, pharmaceutical, and cosmetic

79

industries.8,9 Despite its potential application and urgent requirements in various areas,

80

the efficiency of eugenol is substantially limited for its high volatility, low

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water-solubility, and highly susceptibility to environmental stress.10 The embedding

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and encapsulation technology is a novel method that can markedly improve the

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physicochemical stability of actively volatile agents.11-13 In the recent past, many

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studies have also revealed that encapsulation of eugenol into nanoparticles could

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enhance its stability, permeability, and antimicrobial activity.14-16

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In the last decades, numerous micro- and nano-sized carriers (e.g. capsules,

87

particles, vesicles or liposomes, emulsions and gels) have received a growing

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scientific and industrial interest. Specific studies have been focused on the use of

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liposomes,17 emulsions18 and polysaccharides10,16 to improve the antibacterial activity

90

of eugenol. However, these carriers are unable to penetrate into deeper cortex and

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remain at the upper layer of the stratum corneum.19 As most of the pathogens inhabit

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the deeper cortex layers of fruits and vegetables, it is commonly agreed that these

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conventional formulations are not suitable as carriers for transdermal drug delivery.20

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Herein, ethosomes may provide a promising strategy to deliver eugenol and similar

95

bioactive agents. Typically, ethosomes are modified forms of liposomes, which are

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composed of phospholipids and relatively high concentration of ethanol and

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water–they have been constructed for the administration of both hydrophilic and

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lipophilic drugs.21 Ethanol is able to aid ethosomes in squeezing through the pores and

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permeating deeper into cortex layers, and hence releasing transdermal flux drugs into 4


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deeper layers more efficiently than conventional nano-carriers.22 Nevertheless, to our

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knowledge, there are no studies using nanoethosomal carriers to encapsulate eugenol

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for fruit preservation and antibacterial purposes; moreover, it is necessary to verify

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the enhanced penetrability through fruit cortex and the consequent drug delivery

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efficiency of encapsulated eugenol.

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In this study, to investigate the effect of nanoethosomes particle size on the

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antibacterial activity, we prepared various sizes of nanoethosomes by optimizing the

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proportion of eugenol and ethanol. The antibacterial effects and the anthracnose

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incidence in fresh loquat fruit were compared between the groups treated with

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nanoethosomal or natural eugenol. Furthermore, the penetrability across cortex and

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sustained-release effects of nanoparticles were also assessed by the dye tracer and

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GC-MS analyses. This work provides an insight into the enhancement of eugenol

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antibacterial activities by nanoethosomal system, suggesting that it is a suitable and

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efficient carrier for eugenol encapsulation and delivery. The information gained from

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this study may be useful for designing various nanoethosomal delivery systems to

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fight against postharvest diseases caused by specific bacteria.

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MATERIALS AND METHODS

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Chemicals and reagents. Eugenol, Tween 80, and soyabean lecithin were

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purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA). All Other reagents are

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analytical grade. Deionized water was prepared by filtered through a MILLI-Q water

120

system (Millipore Corp., Bedford, MA, USA). Sucrose esters of fatty acids (SEFA) 5



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were purchased from Hangzhou JinHeLai Food Additive CO. LTD (Hangzhou,

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China).

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Preparation of nanoparticles. Ethanol, tween-80, and eugenol were mixed in

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sealed conical flask, then soybean lecithin and SEFA were added and dissolved in the

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mixture solution. Then the mixture was incubated at 37 °C with agitation at 1,000 rpm

126

for 2 min until a homogeneous mixture formed. Then water was added drop wise at

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37 °C and kept stirring for 5 minutes. This mixture was immediately sonicated for 2

128

min at 500 W in ice-water by Ultrasonicator (Bandelin SONOPULS HD 2200,

129

Germany). The nanoethosomes was filtered through a 0.22 μm filter and then used for

130

the following tests.

131

Physicochemical Characterization of nanoparticles. The mean particle size, size

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distribution and zeta potential of ethosomes nanoparticles were analyzed by dynamic

133

light scattering using a ZetasizerNano ZS 3690 (Malvern Instruments, Malvern, UK),

134

employing a nominal 5 mW He–Ne laser operating at 633 nm wavelength and 173°

135

scattering angle as our previous description.23 1 mL of the nanoethosomes was diluted

136

with 10 mL of water at room temperature. The entrapment efficiency (EE, %) of

137

eugenol was calculated as the percentage of entrapped eugenol to total eugenol. All

138

data were expressed as the mean of 3 independent batches of the samples.

139

Transmission

140

characterization of eugenol nanoethosomes as the process described previously.15

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Samples were prepared by firstly diluted and placed on a film-coated 200-mesh

electron

microscopy

(TEM)

was

6


used

for

morphological

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copper specimen grid for 10 min, and then stained with 3 % phosphotungstic acid and

143

dried prior to TEM observation. TEM micrographs were obtained using a

144

TECNAI-20F (120 kV) FEG microscope (Philips, Tecnai 20F) equipped with the

145

high-angle annular dark-field (HAADF) detector.

146

Strains and cell culture. Colletotrichum musae, Colletotrichum fragariae,

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Colletotrichum gloeosporioides Penz, and Colletotrichum gloeosporioides were

148

isolated from decayed banana and loquat fruits. Strain mycelium were precultured on

149

Luria-Bertani (LB, g/L: tryptone 10, yeast extract 5, and NaCl 10) agar media at 28

150

°C for 7 d as the seed cultures. The spore suspension were prepared by washing the

151

20-day seed cultures with sterile water containing 0.01% (v/v) Tween-80, and then

152

diluted to 1 × 105 spores/mL with the aid of a hemocytometer. To evaluate the effects

153

of particle sizes on antibacterial activity, a diameter of 6 mm C. fragariae mycelium

154

was cut from the seed cultures and placed in the fresh LB agar medium. Then 800 μl

155

of eugenol nanoethosomes solution was dropped into the filter paper (5 mm × 10 mm),

156

which was then placed in the middle of the cover. Finally, the petri dishes were

157

immediately sealed and incubated at 25 °C (mycelial growth inhibition experiment,

158

MGI experiment). For minimum inhibitory concentration (MIC) analysis by eugenol

159

evaporation method, various amounts of free eugenol (1, 2, 3, 4, 5, 5.6 and 6 μl) and

160

equivalent nanoethosomes solution (200, 400, 600, 800, 1000, 1120 and 1200 μl)

161

were added into the filter paper as described above, respectively, and then performed

162

the same manual tests.

7



163

To investigate the inhibitory effect of nanoethosomes on various anthrax bacteria,

164

ELG-NPs (3 μL) solution was added into the LB agar medium with a final eugenol

165

concentration of 0.26 μl/mL, and then the above-mentioned manual tests were

166

performed. The spore germination inhibition effects were evaluated as follows: first,

167

200 ul of spore suspensions (1 × 105 spores/mL) were inoculated into 50 mL flasks

168

containing 5 mL of LB medium, with the initial concentration of eugenol at 0.6 μl/mL

169

in groups treated with either nanoethosomal or free eugenol (dissolved in equal

170

ethanol solution); then, the suspensions were cultured at 28 °C for 30 h (200 rpm);

171

finally, the inhibition rate of mycelium growth was calculated as a percentage of

172

control groups without eugenol.

173

Effects of ELG-NPs on anthracnose disease on loquat fruit. Fresh loquat fruit

174

were wounded (d ≈ 2 mm) in the middle of each fruit with a sterile nail. The wound

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was then inoculated with 10 μL of spore suspensions of C. gloeosporioides Penz. The

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treated fruits were put in sealed fresh-preserved storage container (10 L) with

177

encapsulated or non-encapsulated eugenol solution (5 μL/L) in the bottom. The

178

control was set with equal volume of distilled water and administered in the same

179

protocol. All loquat fruit were stored at 20 °C. The disease incidence and lesion

180

diameter were observed and recorded. The anthracnose incidence was defined as the

181

percentage of loquats (with lesion diameter > 4 mm) in the total amounts of loquats in

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each group. Each treatment contained three replicates and the entire experiment was

183

repeated three times. Statistical analysis was performed with SPSS software (SPSS

184

Inc., Chicago, IL, USA) and using one-way ANOVA analysis. Difference with P < 8


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0.05 was considered statistically significant.

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Penetration test of ELG-NPs on banana cortex. 10 μL of spore suspensions of C.

187

musae were inoculated in the endodermis (sarcocarp surface) of fresh bananas with

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sterile injector with an oblique angle of 30°. Then equivalent eugenol (0.5%) of free

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eugenol, non-nano mixture (which had the same composition as ELG-NPs but without

190

ultrasonic treatment), and ELG-NPs solution (1 mL) was smeared onto the banana

191

surface, respectively. Each group contained three replicates of 20 bananas and the

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control group was correspondingly treated with sterile distilled water. Subsequently,

193

all the treated samples were stored at 20 °C in a sterile room. Disease incidence and

194

lesion diameters were recorded.

195

To trace the penetration process, Nile red, a fluorescent dye, was dissolved in the

196

ethanol phase during the ELG-NPs preparation procedure. Equivalent volume of

197

free-eugenol, non-nano mixture, and ELG-NPs solution (1 mL) with 0.01% (v/w) Nile

198

red were used to smear the banana surface. After a 3-h exposure at ambient

199

temperature, a banana cortex piece (0.8 × 1.0 cm) was taken from the treated

200

epidermis. The obtained samples were rapidly frozen by liquid nitrogen and then

201

trasferred into a metal block. Finally, a vertical cross-section of banana cortex

202

(thickness = 100 μm) was sliced with a cryostat microtome (LE ICACM 1850,

203

Germany). The sliced sections were subjected to fluorescent microscopy using an

204

Olympus CK40 microscope (Leica Germany) with an excitation wavelength of 543

205

nm and emission wavelength of 604 nm. Subsequently, the treated banana cortex was

206

uniformly sliced into two sections–epidermis and endodermis, from which the 9



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permeated eugenol was extracted and quantitatively analyzed by GC-MS (Shimadzu,

208

Japan). All the experiment was replicated three times.

209

Effects of ELG-NPs release studies on the antibacterial activity. Equivalent

210

volume (1200 μl) of free eugenol, non-nano mixture, and ELG-NP solution,

211

containing 6 μl of eugenol, was added onto the filter paper in the middle of the cover.

212

After being exposed for 0, 2, 4, 6, 8 and 10 h in a sterile room, all the filter papers

213

were immediately used for the MGI experiment as described above. Thereafter, the

214

petri dishes were immediately sealed and incubated at 28 °C for 5 days. To further

215

quantitatively analyzing the sustained-release effect of ELG-NPs, the spore

216

suspension of C. fragariae (1 × 105 spores/mL, 200 μl) was inoculated into a flask

217

containing 5 mL of LB medium, with an initial eugenol concentration of 1 μl/mL in

218

both nanoencapsulated and non-encapsulated groups, and then cultured at 28 °C with

219

shaking (200 rpm). The optical density at 600 nm (OD600) was used to monitor cell

220

growth. Samples were periodically withdrawn from the culture and used to quantify

221

the remaining eugenol contents by GC-MS. The control was set without eugenol in

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the culture and the entire experiment contained three replicates.

223

GC–MS analysis. The extracted solution (10 μl) was injected via the autosampler

224

of an HPLC system equipped with a C18 column (300 ×

225

Shimadzu, Kyoto, Japan). Analyses were performed using helium as carrier gas at a

226

flow rate of 1 mL/min. Oven temperature parameters as follows: an initial hold at 80

227

°C for 5 min, and then rise to 250 °C at 10 °C/min, held at 250 °C for 10 min. Mass 10


0.25 mm 0.25 um,

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spectrometry was performed in negative ion mode with EI source temperature at 230

229

°C, electron energy at 70 eV, interface temperature at 250 °C. The ion monitoring

230

mode was set as sim mode, monitoring ions = 62, 74 and 89, quantitative ions = 62,

231

solvent delay = 3 min. The eugenol was identified by comparing their mass spectral

232

fragmentation patterns with the eugenol standard sample and combined with

233

comparison of GC retention indices (RI). The standard curve of eugenol was then

234

constructed for GC-MS quantitative analysis, and the recovery efficiencies of eugenol

235

from banana cortex were 89%–92%, with R2 > 0.993 for all calibration curves.

236

RESULTS AND DISCUSSION

237

Preparation

and

characterization

of

Eugenol-entrapped

soybean

238

lecithin-glycolipids Nanoparticles (ELG-NPs). Ethosomal nanocarriers were widely

239

used as delivery systems for hydrophobic drugs and bioactive compounds due to their

240

hydrophobic nature. In this study, the ethanol and tween 80 were used as the organic

241

solvent for the solubilization of eugenol and soybean lecithin. The final concentration

242

of ethanol and soybean lecithin has direct effect on the droplet size of unloaded

243

ethosome nanoparticles. As shown in Table S1, the sizes of nanoparticles with 2 %

244

lecithin were significantly lower (20-40 nm) than that with 3% lecithin. The opposite

245

effect was observed that with an increase of ethanol concentration from 20% to 30%,

246

resulting in a significant reduction of their sizes by 20-40 nm. This is probably due to

247

the ethanol concentration-dependent steric stabilization of nanoethosome that may

248

finally lead to a decrease in the mean particle size,24,25 while no significant differences 11



249

in the zeta potential. The particle size is one of the most important properties of

250

nano-delivery systems. It has direct relevance with the stability, cellular uptake, in

251

vivo distribution, and drug release of nanomedicines.26,27 Thus, to obtain

252

nanoethosomes with optimal size range, 2% lecithin and 30% ethanol were used to

253

prepare the unloaded nanoparticles (mean diameter = 44.2 ± 1.2 nm) in this study.

254

Eugenol-loaded ethosomal nanoparticles were prepared through the formation of

255

oil-in-water emulsion droplets followed by the ultrasonic treatment of the mixture,

256

resulting in formation of smaller nanoparticles. The effects of eugenol concentration

257

(0.25−3%) on the particle size, polydispersity, Zeta-potential, and entrapment

258

efficiency of eugenol loading in ethosome nanoparticles particles were further studied

259

(Table 1). With the eugenol concentration being increased from 0.5% (w/v) to 3%, the

260

particle size of ELG-NPs was notably increased from 44 nm to 172 nm. Meantime,

261

most particle polydispersity indexes of ELG-NPs (0.050-0.084) were significantly

262

decreased about 4-folds than that of unloaded nanoparticles (0.187-0.245).

263

Unexpectedly, ELG-NPs with eugenol of 0.25% didn’t show smaller particle size,

264

whereas similar to the properties of nanoparticle with eugenol of 1 %. However, it is

265

interesting that specific concentration of eugenol (0.5%) brought about better

266

dispersibility and Zeta-potential to the formulated nanoparticles, and showed no

267

significant influence on the size and distribution of the particles (Table 1).

268

Furthermore, analysis of the particle size distribution with TEM image demonstrated

269

uniform and well-dispersed NP populations (Figure 1a). The nanoethosomal system

270

fabricated here had much smaller particle size than that of other eugenol-loaded 12


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nanosystems,3,17 which may contribute to the favorable anti-aggregation and

272

bioactivity attributes of ELG-NPs as compared with those larger particles. Moreover,

273

the entrapment efficiency of nanoparticles also reached the maximum value of

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82.43% using 0.5% eugenol (Table 1). Nevertheless, with the eugenol rising from

275

0.5% to 3%, the entrapment efficiency dropped from 82.43% to 21.33%. This result

276

was in agreement with the previous reports.16 Our results further indicate that the

277

loading efficiency of ethosome nanoparticle is comparable to liposomes,17 which can

278

be ascribed to the highly lipophilic characteristics of eugenol. Overall, considering the

279

particle size, Zeta-potential, and entrapment efficiency, the ELG−NPs were prepared

280

with 0.5% eugenol, 2% lecithin and 30% ethanol.

281

Assessment of in vitro antibacterial ability was performed by observing the

282

inhibiting effect of ELG-NPs on the mycelial growth of anthracnose on LB agarose

283

plate (Figure 1b). ELG-NPs with various particle sizes (83.8, 77.2, 70.3, 68.3, 55.5

284

and 44.2 nm) with 0.5% eugenol were prepared by controlling the ethanol

285

concentration. There was a linear correlation between ethanol concentration and

286

particle size–the increase of ethanol concentration significantly enlarged the particle

287

size. It can be seen that ELG-NPs with the smallest particle size (44.2 nm) showed the

288

highest antibacterial efficiency against anthracnose after the 4-day inoculation (Figure

289

1b). Interestingly, with the particle size mounted from 44.2 nm to 83.8 nm, the

290

inhibition of mycelial growth was significantly decreased from 93% to 72%.

291

Furthermore, in 7 days, the mycelium gradually turned into gray, rather than black

292

after being treated with ELG-NPs (Figure 1), suggesting that ELG-NPs with smaller 13



293

size suppressed the growth and sporulation of anthracnose more prominently. Herein,

294

there was a negative correlation between particle size and antibacterial efficiency in

295

our study. This result was in agreement with the previous reports.7 The observed

296

size-dependent bioactivity of NPs appears to be a commonly phenomenon within

297

various biological systems,28 especially when cell interactions and tissue permeability

298

were involved.29 This may be ascribed to the increased surface area of NPs with

299

smaller diameter and improved penetrability through the tight junction or adventitial

300

cells. As considering the composition limits, such as maximum ethanol concentration,

301

ELG-NPs with a smallest size (d=44.2 nm) was constructed with 30% ethanol were

302

used in the following work.

303

Antibacterial activity of ELG-NPs. The eugenol evaporation method was used to

304

assess the antibacterial activity and minimum inhibitory concentration (MIC) of

305

eugenol and ELG-NPs. Antibacterial filter films were prepared by incorporating

306

various concentrations (15 - 95 μl/L) of eugenol. The difference in the mycelium

307

diameter of inhibition zone indicates the sensitivity of bacterial strains to filter film

308

with free-eugenol or ELG-NPs. In particular, with higher levels (>87 μl/L) of

309

free-eugenol and ELG-NPs inhibited the growth of bacteria completely. It is

310

interesting that the MIC value of ELG-NPs against bacteria was slightly lower than

311

that of free eugenol (Table S2). This can be attributed to the diffusion speed of

312

eugenol from the particle spheres to specific microbiological environment.

313

Specifically, the antibacterial ability of ELG-NPs was compromised, compared with

314

that of free form of eugenol, at the beginning of antibacterial reactions.3,30 14


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Nevertheless, the nano-encapsulated eugenol showed a sustained release profile for a

316

longer duration, and hence brought about more effective inhibiting effects on the

317

growth of pathogenic bacteria than free eugenol.

318

Furthermore, the antibacterial effects of ELG-NPs were evaluated by calculating

319

the inhibition rate on spore germination and mycelium growth of four fruit pathogen

320

bacteria. As shown in Figure 2a, after 30 h of incubation, the spore germination

321

inhibition rates (%) of unencapsulated eugenol mixture on Colletotrichum musae,

322

Colletotrichum fragariae, Colletotrichum gloeosporioides Penz, and Colletotrichum

323

gloeosporioides were only 83.71%, 82.90%, 86.89% and 83.72%, respectively.

324

However, the inhibition rates of ELG-NPs were all significantly increased to 95.23%,

325

90.08%, 89.43%, and 94.19%, respectively (Figure 2a). Meantime, we performed the

326

inhibition analysis on mycelium growth in the agarose plate by incorporating

327

ELG-NPs or unencapsulated eugenol mixture (final concentration = 0.26 μl/mL). As

328

shown in Figure 2b, ELG-NPs markedly suppressed the mycelium growth of all four

329

bacteria after 6 days in culture, with the inhibitory rates at 96.43%, 93.56%, 94.88%

330

and 93.54%, respectively. Notably, the inhibitiory rates of ELG-NPs against four

331

bacteria were 35.90%, 50.12%, 42.44%, and 32.88% higher than that of

332

unencapsulated eugenol, respectively. These results demonstrated that eugenol

333

embedded into nanoethosomal system by ultrasonic treatment exhibited obviously

334

enhancement effect on its antibacterial capacity. Previous reports have clearly

335

demonstrated that eugenol alters membrane permeability and fluidity due to its

336

lipophilic/hydrophobic characteristics15, resulting in the disintegrating and disturbance 15



337

capability of membrane, and finally induces the cell lysis.8,31 Besides, nanoethosomal

338

formulation had better water-solubility and stability.18 Therefore, the eugenol

339

nanoethosomes with smaller particle sizes and better solubility were positively

340

correlated with the permeability through cell membrane into the bacteria to enhance

341

the antibacterial capacity. In addition, the encapsulation system exerted obvious

342

controlled-release behavior, thereby extending the duration and extent of microbial

343

inhibition effect.31,32

344

In the present study, our interest was to determine if eugenol nanoparticles could be

345

employed to suppress the anthracnose of fruits caused by bacterial strains. We aimed

346

to test and compare the inhibiting effects of both the free and encapsulated eugenol on

347

the loquat anthracnose caused by C. gloeosporioides Penz. 10 ul spore suspension

348

(1x105 cells/mL) was injected into loquat epidermis. Then the infected loquats were

349

incubated in insulated boxes with the volatile eugenol from free or eugenol

350

nanoparticles incorporated filter film, respectively. Compared with the control group,

351

after being inoculated with free eugenol or its mixture for 4 d and 6 d, the lesion

352

diameters of loquats treated with C. gloeosporioides Penz were 1.59 and 1.87 cm,

353

4.21 and 3.81 cm, respectively. Meanwhile, the lesion diameters in the control fruit

354

were 3.45 cm (t = 4 d) and 4.39 cm (t = 6 d). In contrast, the lesion diameters of

355

loquats in the ELG-NP treatment were only 0.33 cm (t = 4 d) and 0.74 cm (t = 6 d)

356

(Figure 3a). In fact, both the free eugenol and eugenol mixture showed nearly 100%

357

inhibition efficiency on bacteria at the first 2 days, but the inhibiting effect decreased

358

steeply at the later period (Figure 3a). It was speculated that without any interfacial 16


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protection, the free eugenol molecules could volatilize fastly, and resulted in the

360

effective inhibition on C. gloeosporioides Penz for only a short period. On contrary,

361

with the ethosomal shell around the eugenol molecules, the degradation and

362

volatilization behaviors are minimized.17,33 Consequently, a prolonged antibacterial

363

activity of ELG-NPs was observed. Notably, the anthracnose of loquats in ELG-NPs

364

treatment groups was almost controlled after 6 d (Figure 3b). It suggested that C.

365

gloeosporioides Penz cells were more susceptible to eugenol, and this strain might be

366

persistently suppressed by eugenol in the whole period. Overall, although the decay

367

degree of fruit treated with free eugenol was much less than those in the control group,

368

the desease incidence reached 100% after 6 d (Figure 3c). However, the anthracnose

369

inoculated in postharvest loquat fruit was significantly suppressed by ELG-NPs, with

370

almost no decay occurred (Figure 3d). These results indicated that ethosomal

371

nanoparticle significantly potentiates the antibacterial efficacy of eugenol in inhibiting

372

anthracnose in loquat fruit. Thus, ELG-NPs may provide a promising and potential

373

application on the control of anthracnose in postharvest fruits.

374

Penetration ability assay. The permeability of particles is the most important

375

aspects in determining the antibacterial effectiveness because it has direct relevance

376

with the drug delivery, bioavailability, cellular uptake, in vivo distribution, and drug

377

release of nanomedicines.12,34 It was found that there was a size-dependent

378

antibacterial activity of ELG-NPs (Figure 1). To further validate the delivery efficacy

379

of the nanoethosomal system, the permeating properties of ELG-NPs (44.2 nm) were

380

assessed across the banana cortex. 17



381

To better evaluate the permeability of nanoparticles, the banana cortex with

382

relatively uniform thickness (~3 mm) was chosen as a barrier to investigate the

383

antibacterial activity of drugs from the outer surface. C. musae spores were inoculated

384

into the endodermis, and then unencapsulated or encapsulated eugenol was uniformly

385

smeared on the surface of bananas. After incubation for 2 d, bananas treated with

386

unencapsulated eugenol reduced the decay process of spots caused by anthracnose

387

compared with those untreated fruits (Figure 4a). As expected, in the ELG-NP group,

388

the anthracnose was significantly suppressed and almost no lesion occurred on the

389

pulp of banana. This is probably due to the enhanced permeability of ELG-NPs

390

nanoparticles through the cortex, which resulted in a higher local concentration of

391

eugenol in cortex, and consequently enhanced the antibacterial activity of this type of

392

poorly soluble drug.12,35 After 4 d, the lesion diameters with the free eugenol or

393

mixture treated were 29.5 and 27.5 mm (Figure 4b), respectively, which showed no

394

significant differences compared with the control group (30.5 mm). However, the

395

lesion diameter of ELG-NP-treated bananas was only 3.5 mm. These results indicated

396

that ELG-NPs significantly suppressed the fruit pathogenic bacteria hided in the deep

397

pericarp by increasing the permeation of loaded eugenol.

398

To directly visualize the particle permeation across cortex, Nile red was doped into

399

particles as a tracer. As seen in Figure 5a, we depict representative examples of

400

fluorescence microscopy images of vertically cross-sectioned pericarp following

401

topical application of the nanoparticles. The spatial variations of the above three

402

eugenol formulations showed significant differences in banana pericarp penetration. 18


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403

The fluorescence intensity clearly showed that a majority of unencapsulated eugenol

404

and the mixture remained clumping on the outer surface of the pericarp, and there was

405

almost no distribution of eugenol within the deep pericarp. In contrast, visual results

406

showed that eugenol nanoparticles penetrated deeper and a mass of eugenol reached

407

the pulp interface (Figure 5a). Despite of the penetrability can be clearly identified by

408

broader fluorescence dispersion into the pericarp material; the ratings did not consider

409

the amount of eugenol delivering to the margin. Therefore, the difference in eugenol

410

penetrated

411

chromatography coupled with triple quadrupole tandem mass spectrometry (GC/MS).

412

As shown in Figure 5b, the permeation of eugenol from unencapsulated eugenol

413

(0.0235 ug/g) and the mixture (0.0241 ug/g) were less in comparison with ELG-NPs

414

(0.0277 ug/g) in the outer pericarp. Furthermore, the amounts of eugenol present into

415

the inner pericarp from unencapsulated eugenol, mixture and ELG-NPs were found to

416

be approximately 0.0006, 0.0008 and 0.0043 ug/g, respectively (Figure 5b). This

417

result was also in line with the spatial analysis data obtained using Nile red as a tracer

418

(Figure 5a). The amount of eugenol accumulated in the closest-to-pulp layer from

419

ELG-NPs was predominantly higher (6-7 folds) than that of the unencapsulated ones.

420

The improved penetration extent of eugenol through the pericarp from ELG-NPs may

421

be attributed to the fact that the formation of nanoethosomes brought about a fine

422

range of particle size and less surface tension,14,36 which has been shown to be helpful

423

in improving the permeation effects of nanoparticles.37 The small particle size and

424

hydrophobic and hydrophilic units of nanoethosomes facilitate the penetrating process

amount

was

further

determined

quantitatively

19


by

using

gas


425

through the hydrophobic stratum waxy layer as well as the hydrophilic endodermis.

426

Additionally, the ethanol in nanoethosomes causes disturbance of cortex lipid bilayer

427

organization, hence leading to a rapid increase in the permeability due to an alteration

428

in the barrier properties and a greater degree of diffusion of the eugenol into the

429

pericarp.38,39 Although other studies have highlighted the potential of encapsulation

430

system in essential oils for antibacterial effects,3 for example, improving both

431

solubility and permeability through pathogenic bacteria membrane.9,40 Unfortunately,

432

decay of fresh fruits can easily happen after being contaminated by the pathogenic

433

bacteria. As those bacteria typically hide within the pericarp interior, the antibacterial

434

efficiency of eugenol is difficult to harness because of the existence of pericarp

435

barrier.41 To the best of our knowledge, this is the first report that provides clear

436

evidences of nanoethosomal permeation across fruit pericarp.

437

Analysis of sustained -release effects. During the fruit and vegetable preservation,

438

essential oils were commonly sprayed onto the surfaces. In this case, due to the high

439

volatility of essential oils, the protective effects and antibacteria efficacy usually

440

dissipate quickly.42 This problem could be tackled by developing sustained

441

formulations to keep the active ingredients retention for longer time. Thus, the release

442

of the encapsulated eugenol from the ELG-NPs was characterized to determine

443

whether this formulation would provide the desired depot delivery for antibacterial

444

effect. As shown in Figure 6a, after exposure to air for 0-10 h, the antibacterial

445

activity of remaining eugenol from unencapsulated- and encapsulated-ones was

446

determined on the mycelium diameter of inhibition (7 days of incubation) by 20


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447

evaporation method. Compared to the control, the free-eugenol and mixtures groups

448

showed only 31% and 46% mycelium inhibition activity after exposure to air for 4 h,

449

respectively. In contrast, ELG-NPs exhibited 100% inhibition rates and slowly

450

declined to 37% with the increased exposure times from 4 h to 8 h (Figure 6a).

451

Obviously, the difference in volatility capability of eugenol had relevant in fluence on

452

microbial growth. Burst effect release pattern usually occurs with unmodified eugenol,

453

and resulted in rapid dissipation at first 4 h. The bacterial inhibition effect of

454

ELG-NPs was stronger indicating better control of the release process due to the

455

nanoemulsion formulation strategy adopted.

456

To dynamically investigate the relevance of the antibacterial efficiency and eugenol

457

release process, C. fragariae was chosen as the representive microorganism to

458

determine the inhibition effect of spore germination in liquid medium between

459

unencapsulated- and encapsulated ones. A relative growth inhibition effect of >92%

460

was observed with the addition of free eugenol at the first 12 h, and slight higher

461

inhibition effect of >95% for cell growth with eugenol mixtures treatment may be due

462

to the effects of organic solvent components (Figure 6b). After 24 h cultures, the

463

antibacterial effects of unencapsulated-eugenol disappeared entirely, while ELG-NPs

464

remained the sustained inhibition effectiveness with more than 96% at 24 h (Figure

465

6b). This antibacterial efficiency of encapsulated-eugenol decreased slightly and

466

eventually disappeared from 24-42 h. In contrast, unencapsulated-eugenol groups

467

fleetly lost all the antibacterial activity form 12-24 h, which may be due to a rapid loss

468

of eugenol in media. Therefore, the remaining content of eugenol was determined by 21



469

GC-MS. As shown in Figure 6c, in the unencapsulated-eugenol and mixture groups

470

possessed remaining eugenol contents of 1.12 and 1.32 mg/mL, respectively in the

471

first 4 h, and the eugenol consumed in 4 h was about 80% of the initial amount (5 mg).

472

Notably, the higher amount of eugenol remaining in media containing

473

encapsulated-eugenol nanoparticles ELG-NPs was 4.41 mg/mL, with only 11.8% of

474

eugenol consumed in the first 4 h. This result was further clearly confirmed that free

475

eugenol usually occurs the burst effect dissipation and resulted in the short-lived

476

antibacterial effect. The remaining eugenol contents of ELG-NPs group showed a

477

slow descent in the culture media, dropping from 88.2% at 4 h to 20% at 24 h, but

478

significantly higher than that of unencapsulated-eugenol and mixture groups, which

479

both rapidly consumed the remaining eugenol from 20% to

Enhancement in Antibacterial Activities of Eugenol-Entrapped

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