Subscriber access provided by WESTERN SYDNEY U
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 36
Journal of Agricultural and Food Chemistry
1
Enhancement in antibacterial activities of eugenol-entrapped
2
ethosome nanoparticles via strengthening its permeability and
3
sustained-release
4 5 6 7
Peng Jin, Rui Yao, Dingkui Qin, Qing Chen, Qizhen Du*
8
The Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang Province, The
9
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
25
The College of Agricultural and Food Sciences, Zhejiang A & F University, Linan
26
311300, China. Tel.: +86-571-15958126861; Fax: +86-571-88218710; Email:
27
[email protected] (Q. Du)
28 29 30 31 32 33 1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
34
ABSTRACT
35
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
42
exhibited greater antibacterial activity (>93%) against fruit pathogens than that of
43
free-eugenol, showed 100% inhibition of the anthracnose incidence in postharvest
44
loquat after 6-d. The permeability study firstly visualized in banana cortex with
45
fluorescent indicators, demonstrated that eugenol delivered to the interior with
46
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
49
areas.
50
KEY WORDS: Eugenol; Nanoethosomes; Anthracnose; Antibacterial activity;
51
Permeability; Postharvest diseases
52 53 54 55 2
ACS Paragon Plus Environment
Page 2 of 36
Page 3 of 36
Journal of Agricultural and Food Chemistry
56
INTRODUCTION
57
Fresh fruits and vegetables are susceptible to anthracnose caused by pathogens
58
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
62
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
72
phytosterols6 are reported to have significant antimicrobial, antiviral, and (or)
73
antioxidant potentials due to the presence of saponins, flavonoids, ketones, and
74
aldehydes.
75
(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
ACS Paragon Plus Environment
essential
oil,
eugenol
Journal of Agricultural and Food Chemistry
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
81
water-solubility, and highly susceptibility to environmental stress.10 The embedding
82
and encapsulation technology is a novel method that can markedly improve the
83
physicochemical stability of actively volatile agents.11-13 In the recent past, many
84
studies have also revealed that encapsulation of eugenol into nanoparticles could
85
enhance its stability, permeability, and antimicrobial activity.14-16
86
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
88
scientific and industrial interest. Specific studies have been focused on the use of
89
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
91
remain at the upper layer of the stratum corneum.19 As most of the pathogens inhabit
92
the deeper cortex layers of fruits and vegetables, it is commonly agreed that these
93
conventional formulations are not suitable as carriers for transdermal drug delivery.20
94
Herein, ethosomes may provide a promising strategy to deliver eugenol and similar
95
bioactive agents. Typically, ethosomes are modified forms of liposomes, which are
96
composed of phospholipids and relatively high concentration of ethanol and
97
water–they have been constructed for the administration of both hydrophilic and
98
lipophilic drugs.21 Ethanol is able to aid ethosomes in squeezing through the pores and
99
permeating deeper into cortex layers, and hence releasing transdermal flux drugs into 4
ACS Paragon Plus Environment
Page 4 of 36
Page 5 of 36
Journal of Agricultural and Food Chemistry
100
deeper layers more efficiently than conventional nano-carriers.22 Nevertheless, to our
101
knowledge, there are no studies using nanoethosomal carriers to encapsulate eugenol
102
for fruit preservation and antibacterial purposes; moreover, it is necessary to verify
103
the enhanced penetrability through fruit cortex and the consequent drug delivery
104
efficiency of encapsulated eugenol.
105
In this study, to investigate the effect of nanoethosomes particle size on the
106
antibacterial activity, we prepared various sizes of nanoethosomes by optimizing the
107
proportion of eugenol and ethanol. The antibacterial effects and the anthracnose
108
incidence in fresh loquat fruit were compared between the groups treated with
109
nanoethosomal or natural eugenol. Furthermore, the penetrability across cortex and
110
sustained-release effects of nanoparticles were also assessed by the dye tracer and
111
GC-MS analyses. This work provides an insight into the enhancement of eugenol
112
antibacterial activities by nanoethosomal system, suggesting that it is a suitable and
113
efficient carrier for eugenol encapsulation and delivery. The information gained from
114
this study may be useful for designing various nanoethosomal delivery systems to
115
fight against postharvest diseases caused by specific bacteria.
116
MATERIALS AND METHODS
117
Chemicals and reagents. Eugenol, Tween 80, and soyabean lecithin were
118
purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA). All Other reagents are
119
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 6 of 36
121
were purchased from Hangzhou JinHeLai Food Additive CO. LTD (Hangzhou,
122
China).
123
Preparation of nanoparticles. Ethanol, tween-80, and eugenol were mixed in
124
sealed conical flask, then soybean lecithin and SEFA were added and dissolved in the
125
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
127
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
132
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
141
Samples were prepared by firstly diluted and placed on a film-coated 200-mesh
electron
microscopy
(TEM)
was
6
ACS Paragon Plus Environment
used
for
morphological
Page 7 of 36
Journal of Agricultural and Food Chemistry
142
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,
147
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
175
was then inoculated with 10 μL of spore suspensions of C. gloeosporioides Penz. The
176
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
182
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
ACS Paragon Plus Environment
Page 8 of 36
Page 9 of 36
185
Journal of Agricultural and Food Chemistry
0.05 was considered statistically significant.
186
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
188
sterile injector with an oblique angle of 30°. Then equivalent eugenol (0.5%) of free
189
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
192
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 10 of 36
207
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
222
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
ACS Paragon Plus Environment
0.25 mm 0.25 um,
Page 11 of 36
Journal of Agricultural and Food Chemistry
228
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 12 of 36
Page 13 of 36
Journal of Agricultural and Food Chemistry
271
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
274
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 14 of 36
Page 15 of 36
Journal of Agricultural and Food Chemistry
315
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 16 of 36
Page 17 of 36
Journal of Agricultural and Food Chemistry
359
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 18 of 36
Page 19 of 36
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
by
using
gas
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 20 of 36
Page 21 of 36
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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