Enhancement of Antifungal Activity of Juglone (5-Hydroxy-1,4

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Enhancement of Antifungal Activity of Juglone (5-Hydroxy-1,4Naphthoquinone) Using PLGA Nanoparticles System Tulin Arasoglu, Banu Mansuroglu, Serap Derman, Busra Gumus, Busra Kocyigit, Tayfun Acar, and Ismail Kocacaliskan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03309 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 7, 2016

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 32

Journal of Agricultural and Food Chemistry

(A) The particle size distribution of JNP5 179x134mm (300 x 300 DPI)

ACS Paragon Plus Environment


(B) The zeta-potential distribution of JNP5 184x131mm (300 x 300 DPI)


Page 2 of 32

Page 3 of 32


(C) The SEM image of JNP5 76x58mm (220 x 220 DPI)



(D) time-dependent release pattern of free juglone (square) and JNP5 (circle) 237x177mm (300 x 300 DPI)


Page 4 of 32

Page 5 of 32


(E) FT-IR spectrums of Juglone, free NP and JNP5. 741x584mm (96 x 96 DPI)



According to agar dilution method, the petri images of growth inhibition effects of juglone-PLGA nanoparticles and free juglone due to MIC values of JNP5 (250 µg/mL, 31.25 and 62.5 for A. flavus, C. albicans and Fusarium spp. respectively ) in the agar dilution method. (a) control, (b) JNP5, (c) Free juglone 137x140mm (96 x 96 DPI)


Page 6 of 32

Page 7 of 32


The petri images of growth inhibition effects of juglone-PLGA nanoparticles and free juglone due to MIC values of JNP5 (125 µg/mL, 62.5 and 31.25 for A. flavus, C. albicans and Fusarium spp. respectively ) in the top agar dilution method (a) control, (b) JNP5, (c) Free juglone 145x140mm (96 x 96 DPI)



Graphical abstract 357x152mm (96 x 96 DPI)


Page 8 of 32

Page 9 of 32


1

Enhancement of Antifungal Activity of Juglone (5-Hydroxy-1,4-Naphthoquinone) Using

2

PLGA Nanoparticles System

3 4 5

Tulin Arasoglu†, Banu Mansuroglu†, Serap Derman‡,*,

6

Busra Gumus†, Busra Kocyigit†, Tayfun Acar‡ and Ismail Kocacaliskan†,

7 8

†

9

Department, 34220, Istanbul/Turkey

Yildiz Technical University, Science and Letters Faculty, Molecular Biology and Genetics

10

‡

11

34220, Istanbul/Turkey

Yildiz Technical University, Chemical and Metallurgy Faculty, Bioengineering Department,

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 1 ACS Paragon Plus Environment


27

ABSTRACT

28 29

In this study, it was aimed to synthesize and characterize juglone entrapped PLGA

30

nanoparticle and compare the antifungal properties of free juglone with its PLGA nanoparticle

31

formulation for the first time in the literature. The juglone loaded nanoparticles prepared

32

using the oil-in-water (o-w) single-emulsion solvent evaporation method were characterized

33

by the reaction yield (RY), encapsulation efficiency (EE), polydispersity index (PDI), particle

34

size, zeta potential (ZP), FT-IR, in vitro release properties and evaluated their morphological

35

features using SEM. The nanoparticle formulation had a size, RY, ZP, EE and PDI value of

36

212 nm, 66.91 ± 2.4 %, −16.3 ± 0.7 mV, 70.66 ± 3.1 % and 0.083 ± 0.024 respectively. In

37

vitro release showed triphasic pattern with initial burst followed by sustained release and

38

dormant phase over the study period, total releasing about 72.8 % after 42 days. The

39

antifungal studies against A.flavus, C. albicans and Fusarium spp. using agar dilution and top

40

agar dilution methods indicated that the juglone encapsulated nanoparticle was more effective

41

than free juglone. In this study, it is showed that the top agar method which applied for the

42

first time on the antifungal activity is more suitable for the nanoparticular system based on

43

sustained release. So that, the PLGA nanoparticle formulations may be an important tool for

44

application in many areas from the point of the effective and beneficial use for hydrophobic

45

compounds such as juglone.

46 47

Keywords

48

5-hydroxy-1,4-naphthoquinone, Juglone, nanoparticle, antifungal activity

49 50 51

2 ACS Paragon Plus Environment

Page 10 of 32

Page 11 of 32

52


INTRODUCTION

53 54

Allelopathy is direct or indirect, harmful or beneficial effect of a plant on other plants, insects

55

and microbes due to chemical compounds synthesized and secreted into the environment.

56

These compounds are called as allelochemicals and they can usually be found in every tissue

57

of a plant, especially in leaves and roots. These compounds are released into the environment

58

through volatilization, water contact, exudation from root and plant residues. Allelochemicals

59

can have effects on cell division, respiration, photosynthesis, membrane permeability, protein

60

synthesis and seed germination 1, 2.

61

Juglone is an allelochemical that is located in the naphthoquinone subgroup within the

62

quinones, and a secondary metabolite produced by the walnut tree (Juglandaceae)3. Juglone is

63

selectively toxic towards some animals, microorganisms and plants and has been used for

64

many other purposes through long ages

65

other organisms but not the plant itself, Duroux et al. reported that this compound is also

66

necessary for the plant development7. Besides, it was reported that juglone has anticancer 8, 9,

67

cytotoxic and genotoxic

68

antifungal

69

poor dissolution property due to its hydrophobic character.

70

In order to provide sustained release of active substances, polymers that convert into nontoxic

71

metabolites are usually preferred

72

polyglycolic acid (PGA) and copolymer of these, poly (d,l-lactic-co-glycolic acid) (PLGA)

73

are most commonly used to synthesize active substance encapsulated nanoparticle systems to

74

provide sustained release 26-30. PLGA, one of the FDA approved biopolymers used frequently

75

for drug delivery systems, was an appropriate carrier to prepare nanoparticle systems with

76

substances soluble in water or oil 31. In recent years, the use of nanoparticle systems produced

20-24

10, 11

4-6

. Although it was thought that juglone only affects

, antioxidant

9, 12-15

, antiviral

16

, antibacterial

8, 9, 14, 17-20

and

effects. However, its application and bioavailability is limited because of its

25

. Biodegradable polymers such as polylactic acid (PLA),



Page 12 of 32

77

by encapsulating antimicrobial agents into PLGA biopolymer has become very prevalent.

78

Thus;

79

biocompatibilities of these agents increased remarkably in comparison with the free form of

80

the same drug 32, 33. Additionally, nanotechnology studies on developing antifungal agents are

81

still in the early stages and there is an urgent need for the development of eco-safe fungicides.

82

Bionanomaterials are a promising alternative with high efficacy and relevant cost34. Due to

83

their high surface to volume ratio and small size; they can surpass the physiological barriers,

84

reach the target and interact well with the pathogens. Although the introduction of

85

nanomaterials to the field is recent, it has already been agreed that the use of bionanomaterials

86

are a necessity for defeating resistant pathogenic organisms 35.

87

In the literature, researches about the antifungal activity of juglone compound are few in

88

number; but instead, a large number of studies published about walnut tree extracts. In our

89

research, we aimed to encapsulate juglone molecule into PLGA nanoparticle system for the

90

first time and to evaluate its antifungal activity comparatively with free juglone. It should be

91

stated that this is the first research in the literature about the antifungal activity of juglone

92

encapsulated PLGA nanoparticles. To assess this antifungal activity, two different methods

93

were used and compared against Aspergillus flavus, Candida albicans and Fusarium spp.

it

was

stated

that

pharmacokinetics

properties,

therapeutic

indexes

and

94 95

METHODS

96

Preparation of Nanoparticles

97

In this study, juglone, PVA, PLGA (lactide:glicolide = 50:50; inherent viscosity 0.45-0.60

98

dL/g, Mw ~ 38-54 kDa P50/50), and DCM were purchased from Sigma Aldrich (St. Louis,

99

USA). The modified single emulsion solvent evaporation method was used for preparation of 36, 37

100

Juglone loaded PLGA nanoparticles which described by our previous studies

101

Juglone (50 mg/mL) and PLGA (40 mg/mL) were firstly dissolved in water immiscible DCM,


. The

Page 13 of 32


102

mixed together then suitably stirred to ensure that all materials were dissolved. This mixture

103

was emulsified with aqueous PVA solution (3 % w/v), over an ice bath, using microtip probe

104

sonicator (output power 100 W, power of % 80 and 2 minutes, Bandelin Sonopuls, Germany).

105

The obtained o/w single emulsion was then diluted in 35 mL of PVA solution (0.1 % w/v) and

106

left on magnetic stirring for overnight at room temperature. The nanoparticles were collected

107

by centrifugation at 12.000 x rpm for 40 minutes (Beckman Coulter, Allegra-X-30R) and

108

washed three times with ultra-pure water then the obtained suspensions were lyophilized. The

109

free nanoparticles were prepared with similar method without using Juglone and lyophilized

110

nanoparticles were stored at -80 °C until used.

111

Characterization of Nanoparticles

112

The reaction yield and encapsulation efficiency of nanoparticles were determined in triplicate.

113

The reaction yield was calculated gravimetrically as follow 36.

114

% =

115

The indirect quantification method was used to analyze encapsulation efficiency of

116

nanoparticles using UV-Vis Spectroscopy at 424 nm in triplicate. The juglone concentration

117

in the supernatant was determined by using previously constructed standard calibration curve.

118

The encapsulation efficiency of juglone was calculated as follow:

119

% =

120

Both zeta potential (ζ) and mean diameter of particles were analyzed using a Zetasizer

121

(Zetasizer Nano ZS, Malvern, UK) instrument with electrophoretic light scattering (ELS) and

122

dynamic light scattering (DLS) techniques, respectively 38.

123

Morphology and FT-IR Study of Nanoparticles

124

Scanning electron microscopy (SEM, A JSM-7001FA, Jeol, Japan) was used to observe the

125

shape and surface morphology of produced nanoparticles as previously described in our

126

studies

100

100

36

(1)

(2)

. Lyophilized nanoparticles were fixed on metallic studs using adhesive tapes and 5 ACS Paragon Plus Environment


Page 14 of 32

127

then coated with gold under vacuum. SEM photomicrographs were taken at an acceleration

128

voltage of 10–30 kV.

129

FT-IR (Fourier Transform Infrared Spectroscopy) measurements were carried out by IR-

130

Prestige 21 FTIR spectrophotometer (Shimadzu, Japan) in ATR mode

131

ranging between 600 cm-1 and 4000 cm-1 were obtained with resolution of 4 cm−1.

132

Release Study

133

The in vitro drug release study was carried out in triplicate using modified dissolution method

134

36

135

saline) and incubated 37 °C in a shaking incubator (150 rpm) at pH 7.4. At specified time

136

interval, the samples were centrifuged at 9000 rpm for 20 minutes, then the supernatant was

137

collected and the precipitates were suspended with 2 mL of fresh PBS. The juglone

138

concentration in the supernatant was determined with UV-Vis Spectroscopy at 424 nm by

139

comparing the concentration to a previously constructed standard calibration curve.

140

Determination of Antifungal Activities of Free Juglone and Synthesized Nanoparticles

141

The antifungal activity of free juglone and juglone-PLGA-NPs were assessed by the agar

142

dilution method, according to the recommendations of the National Committee for Clinical

143

Laboratory Standards 39, and top agar dilution method which was applied for the first time in

144

this study. Two methods were performed separately for Candida albicans (ATCC 10231),

145

Aspergillus flavus (ATCC 204304) and Fusarium spp.(ATCC 20883) (provided by

146

Bacteriological Laboratory, Faculty of Science, at Ataturk University, Erzurum, Turkey).

147

Three different fungi isolates were subcultured at 27 oC on Sabouraud Dextrose Agar with

148

2 % dextrose (SDA- from Dıfco, BD Diagnostic Systems, United States) until good growth

149

was obtained. Spores were harvested in 5 mL Sabouraud Dextrose Broth (SDB- from Dıfco,

150

BD Diagnostic Systems, United States) and the tubes were incubated at 27 oC for three days.

151

After the incubation period, the tubes were centrifuged at 5000 rpm for 8 minutes. The pellet

36

. The FTIR spectra

. Juglone-PLGA-NPs (5 mg) were suspended in 2 mL of 150 mM PBS (phosphate buffer


Page 15 of 32


152

was washed three times in 0.01 M sodium phosphate buffer (pH 7.4), resuspended in PBS.

153

The spore suspensions in buffer were then adjusted spectrophotometrically to 78-82 %

154

transmittance for C. albicans and 68-70 % transmittance for Fusarium spp. and A. flavus at a

155

wavelength of 600 nm 40. These suspensions were used in the top agar method.

156

Agar Dilution Method

157

This method involves the incorporation of varying desired concentrations of the antifungal

158

agent into a molten agar medium, followed by the inoculation of fungal cultures onto the agar

159

plate surface. The minimum inhibition concentration (MIC) value was determined as the

160

lowest concentration of antifungal agent that inhibits growth after incubation time. The

161

minimum fungicidal concentration (MFC) was regarded as the lowest concentration doesn’t

162

yield any fungal growth on the solid medium used. Briefly, appropriate amounts of the free

163

juglone and the juglone-PLGA-NPs were dissolved in sterile distilled water and added

164

aseptically into sterile molted SDA medium, ending up with a concentration range of 15.33-

165

500 µg/mL. The resulting SDA mediums were immediately poured into Petri plates after

166

vortexing. Small blocks of agar (2x2 mm) from growing fungal cultures were cut and placed

167

on the SDA plates. The inoculated plates were incubated at 27 oC for seven days. At the end

168

of the incubation period, the plates were evaluated for the presence or absence of fungal

169

growth. In this study, two control plates were used: the first one containing only fungal

170

culture, the latter containing empty nanoparticles and fungal culture. Each test was repeated

171

three times.

172

Top Agar Dilution Method

173

Top agar dilution method has been developed by modeling AMES method

174

(Salmonella/microsome assay) for this study. In this method, the fungal spores were directly

175

exposed to the test chemical for a certain time in a sterile tube containing buffered medium.

176

Then, it was spread over the surface of SDA after adding the molten top agar into the same


41, 42


177

tube. The most important point in the use of this method is the direct interaction between the

178

test substance and the tested organism for a short period or preincubation time. The antifungal

179

activity was examined according to the fungal growth on the plates.

180

In the top agar method; the stock solutions were prepared by dissolving of the free juglone

181

and the juglone-PLGA-NPs in sterile distilled water (1.38 mg/mL for the free juglone; 5

182

mg/mL for the juglone-PLGA-NPs, containing the same amount of active ingredient with free

183

juglone). Six different concentrations for each material were prepared (500 µg/mL, 250

184

µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL, 15.33 µg/mL) and distributed to the sterile

185

tubes. After that, 100 µL of the fungal cultures (prepared as mentioned above) was added into

186

the tubes and the tubes were preincubated for 30 minutes. Finally, the tubes were shaken

187

gently by adding 2 mL of molten top agar containing 0.5 mM NaCl and the mixtures were

188

poured and spreaded quickly onto the plates containing SDA. After incubation at room

189

temperature for 7 days, the antifungal activity was evaluated based on the presence or absence

190

of fungal growth. Two control plates were included in each test run. One control plate

191

contained only fungal culture; and the other one contained the free PLGA nanoparticles

192

dissolved in sterile distilled water, in addition to the molten top agar. Each test was repeated

193

three times.

194 195

RESULTS

196

Nanoparticle Preparation and Characterization

197

The o/w single-emulsion solvent evaporation method was used for the preparation of free and

198

juglone loaded nanoparticles. The juglone-PLGA-NPs were analyzed for R.Y., E.E., Z-Ave,

199

PDI and Z.P.


Page 16 of 32

Page 17 of 32


200

After weighing of nanoparticles, the reaction yield was calculated to 50.47 ± 2.6 % and 66.91

201

± 2.4 % for free nanoparticles and juglone-PLGA-NPs, respectively. The encapsulation

202

efficiency of Juglone was calculated as 70.66 ± 3.1 % using indirect method.

203 204

Table 1. R.Y., E.E., Z-Ave, PDI and Zeta potential of nanoparticles (JNP5)

205

Particle Size, Zeta Potential and Polydispersity of Nanoparticles

206

The particle size and zeta potential of nanoparticles were measured using dynamic and

207

electrophoretic light scattering, respectively. Figure 1 shows particle size (A) and zeta

208

potential (B) distribution of juglone-PLGA-NPs. The Z-average of nanoparticles were

209

measured as 183.1 ± 1.35 nm and 212.2 ± 4.65 nm for free nanoparticles and juglone-PLGA-

210

NPs, respectively. It was seen that the mean diameter of juglone-PLGA-NPs increased up to

211

212.2 ± 4.65 nm by encapsulation of juglone. Both Z-Ave of the nanoparticles were measured

212

less than 220 nm in diameter with monodisperse size distribution. As known, the smaller

213

particle size (100-250 nm) is required for the high cellular uptake of nanoparticles

214

formulations were part of a single population (100 % intensity) and had low values of

215

polydispersity (0.092 ± 0.020, and 0.083 ± 0.024) which indicates the uniformity of particle

216

size.

217

Results of the zeta potential measurement of nanoparticles were shown in Table 1. Free

218

nanoparticles and juglone-PLGA-NPs showed negative zeta potential values which indicate

219

the stable state of nanoparticles owing to electrostatic repulsion 44. In the present study, free

220

juglone and juglone-PLGA-NPs had -23,6 ± 1.6 mV and -16.3 ± 0.7 mV, respectively due to

221

the use of nonionic emulsifier PVA

222

negative zeta potential provides the nanoparticle suspensions to be stable by preventing the

223

agglomeration of nanoparticles 36, 47.

45

43

. All

and the terminal carboxylic group of polymer 46. This

224 9 ACS Paragon Plus Environment


225

Figure 1. (A) The particle size distribution of JNP5, (B) zeta-potential distribution of JNP5,

226

(C) SEM image of JNP5, (D) time-dependent release pattern of free juglone (square) and

227

JNP5 (circle) and (E) FT-IR spectrums of Juglone, free NP and JNP5.

228 229

Morphology and FT-IR Study of Nanoparticles

230

The image of juglone-PLGA-NPs was observed using SEM as shown in Figure 1 C. The

231

nanoparticles had smooth texture of surface morphology, spherical topography and uniform

232

size distributions without any agglomeration. The SEM image is in good agreement with the

233

result measured by DLS, indicates clearly that the produced nanoparticles had narrow size

234

distribution.

235

The FT-IR spectra of juglone, free nanoparticles and juglone loaded PLGA nanoparticles

236

were demonstrated in the Figure 1E. In the FT-IR spectra, absorption peaks are assigned to

237

specific functional groups. The pure juglone showed the main characteristic peak in the region

238

of 1665-1630 cm-1 due to carbonyl groups (C=O) and the other important peaks are C=C

239

stretching that is observed at 1592 cm-1 and C-O stretching which is observed at 1288 cm-1. It

240

is understood from the spectra of free NPs and JNP5 that the 1750 cm-1 peak is corresponding

241

to C=O group and the peaks between 1250-1100 cm-1 is due to C-O stretch of the PLGA

242

polymer. When the spectra of free NPs and JNP5 are compared, it is seen that specific

243

functional groups in the surface of JNP5 have almost the same chemical characteristics of the

244

free nanoparticles. The FT-IR spectra show that juglone was successfully encapsulated to

245

PLGA nanoparticles without having any physical or chemical interaction.

246

Release Study

247

On the other hand, the release study was performed in 150 mM PBS (pH 7.4). The results

248

presented in Figure 1 D show that free juglone was dissolved in the media within 48 h period.

249

Whereas, juglone loaded PLGA nanoparticles showed sustained triphasic release pattern up to

250

42 days. In vitro release profile of juglone-PLGA-NPs over 40 days shows that after 4 h, 10 ACS Paragon Plus Environment

Page 18 of 32

Page 19 of 32


251

approximately 30 % of juglone had been released from nanoparticle which is indicative of

252

initial burst release. Then, in the intermediate phase, the released juglone amount had

253

increased up to 69.9 % within the following 10 days. Next, the release became very slow and

254

the release curve slope reaches to a plateau corresponding to a maximum 72.8 % at the end of

255

42 days.

256

Antifungal Activity of Free Juglone and Synthesized Nanoparticles

257

In the agar dilution method, the antifungal activity of free juglone and juglone-PLGA-NPs

258

were evaluated on the basis of fungal growth. The MIC and MFC values are shown in Table

259

2. It was observed that the PLGA nanoparticle systems caused a remarkable decrease in MIC

260

values against C. albicans, Fusarium spp. Among the three fungal species, the lowest MIC

261

value of the juglone encapsulated PLGA nanoparticles were 31.25 µg/mL, while the

262

lowest MIC value of the free juglone was found 62.5 µg/mL against C. albicans. The MIC

263

value of the free juglone was determined as 125 µg/mL for Fusarium spp., whereas it was

264

twofold higher for the juglone-PLGA-NPs. According to the MFC values, the free juglone

265

was higher four times for C. albicans, two times for Fusarium spp than its nanoparticle

266

formulations. In the case of A. flavus, the MIC and MFC values for the juglone encapsulated

267

PLGA nanoparticles was obtained as 250 µg/mL, as did for the free juglone.

268 269

Table 2. The MFC and MIC values according to agar dilution method

270 271

Figure 2 shows the Petri images of the growth inhibition ratios between juglone-PLGA-NPs,

272

free juglone and control groups according to the MIC values detected for JNP5 by agar

273

dilution method against all strains. As shown in this figure, the growth difference between

274

juglone-PLGA-NPs and the free juglone for C. albicans and Fusarium spp. was slightly



275

observed. Also, this difference appears to be much higher for both groups compared with the

276

control.

277 278

Figure 2. According to agar dilution method, the Petri images of growth inhibition effects of

279

juglone-PLGA nanoparticles and free juglone due to MIC values of JNP5 (250 µg/mL, 31.25

280

µg/mL and 62.5 µg/mL for A. flavus, C. albicans and Fusarium spp respectively ) in the agar

281

dilution method. (a) control, (b) Juglone-PLGA-NPs, (c) Free juglone

282 283

The top agar dilution method was performed by adding the molten top agar on fungal spores

284

being exposed directly to juglone and its nanoparticle formulation in the buffered medium for

285

a short time. In this method, the decrease in visible fungal growth was interpreted as

286

antifungal activity or fungicidal activity. According to our results, the MIC and MFC value of

287

free juglone was higher four times for C. albicans, four times for Fusarium spp. and two

288

times for A. flavus, when compared to the nanoparticle formulations (Table 3). The empty

289

NPs were evaluated as the control group showed no effect in both studies (data not shown).

290 291

Table 3. The MFC and MIC values according to top agar dilution method

292 293

In the Figure 3, strong visual evidence of growth inhibitiory effect was observed in the

294

juglone-PLGA-NPs exposed to Petri plates compared with both free juglone and control

295

groups in top agar dilution method.

296 297

Figure 3. The Petri images of growth inhibition effects of juglone-PLGA nanoparticles and

298

free juglone due to MIC values of JNP5 (125 µg/mL, 62.5 µg/mL and 31.25 µg/mL for A.


Page 20 of 32

Page 21 of 32


299

flavus, C. albicans and Fusarium spp. respectively) in the top agar dilution method (a)

300

control, (b) JNP5, (c) Free juglone

301 302

DISCUSSION

303

Fungal organisms cause a significant problem in health sector by developing antifungal

304

resistance mechanisms recently and in food industry by decreasing the food quality, quantity

305

and storage time 3. Therefore, there is an urgent need for exploring new antifungal agents that

306

can be used in both sectors. When the harmful effects of synthetically produced chemical

307

agents in human health considered, the demand for natural antifungal agents obtained from

308

plants increases gradually

309

antifungal agents 49. PLGA nanoparticle systems are widely used to improve the efficacy and

310

the impact time of natural bioactive compounds. Considering this situation, PLGA

311

nanoparticle systems are widely used to improve the efficacy and the impact time of natural

312

bioactive compounds in recent years

313

example gene therapy, targeted drug delivery, and the effective usage of active agents such as

314

seconder metabolites and pharmaceutical drugs. In antifungal treatments, a major concern

315

with currently available antifungal drugs is the low systemic bioavailability arising from low

316

aqueous solubility and early drug degradation. Keeping this in perspective, the present study

317

was aimed that the juglone loaded PLGA nanoparticle systems were successfully synthesized

318

using the oil-in-water single-emulsion solvent evaporation method, characterized in detailed

319

and evaluated its antifungal activity in comparison with free juglone. The antifungal activity

320

of the synthesized JNP5 was also evaluated by two different methods against Aspergillus

321

flavus, Candida albicans and Fusarium spp. for the first time in literature.

322

Juglone is a naphthoquinone synthesized in walnut tree and the antifungal properties of this

323

compound have been known for many years 3. When literature reviewed; it was observed that

48

. Also, plants are excellent resources for the supply of new

50

. Applications of PLGA-NPs are greatly broad, for



Page 22 of 32

51-53

324

the antifungal activity research about naphthoquinone derivatives are predominant

325

there are only a few studies about the juglone molecule 20. Although it was reported in these

326

research that juglone has an apparent antifungal activity, differences found between the MIC

327

values that was thought to be caused by reasons such as methods selected, solvents, juglone

328

source and fungal strains.

329

Furthermore; based on our knowledge, there was no reported data about the synthesis and

330

characterization of the juglone loaded PLGA nanoparticles and the evaluation of the

331

antifungal activity of the juglone encapsulated nanoparticles, as well. Therefore, in our study,

332

the antifungal activity of the juglone-PLGA NPs was evaluated for the first time in literature.

333

According to the our results, the juglone loaded PLGA nanoparticles proved to be more

334

effective in inhibiting fungal growth, when compared to free juglone by both methods used.

335

In former studies, it has also been shown that amphotericin b 54, voriconazole 55 and ITZ 56, 57

336

entrapped PLGA nanoparticles showed more potent antifungal efficacy than their free

337

formulations congruently. In our study, one of the main reason behind the enhancement in

338

antifungal activity may be the solubility increase of the hydrophobic juglone molecule which

339

provided by the encapsulation into the nanoparticle system.

340

Another important reason may be existence of chitin that is the main component of the fungal

341

cell wall structure. β-1,4- linked N-acetylglucosamine unites of the chitin provide a strong

342

positive charge to fungal cells. PLGA nanoparticles, due to their negative charge, form an

343

electrostatic interaction with the fungal cell walls. This situation provides the access of the

344

nanoparticles to the cell surfaces, their accumulation and later on, the entrance of the released

345

active substances or the nanoparticles into the cells and therefore increases the antifungal

346

activity by forming an electrostatic attraction between the organisms and the PLGA-NPs

347

Besides, the hydrophobins secreted by fungal organisms form an amphipathic membrane

348

structure and so provide contact between the hydrophilic medium and hydrophobic agents


, but

58

.

Page 23 of 32


57

349

such as juglone due to their hydrophobic and hydrophilic regions

350

limited and the drug activity is low. In contrast to that; because of their hydrophilic structure,

351

the juglone loaded PLGA nanoparticles interact well with the fungal surfaces and reveal a

352

stronger growth inhibitory effect.

353

As reported in previous researches, it is easier for small sized particles to enter the cells and

354

the size of the juglone-PLGA nanoparticles synthesized in this study (212.2 ± 4.65 nm) is

355

within the range described as «small sized nanoparticle» in the literature 43, 57, 59. In addition to

356

these; despite the fact that the initial dose of the free juglone provides growth inhibition, drug

357

concentration decreases based upon the consumption or degradation by the spores as time

358

progresses. On the contrary, nanoparticle systems burst in first 4 hours (the moment when the

359

most of the active substance is released) and then continue to release active material in the

360

process of time. This continuous exposure prevents the regrowth of fungal spores and that

361

way provides a longer effect 57.

362

In the present study; we examined the antifungal activity of the free juglone and its

363

nanoparticle formulation by using two different methods: agar dilution and top agar dilution.

364

The effect of the time dependent release of the active material on the fungal organisms was

365

observed more specifically in the top agar dilution method due to the interaction occurred

366

between the fungal organisms and nanoparticles in the liquid medium for a while (30 minutes

367

– preincubation time). Moreover, it was thought that the effect of the pH change -caused by

368

this interaction- on the surface charges, has an effect again on the interaction itself as well 60.

369

It was also thought that the interaction time may be important for the method’s efficacy.

370

Because of that, further research will be performed to evaluate the correlation between the

371

antifungal activity and the interaction time in detail. Since the top agar dilution method allows

372

using far less material than the agar dilution method, it is planned to perform validation


. Still, this interaction is


373

studies to improve the methodology and provide the efficient usage of the method in release-

374

based nanoparticle systems.

375

In conclusion, the juglone’s antifungal activity improved by the entrapment into the PLGA

376

nanoparticle system has been shown visually in Petri plates of agar dilution and top agar

377

dilution methods (Figure 2 and 3). Moreover; in this study, more effective results were found

378

in the top agar dilution method which was used to detect the antifungal activity for the first

379

time in literature when compared to the agar dilution method. A stronger inhibition effect on

380

the fungal growth was observed clearly for the juglone-PLGA-NPs than the free juglone

381

molecules by the top agar method. This suggests increased sensitivity for the top agar dilution

382

method in the antimicrobial and antifungal activity studies of the PLGA nanoparticle systems,

383

particularly. Additionally, to provide a more detailed explanation of the enhanced antifungal

384

effect detected in this study, we suggest performing further in vivo and in vitro studies on the

385

cellular uptake mechanisms and the interactions between the juglone-PLGA-NPs and fungal

386

species.

387 388

ACKNOWLEDGEMENTS

389

The authors wish to express their thanks to TUBITAK for supplying the financial support

390

(project numbers; 114Z093). Also, the authors are grateful to Prof. Dr. Medine Güllüce and

391

Assistant Professor Dr. Zeynep Mustafaeva Akdeste for providing laboratory facilities.

392 393

AUTHOR INFORMATION

394

Corresponding Author;

395

Serap DERMAN (Ph.D)

396

Telephone: +090 212 483 4643 E-mail: [email protected]

397


Page 24 of 32

Page 25 of 32


398

Funding

399

This research has been supported by TUBITAK, project numbers; 114Z093.

400

Notes

401

The authors declare no competing financial interest.

402 403

REFERENCES

404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440

1. Seigler, D. S., Chemistry and mechanisms of allelopathic interactions. Agronomy Journal. 1996. 88, 876-885. 2. Halbrendt, J., Allelopathy in the management of plant-parasitic nematodes. Journal of Nematology. 1996. 28, 8. 3. Strugstad, M.; Despotovski, S., A summary of extraction, synthesis, properties, and potential uses of juglone: A literature review. Journal of Ecosystems and Management. 2013. 13,1-16. 4. Kocacaliskan, I.; Terzi, I., Allelopathic effects of walnut leaf extracts and juglone on seed germination and seedling growth. The Journal of Horticultural Science and Biotechnology. 2001. 76, 436-440. 5. Terzi, I.; Kocaçalişkan, I.; Benlioğlu, O.; Solak, K., Effects of juglone on growth of cucumber seedlings with respect to physiological and anatomical parameters. Acta Physiologiae Plantarum. 2003. 25, 353-356. 6. Topal, S.; Kocacaliskan, I.; Arslan, O.; Tel, A. Z., Herbicidal effects of juglone as an allelochemical. Phyton. 2007. 46, 259-269. 7. Duroux, L.; Delmotte, F. M.; Lancelin, J.-M.; Keravis, G.; Christian, J.-A., Insight into naphthoquinone metabolism: β-glucosidase-catalysed hydrolysis of hydrojuglone β-Dglucopyranoside. Biochemical Journal. 1998. 333, 275-283. 8. Xu, H.; Yu, X.; Qu, S.; Sui, D., Juglone, isolated from Juglans mandshurica Maxim, induces apoptosis via down-regulation of AR expression in human prostate cancer LNCaP cells. Bioorganic & Medicinal Chemistry Letters. 2013.23, 3631-3634. 9. Zakavi, F., Antibacterial effect of Juglansregia bark against oral pathologic bacteria. International Journal of Dentistry. 2013. 2013, 1-5. 10. Kiran Aithal, B.; Sunil Kumar, M.; Nageshwar Rao, B.; Udupa, N.; Satish Rao, B., Juglone, a naphthoquinone from walnut, exerts cytotoxic and genotoxic effects against cultured melanoma tumor cells. Cell Biology International. 2009. 33, 1039-1049. 11. Paulsen, M. T.; Ljungman, M., The natural toxin juglone causes degradation of p53 and induces rapid H2AX phosphorylation and cell death in human fibroblasts. Toxicology and Applied Pharmacology. 2005. 209, 1-9. 12. Chobot, V.; Hadacek, F., Milieu-dependent pro-and antioxidant activity of juglone may explain linear and nonlinear effects on seedling development. Journal of Chemical Ecology. 2009. 35, 383-390. 13. Yiğit, D.; Yiğit, N.; Aktaş, E.; Özgen, U., Ceviz (Juglans regia L.)'in antimikrobiyal aktivitesi. Türk Mikrobiyol Cem Derg. 1993. 39,7-11. 14. Tan, D. T. C.; Osman, H.; Mohamad, S.; Kamaruddin, A. H., Synthesis and Antibacterial Activity of Juglone Derivatives. Journal of Chemistry and Chemical Engineering. 2012. 6, 84-89.



441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490

15. Altikat, S.; Terzi, I.; Kuru, H.; Kocacaliskan, I., Allelopathıc Effects Of Juglone On Growth Of Cucumber And Muskmelon Seedlıngs Wıth Respect To Antıoxıdant Enzyme Actıvıtıes And Lıpıd Peroxıdatıon. Journal Of Envıronmental Protectıon And Ecology. 2013. 14, 1244-1253. 16. Montenegro, R. C.; Araújo, A. J.; Molina, M. T.; Rocha, D. D.; Lopéz-Montero, E.; Goulart, M. O.; Bento, E.; Alves, A. P. N. N.; Pessoa, C.; de Moraes, M. O., Cytotoxic activity of naphthoquinones with special emphasis on juglone and its 5-methyl derivative. Chemico-Biological Interactions. 2010. 184, 439-448. 17. Dama, L.; Poul, B.; Jadhav, B., Antimicrobial activity of naphthoquinonic compounds. Journal of Ecotoxicology & Environmental Monitoring. 1998. 8, 213-215. 18. Albayrak, A. Ceviz Yaprak Özütleri ve Juglonun Bazı Mikroorganizmalar Üzerine Etkileri. Yüksek Lisans Tezi. Dumlupınar Üniversitesi, Kütahya, 2006. 19. Oliveira, I.; Sousa, A.; Ferreira, I. C.; Bento, A.; Estevinho, L.; Pereira, J. A., Total phenols, antioxidant potential and antimicrobial activity of walnut (Juglans regia L.) green husks. Food and Chemical Toxicology. 2008. 46, 2326-2331. 20. Clark, A. M.; Jurgens, T. M.; Hufford, C. D., Antimicrobial activity of juglone. Phytotherapy Research. 1990. 4, 11-14. 21. Bonjar, G. S.; Aghighi, S.; Nik, A. K., Antibacterial and antifungal survey in plants used in indigenous herbal-medicine of south east regions of Iran. Journal of Biological Sciences. 2004. 4, 405-412. 22. Bonjar, G. S., Anti yeast activity of some plants used in traditional herbal medicine of Iran. J Biol Sci. 2004. 4, 212-5. 23. Nahrstedt, A.; Vetter, U.; Hammerschmidt, F., Composition of the steam distillation product from leaves of Juglans regia [walnuts]. Planta Medica (Germany, FR). 1981. 24. Sharma, N.; Ghosh, P.; Sharma, U. K.; Sood, S.; Sinha, A. K.; Gulati, A., Microwaveassisted efficient extraction and stability of juglone in different solvents from Juglans regia: quantification of six phenolic constituents by validated RP-HPLC and evaluation of antimicrobial activity. Analytical Letters. 2009. 42, 2592-2609. 25. Hans, M.; Lowman, A., Biodegradable nanoparticles for drug delivery and targeting. Current Opinion in Solid State and Materials Science. 2002. 6, 319-327. 26. Lemoine, D.; Francois, C.; Kedzierewicz, F.; Preat, V.; Hoffman, M.; Maincent, P., Stability study of nanoparticles of poly (ɛ-caprolactone), poly (d, l-lactide) and poly (d, llactide-co-glycolide). Biomaterials. 1996. 17, 2191-2197. 27. Akagi, T.; Baba, M.; Akashi, M., Biodegradable Nanoparticles as Vaccine Adjuvants and Delivery Systems: Regulation of Immune Responses by Nanoparticle-Based Vaccine. Adv. Polym. Sci. 2011. 1-34. 28. Jiang, W.; Gupta, R. K.; Deshpande, M. C.; Schwendeman, S. P., Biodegradable poly (lactic-co-glycolic acid) microparticles for injectable delivery of vaccine antigens. Advanced Drug Delivery Reviews. 2005. 57, 391-410. 29. Mohamed, F.; Van Der Walle, C. F., Engineering biodegradable polyester particles with specific drug targeting and drug release properties. Journal of Pharmaceutical Sciences. 2008. 97, 71-87. 30. Kumari, A.; Yadav, S. K.; Yadav, S. C., Biodegradable polymeric nanoparticles based drug delivery systems. Colloids and Surfaces B: Biointerfaces. 2010. 75, 1-18. 31. Çırpanlı, Y. Kamptotesin Đçeren Polimerik Ve Oligosakkarit Bazlı Nanopartiküler Formülasyonların Geliştirilmesi Ve Đn Vitro-Đn Vivo Değerlendirilmesi. Hacettepe Üniversitesi, Ankara, 2009. 32. Soppimath, K. S.; Aminabhavi, T. M.; Kulkarni, A. R.; Rudzinski, W. E., Biodegradable polymeric nanoparticles as drug delivery devices. Journal of Controlled Release. 2001. 70, 1-20. 18 ACS Paragon Plus Environment

Page 26 of 32

Page 27 of 32

491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540


33. Desai, M. P.; Labhasetwar, V.; Amidon, G. L.; Levy, R. J., Gastrointestinal uptake of biodegradable microparticles: effect of particle size. Pharmaceutical research. 1996. 13, 1838-1845. 34. Abd-Elsalam, K.; Alghuthaymi, M., Nanobiofungicides: are they the Next-Generation of Fungicides?. J Nanotech Mater Sci. 2015. 2, 1-3. 35. Blecher, K.; Nasir, A.; Friedman, A., The growing role of nanotechnology in combating infectious disease. Virulence. 2011. 2, 395-401. 36. Arasoglu, T.; Derman, S.; Mansuroglu, B., Comparative evaluation of antibacterial activity of caffeic acid phenethyl ester and PLGA nanoparticle formulation by different methods. Nanotechnology. 2015. 27, 025103. 37. Derman, S.; Akdeste, Z. M., Particle size and zeta potential investigation of synthetic peptide-protein conjugates/Sentetik peptid-protein konjugatlarının parçacık boyutu ve zeta potensiyel analizi. Turkish Journal of Biochemistry. 2015. 40, 282-289. 38. Derman, S.; Mustafaeva, Z. A.; Abamor, E. S.; Bagirova, M.; Allahverdiyev, A., Preparation, characterization and immunological evaluation: canine parvovirus synthetic peptide loaded PLGA nanoparticles. Journal of Biomedical Science. 2015. 22, 1-12. 39. Institute, C. A. L. S., Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Ninth Edition. 2012. In M07-A9, 32, 2 USA. 40. Al-Hatmi, A. M.; Meletiadis, J.; Curfs-Breuker, I.; Bonifaz, A.; Meis, J. F.; De Hoog, G. S., In vitro combinations of natamycin with voriconazole, itraconazole and micafungin against clinical Fusarium strains causing keratitis. Journal of Antimicrobial Chemotherapy .2015. doi:10.1093/jac/dkv421. 41. Ozbek, T.; Gulluce, M.; Agar, G.; Adiguzel, A.; Ozkan, H.; Baris, O.; Sahin, F., Antimutagenic activities of methanol extracts of some endemic Astragalus species evaluated by Ames Salmonella/Microsomal Test. Asian Journal of Chemistry. 2009. 21, 451-458. 42. Mortelmans, K.; Zeiger, E., The Ames Salmonella/microsome mutagenicity assay. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 2000. 455, 2960. 43. Win, K. Y.; Feng, S.-S., Effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticancer drugs. Biomaterials. 2005. 26, 2713-2722. 44. Gaur, P. K.; Mishra, S.; Bajpai, M.; Mishra, A., Enhanced oral bioavailability of efavirenz by solid lipid nanoparticles: in vitro drug release and pharmacokinetics studies. BioMed Research International. 2014. 2014, 1-9. 45. Zigoneanu, I. G.; Astete, C. E.; Sabliov, C. M., Nanoparticles with entrapped αtocopherol: synthesis, characterization, and controlled release. Nanotechnology. 2008. 19, 105606. 46. Turk, C. T. S.; Oz, U. C.; Serim, T. M.; Hascicek, C., Formulation and optimization of nonionic surfactants emulsified nimesulide-loaded PLGA-based nanoparticles by design of experiments. AAPS PharmSciTech. 2014. 15, 161-176. 47. Derman, S., Caffeic Acid Phenethyl Ester Loaded PLGA Nanoparticles: Effect of Various Process Parameters on Reaction Yield, Encapsulation Efficiency, and Particle Size. Journal of Nanomaterials. 2015. 2015,1-6. 48. Das, K.; Tiwari, R.; Shrivastava, D., Techniques for evaluation of medicinal plant products as antimicrobial agents: Current methods and future trends. Journal of Medicinal Plants Research. 2010. 4, 104-111. 49. Sardi, J.; Scorzoni, L.; Bernardi, T.; Fusco-Almeida, A.; Giannini, M. M., Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. Journal of Medical Microbiology. 2013. 62, 10-24. 19 ACS Paragon Plus Environment


541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574

50. Li, Z.; Jiang, H.; Xu, C.; Gu, L., A review: Using nanoparticles to enhance absorption and bioavailability of phenolic phytochemicals. Food Hydrocolloids. 2015. 43, 153-164. 51. Sasaki, K.; Abe, H.; Yoshizaki, F., In vitro antifungal activity of naphthoquinone derivatives. Biological and Pharmaceutical Bulletin. 2002. 25, 669-670. 52. Errante, G.; La Motta, G.; Lagana, C.; Wittebolle, V.; Sarciron, M.-É.; Barret, R., Synthesis and evaluation of antifungal activity of naphthoquinone derivatives. European Journal of Medicinal Chemistry. 2006. 41, 773-778. 53. Tandon, V. K.; Singh, R. V.; Yadav, D. B., Synthesis and evaluation of novel 1, 4naphthoquinone derivatives as antiviral, antifungal and anticancer agents. Bioorganic & Medicinal Chemistry Letters. 2004. 14, 2901-2904. 54. Jung, S. H.; Lim, D. H.; Jung, S. H.; Lee, J. E.; Jeong, K.-S.; Seong, H.; Shin, B. C., Amphotericin B-entrapping lipid nanoparticles and their in vitro and in vivo characteristics. European Journal of Pharmaceutical Sciences. 2009. 37, 313-320. 55. Peng, H.-s.; Liu, X.-j.; Lv, G.-x.; Sun, B.; Kong, Q.-f.; Zhai, D.-x.; Wang, Q.; Zhao, W.; Wang, G.-y.; Wang, D.-d., Voriconazole into PLGA nanoparticles: Improving agglomeration and antifungal efficacy. International Journal of Pharmaceutics. 2008. 352, 29-35. 56. Cunha-Azevedo, E. P.; Silva, J. R.; Martins, O. P.; Siqueira-Moura, M. P.; Bocca, A. L.; Felipe, M. S. S.; Tedesco, A. C.; Azevedo, R. B., In vitro antifungal activity and toxicity of itraconazole in DMSA-PLGA nanoparticles. Journal of nanoscience And Nanotechnology. 2011. 11, 2308-2314. 57. Patel, N. R.; Damann, K.; Leonardi, C.; Sabliov, C. M., Itraconazole-loaded poly (lactic-co-glycolic) acid nanoparticles for improved antifungal activity. Nanomedicine. 2010. 5, 1037-1050. 58. Patel, N. R.; Damann, K.; Leonardi, C.; Sabliov, C. M., Size dependency of PLGAnanoparticle uptake and antifungal activity against Aspergillus flavus. Nanomedicine. 2011. 6, 1381-1395. 59. Rejman, J.; Oberle, V.; Zuhorn, I. S.; Hoekstra, D., Size-dependent internalization of particles via the pathways of clathrin-and caveolae-mediated endocytosis. Biochemical Journal. 2004. 377, 159-169. 60. Dimkpa, C. O.; McLean, J. E.; Britt, D. W.; Anderson, A. J., Antifungal activity of ZnO nanoparticles and their interactive effect with a biocontrol bacterium on growth antagonism of the plant pathogen Fusarium graminearum. Biometals. 2013. 26, 913-924.

575 576 577 578 579 580 581 582 20 ACS Paragon Plus Environment

Page 28 of 32

Page 29 of 32

583


Tables

584 585

Table 1. R.Y., E.E., Z-Ave, PDI and Zeta potential of nanoparticles (JNP5)

586 Name

R.Y %

E.E %

Z-Ave (nm)

PDI

Z.P (mV)

Free NP

50.47 ± 2.6

-

183.1 ± 1.35

0.092 ± 0.020

-23.6 ± 1.6

212.2 ± 4.65

0.083 ± 0.024

-16.3 ± 0.7

Juglone NP

66.91 ± 2.4 70.66 ± 3.1

587 588 589

Table 2. The MFC and MIC values according to agar dilution method

Test samples

MFC values (µg/mL)

MIC values (µg/mL)

Fungal organisms

Fungal organisms

A. flavus C. albicans Fusarium spp. A. flavus C. albicans Fusarium spp.

Juglone-NPs

500

62.5

125

250

31.25

62.5

Free juglone

500

250

250

250

62.5

125

590 591

Table 3. The MFC and MIC values according to top agar dilution method

Test samples

MFC values (µg/mL)

MIC values (µg/mL)

Fungal organisms

Fungal organisms

A. flavus C. albicans Fusarium spp. A. flavus C. albicans Fusarium spp.

Juglone-NPs

250

125

62.5

125

62.5

31.25

Free juglone

>500

500

250

250

250

125

592 593 594 595 596 597 598 21 ACS Paragon Plus Environment


599

Figures

600

601

602

603 604 605

Figure 1. (A) The particle size distribution of JNP5, (B) zeta-potential distribution of JNP5,

606

(C) SEM image of JNP5, (D) time-dependent release pattern of free juglone (square) and

607

JNP5 (circle) and (E) FT-IR spectrums of Juglone, free NP and JNP5. 22 ACS Paragon Plus Environment

Page 30 of 32

Page 31 of 32


608

609 610

Figure 2. According to agar dilution method, the petri images of growth inhibition effects of

611

juglone-PLGA nanoparticles and free juglone due to MIC values of JNP5 (250 µg/mL, 31.25

612

and 62.5 for A. flavus, C. albicans and Fusarium spp respectively ) in the agar dilution

613

method. (a) control, (b) JNP5, (c) Free juglone

614



615 616

Figure 3. The petri images of growth inhibition effects of juglone-PLGA nanoparticles and

617

free juglone due to MIC values of JNP5 (125 µg/mL, 62.5 and 31.25 for A. flavus, C. albicans

618

and Fusarium spp respectively ) in the top agar dilution method (a) control, (b) JNP5, (c) Free

619

juglone

620


Page 32 of 32

Enhancement of Antifungal Activity of Juglone (5-Hydroxy-1,4

Recommend Documents