Bioactive Constituents, published by the Metabolites,isand American Chemical 1155 Functions Society. Sixteenth Street N.W., Washington, DC 20036
BioactivityPublished by American Chemical Society. Subscriber access metabolite provided by©University Copyright American of guided Michigan-Flint Chemical Society. However, no copyright
profiling of feijoa (Acca sellowiana) is published by the American Chemical Society. 1155 cultivars identifies 4Sixteenth Street N.W., Washington, DC 20036 cyclopentene-1,3Published by American Chemical Society. dione as a potent Subscriber access provided by University of
Copyright © American ChemicalMichigan-Flint Society. However, no copyright
antifungal inhibitor of chitin synthesis is published by the American Chemical
Mona Mokhtari, Michael Society. 1155 Sixteenth Street N.W., Jackson, AlistairWashington, Brown,DCDavid 20036 Published by AmericanA Ackerley, Nigel Ritson, Robert Chemical Society. Keyzers, and Andrew Subscriber access provided byMunkacsi Copyright ©University American of ChemicalMichigan-Flint Society. However, no copyright
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): is 16published Mar 2018 by the American Chemical
DownloadedSociety. from http:// 1155 pubs.acs.org onSixteenth March Street 16, 2018 N.W., Washington, DC 20036 Published by American Chemical Society. Subscriber access provided by©University Copyright American of ChemicalMichigan-Flint Society. However, no copyright
Just Accepted
published by the “Just Accepted” is manuscripts have been pe American Chemical online prior to technical editing, formatting Society. 1155 Street N.W., as a serv Society providesSixteenth “Just Accepted” Washington, DC 20036 of scientific material asbysoon as possible Published American Chemical Society. full in PDF format accompanied by an HT Subscriber access provided by University of Copyright © American ChemicalMichigan-Flint Society. However, no copyright
peer reviewed, but should not be conside Digital Object Identifier (DOI®). “Just Acc is published the may not inc the “Just Accepted” Webbysite American Chemical a manuscript is Society. technically edited and fo 1155 Sixteenth N.W., site and published as Street an ASAP article. Washington, DC 20036 to the manuscript text and/or graphics w Published by American Chemical Society.
Subscriber access provided by©University Copyright American of ChemicalMichigan-Flint Society. However, no copyright
ethical guidelines that apply to the journ consequences arising from the use of in is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Subscriber access provided by©University Copyright American of ChemicalMichigan-Flint Society. However, no copyright
PageJournal 1 of 36of Agricultural and Food Chemistry
CH2OH H
Multivariate analysis of metabolites from 16 feijoa cultivars
H OH H
H
O O H
H NHCOH3
CH2OH
O H OH H
H
O
H NHCOH3
ACS Paragon Plus Environment 4-cyclopentene1,3-dione
Antifungal activity via chitin synthesis inhibition
Journal of Agricultural and Food Chemistry
1
Bioactivity-guided metabolite profiling of feijoa (Acca sellowiana) cultivars identifies 4-
2
cyclopentene-1,3-dione as a potent antifungal inhibitor of chitin synthesis
Mona Mokhtari1, Michael D. Jackson1, Alistair S. Brown1, David F. Ackerley1,2, Nigel J. Ritson3, Robert A. Keyzers2,4,§ and Andrew B. Munkacsi1,2,§,*
1
School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand
2
Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand
3
Foretaste Feijoa Fruit Ltd., Takaka, New Zealand
4
School of Chemical and Physical Sciences, Victoria University of Wellington, Wellington,
New Zealand §
Co-senior author
*
Corresponding author
1 ACS Paragon Plus Environment
Page 2 of 36
Page 3 of 36
Journal of Agricultural and Food Chemistry
3
ABSTRACT
4
Pathogenic fungi continue to develop resistance against current antifungal drugs. To explore
5
the potential of agricultural waste products as a source of novel antifungal compounds, we
6
obtained an unbiased GC-MS profile of 151 compounds from 16 commercial and
7
experimental cultivars of feijoa peels. Multivariate analysis correlated 93% of the compound
8
profile with antifungal bioactivity. Of 18 compounds that significantly correlated with
9
antifungal activity, five had not previously been described from feijoa. Two novel cultivars
10
were the most bioactive, and 4-cyclopentene-1,3-dione detected in these cultivars was
11
potently antifungal (IC50 = 1-2 µM) against human pathogenic Candida species.
12
Haploinsufficiency and fluorescent microscopy analyses determined that the synthesis of
13
chitin, a fungal cell wall polysaccharide, was the target of 4-cyclopentene-1,3-dione. This
14
fungal-specific mechanism was consistent with 22-70-fold reduction in antibacterial activity.
15
Overall, we identified the agricultural waste product of specific cultivars of feijoa peels as a
16
source of potentially high value antifungal compounds.
17
KEYWORDS: antifungal / chitin synthesis / cyclopentenedione / feijoa / metabolite profiling
18 19 20 21 22 23
2 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
24 25
INTRODUCTION
26
Nature has been a rich source for pharmaceutical compounds, underlying 80% of our
27
currently prescribed drugs.1 In particular, many noteworthy drugs in the history of human
28
civilization have been derived from plants (e.g., morphine and aspirin to treat pain and
29
headaches, artemisinin to treat malaria). Just as there is a potential epidemic due to bacteria
30
developing resistance to current antibiotics and delays in the development of new antibiotics,2
31
the same applies to pathogenic fungi. In recent years, the limited nature of our current arsenal
32
of antifungal drugs has been manifest in the resistance of pathogenic fungi to current
33
antifungal drugs, increases in rates of fungal diseases in immunocompromised patients (e.g.,
34
chemotherapy, transplant, and HIV patients), and increases in the reports of new pathogenic
35
species.3 Like antibiotics, most antifungal drugs have historically been based on natural
36
products isolated or derived from cultured microbes. However, given that plants are
37
consistently regulating infections by fungi (i.e., permitting infection by mutualistic fungi and
38
resisting infection by pathogenic fungi), extracts of plants including fruits are also a
39
promising source of novel antifungal compounds.4
40
Feijoa (Acca sellowiana, Myrtacaeae) is native to Southern Brazil, Northern
41
Argentina and Western Paraguay, where it is also known as pineapple guava. It is actively
42
cultivated as an annual multi-million dollar (>$8M USD) agricultural crop in temperate and
43
subtropical climates in the United States of America, South America, Europe, Australia and
44
New Zealand. The pulp of the fruit (Figure 1) is consumed in its entirety or as a juice, as well
45
as in food products such as ice cream, chutney, chocolate, and wine. The fruit pulp has a
46
gritty texture with a distinctive highly perfumed odor and a flavor that is is sweet to mid-sour.
47
Bioactive compounds with anticancer, antioxidant, antiviral, anti-inflammatory, anti-diabetic,
48
probiotic and antibacterial activities have been isolated from the leaves, pulp and peels of
3 ACS Paragon Plus Environment
Page 4 of 36
Page 5 of 36
Journal of Agricultural and Food Chemistry
49
commercial cultivars of the thick-skinned feijoa fruit. However, antifungal compounds in
50
feijoa are dramatically understudied with only four reports to date,12-15 none of which has
51
identified mechanism of action or the bioactive compound.
52
The presence of antifungal compounds in feijoa is particularly compelling given the
53
longstanding observation by feijoa breeders that feijoa fruit is rarely penetrated by fungal
54
pathogens. There is thus great potential to explore feijoa peels for antifungal compounds and
55
turn this waste product into a high value antifungal pharmaceutical. Out of the annual global
56
feijoa crop (105,000 tons), approximately 40% is used in juice production that generates
57
approximately 42,000 tons of fejoa peel waste. This is a significant amount of waste
58
production in the feijoa industry that could potentially be used to generate additional
59
commercial gain. While there has been extensive success in the citrus industry making use of
60
citrus processing byproduct streams (e.g., the production of phytochemicals and
61
nutraceuticals such as citrus molasses, D-limonene and bioflavonoids),16 such efforts have
62
not yet been explored for feijoa.
63
Since bioactivity has been documented without reference to specific feijoa cultivars
64
and antifungal activity has not often been investigated,5-15 here we hypothesize the potential
65
of developing high value antifungal drugs from feijoa peels via compound profiling of 16
66
cultivars of feijoa including commercial and non-commercial cultivars. We correlated 151
67
volatile compounds with antifungal activity, identified two novel cultivars as those with the
68
most antifungal activity, and determined that the compound 4-cyclopentene-1,3-dione,
69
present in both of these cultivars was potently antifungal via the inhibition of chitin synthesis.
70
As the antifungal mechanism of action targets chitin, which does not exist in humans, animals
71
or plants, this antifungal compound with pharmaceutical- and agrichemical-grade potency as
72
well as specificity has the potential to turn feijoa peel waste into a high-value product.
73
4 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
74
MATERIALS AND METHODS
75
Plant Material. Fruits of 16 feijoa cultivars were grown and harvested in one location
76
(Takaka, New Zealand): Apollo, FFF5, FFF6, FFF7, FFF8, FFF9, Kakapo, Karamea,
77
Kawatiri, Opal Star, R263, Tagan, Triumph, Unique, Waingaro, and Waitui. Peels were
78
surface-sterilized with ethanol, separated from pulp with a sterile scalpel, homogenized
79
(Breville BLR50S Motiv Blender, Melbourne, Australia) at maximum speed for 5 min,
80
centrifuged at 4000 rpm for 5 min, and the pellet was used for methanolic extraction.
81
Compound Extraction. Compounds were extracted with methanol at a ratio of 700 g
82
of homogenized peel to an equal volume of methanol. The peel:methanol mixture was
83
extracted through shaking at 230 rpm at 30ºC for 24 h, and then centrifuged at 4000 rpm for 5
84
min. The supernatant was filtered through 0.22 µm filters to ensure sterility. Methanol was
85
removed using a cold trap system (Labconco, Kansas City, MO, USA), and the solid
86
methanol extract was resuspended in DMSO (at a ratio of 1 mL DMSO to 1 mL methanol)
87
and stored at -20ºC.
88
Compound Profiling. Volatile and semi-volatile compounds were analyzed on a
89
Shimadzu QP2010-Plus gas chromatograph (Shimadzu, Kyoto, Japan) fitted with a RXI-
90
5SilMS column (30 m x 0.25 mm i.d. x 0.25 µm film thickness) (Restek, Bellefonte, PA,
91
USA) and attached to an electron impact mass spectrometer operating at 70 eV in positive ion
92
mode, scanning from m/z 42-600 every 300 msec. Samples were introduced (1 µL) using an
93
AOC-20i auto-sampler with a split injection (20:1) at an injector temperature of 270°C using
94
helium as the carrier gas at a linear velocity of 43.4 cm/s (1.38 mL/min) at constant flow.
95
Each sample was injected with an initial oven temperature of 50°C for 2 min, followed by a
96
ramp at 10°C/min to 300°C with a final hold for 5 min. Compounds were identified by
97
comparison of fragmentation mass spectra with those contained in the NIST11 MS library
98
with a spectral matching similarity index (SI) >85% in at least two of three experimental
5 ACS Paragon Plus Environment
Page 6 of 36
Page 7 of 36
Journal of Agricultural and Food Chemistry
99
replicates. Compound annotation was performed using Linear Retention Indices (LRIs)
100
compared with freely accessible LRI17 (± 50 LRI units) values using similar column-types
101
while compound identification was achieved by comparison of LRI and mass spectral
102
fragmentation patterns with those of authentic standards based on the proposed minimum
103
reporting standards according to the Chemical Analysis Working Group.18 According to this
104
standard, we report compounds as the following: a, identified compound based on matched
105
mass spectrum and LRI of an authentic standard; b, annotated compound with matched LRI
106
and mass spectrum; c, annotated compound with matched mass spectrum; d, unknown.
107
Antifungal Activity and Haploinsufficiency. All strains of S. cerevisiae were
108
obtained from Open Biosystems (Huntsville, AL, USA) in the haploid BY4741 background
109
and diploid BY4743 background. Pathogenic Candida strains were obtained from American
110
Type Culture Collection (Manassas, VA, USA) and included C. albicans (ATCC 10231), C.
111
glabrata (ATCC 90030), C. parapsilosis (ATCC 90018), and C. tropicalis (ATCC 13803).
112
Fungal growth was measured in a liquid assay as previously described.19 Fungal cultures at 5
113
x 105 cells/mL were grown in Synthetic Complete (SC) media with varying concentrations of
114
feijoa extract or compound, incubated at 30ºC for 14 h, and quantified via optical density at
115
600 nm using a Perkin Elmer Envision plate reader (Perkin Elmer, Waltham, MA, USA).
116
Growth inhibition was calculated using the following formula:
117
Growth inhibition (%) = 100 – [(OD600 of treatment/OD600 of control) x 100]
118
Multivariate Statistical Analysis. Compound profiles and bioactivity were compared
119
using a partial least squares regression (PLSR) analysis incorporated in UNSCRAMBLER
120
(version X10.4, CAMO, Oslo, Norway) as previously described.20 The data were arranged in
121
an X x Y matrix, where the response variable Y (bioactivity) corresponded to growth
122
inhibition of S. cerevisiae of the 16 cultivar extracts and the predictor variable X
123
corresponded to the largest peak area for each compound. All X variables were log
6 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
124
transformed, mean centred and scaled to one standard deviation. All Y variables were mean
125
centred. The PLSR analysis used the Non-Linear Iterative Partial Least Squares (NIPALS)
126
algorithm. Compound lists for each cultivar were generated using the weighted regression
127
coefficient and validated using random cross-validation. The Martens Uncertainty Test based
128
on cross-vaidation used factors 1 and 2 to identify compounds statistically significant to the
129
model.
130
Antibacterial Activity. Growth inhibition of Bacillus subtilis (ATCC 6051), a tolC
131
mutant efflux pump-deficient Escherichia coli (W3110), an extended spectrum β-lactamase
132
Klebsiella pneumoniae (NZRM 4387), and methicillin-resistant Staphylococcus aureus
133
(MRSA; ATCC 43300) were measured in liquid media via optical density at 600 nm using an
134
Envision plate reader (Perkin Elmer, Waltham, MA, USA) as previously described.21
135
Percentage growth inhibition was then calculated using the following formula:
136
Growth inhibition (%) = 100 – [(OD600 of treatment/OD600 of control) × 100]
137
Fluorescent Microscopy. Chitin was visualized using the fluorescent stain Calcafluor
138
White (Sigma) as previously described.22 Cells were grown overnight in SC media and then
139
grown to mid-log in SC starting at a common density (OD600 = 0.1). Calcafluor White (1 g/L)
140
was added on the top of the cells and visualized after 5 min with an Evo-Tec OPERA high-
141
throughput confocal microscope (Perkin Elmer) at 60X using the DAPI filter.
142
Standards. 4-Cyclopentene-1,3-dione, β-caryophyllene, and (E)-3-hexenyl butyrate
143
were purchased from Sigma Aldrich. Flavone was isolated from natural sources and provided
144
as a gift (Stephen Bloor, Callaghan Innovation).
145 146
RESULTS
147
Bioactivity of Feijoa Cultivar Extracts. The bioactivity of feijoa extracts has been
148
examined in an undocumented set of commercial cultivars.5-15 To further understand the
7 ACS Paragon Plus Environment
Page 8 of 36
Page 9 of 36
Journal of Agricultural and Food Chemistry
149
chemical diversity that exists within the genetic diversity of feijoa, here we investigated the
150
bioactivity of ten commercial cultivars (Apollo, Kakapo, Karamea, Kawatiri, Opal Star,
151
Tagan, Triumph, Unique, Waingaro, Waitui) and six novel cultivars (FFF5, FFF6, FFF7,
152
FFF8, FFF9, R263). We quantified the efficacy of methanolic extracts derived from peels of
153
these 16 feijoa cultivars to inhibit the growth of S. cerevisiae (Baker’s yeast), an organism
154
that has extensively been used to examine bioactivity of compounds and extracts.23,24
155
Specifically, we measured the percentage of inhibition of growth of S. cerevisiae over a
156
period of 14 hours, the amount of time that it takes an untreated wild-type strain of yeast to
157
reach the mid-log phase of growth. Based on growth in the presence of a range of extract
158
concentrations, we determined that 0.01% v/v of an extract significantly reduced growth of a
159
wild-type (WT) strain of yeast (BY4741) by 18-85% compared to untreated WT (Figure 2).
160
Methanolic extracts of the commercial cultivars inhibited growth by 18-70% compared to
161
untreated cells, while those from the novel cultivars inhibited growth by 30-85% compared to
162
untreated cells. The most bioactive cultivars were the novel cultivars FFF5 and FFF6, which
163
were each significantly different from the next most bioactive cultivar Waitui (p < 0.0005).
164
These results indicate that there is extensive variation in bioactivity among feijoa cultivars as
165
well as highlighting that the greatest bioactivity is in two novel feijoa cultivars (FFF5 and
166
FFF6).
167
Compound Profiles of Feijoa Cultivars. Antifungal bioactivity has only been
168
reported in a few studies that explored crude acetone extracts and essential oils of feijoa.12-15
169
To further define the compound diversity of feijoa in terms of antifungal properties, we used
170
GC-MS to obtain unbiased metabolomic profiles in the methanolic extracts of peels from 16
171
feijoa cultivars. The identity of the volatile and semi-volatile compounds was tentatively
172
assigned by comparison of mass spectral fragmentation patterns with those of more than
173
212,000 compounds in the NIST11 database, using a cutoff of 85% spectral similarity. 8 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
174
Across the 16 feijoa cultivars, GC-MS analysis distinguished 151 compounds across all
175
cultivars, of which 68 compounds were tentatively annotated on the basis of >85% similarity
176
to a spectrum in the NIST11 database (Figure S1). Additionally, 79 compounds did not match
177
to compounds in the NIST database, and these compounds were thus classified as unknown
178
(Table S1).
179
Multivariate Analysis of Cultivars, Bioactivity and Compounds. To investigate
180
possible relationships between bioactivity and compound profiles of the 16 feijoa cultivars,
181
we conducted a PLSR analysis incorporated in the software UNSCRAMBLER that is
182
regularly used to correlate compounds and different properties.25 We correlated a large
183
number of dependent (151 compounds) and independent (bioactivity) variables for all 16
184
cultivars. The PLSR analysis generated a β-coefficiency value for each compound (ranging
185
from -1 to +1) wherein compounds with higher positive β-coefficiency values were more
186
correlated to bioactivity and negative β-coefficiency values were negatively correlated to
187
bioactivity. The β-coefficiency values were then used to build seven different models
188
(factors) to link bioactivity to the compounds. Notably, the first two factors explained 92.6%
189
of the variance (i.e., X axis is the model factor 1 that explained 73% of the data variance and
190
Y axis is model factor 2 that explained 20% of the data variance) in our data correlating
191
bioactivity and compound identity (Figure 3), a value that is consistent with results in other
192
studies using just the first two factors.26
193
Next, we sought to correlate each cultivar with bioactivity using the first two factors.
194
In agreement with Figure 2, the FFF5 cultivar was most correlated to bioactivity with FFF6
195
as the second closest cultivar, followed by Waitui and R263 (Figure 3A). Based on the β-
196
coefficiency values, these four cultivars were positively correlated with bioactivity in both
197
factor 1 and 2. In contrast, three cultivars (FFF7, Apollo, Kawatiri, FFF9) were positively
198
correlated to factor 1 and negatively correlated to factor 2, while four cultivars (FFF8, Tagan,
9 ACS Paragon Plus Environment
Page 10 of 36
Page 11 of 36
Journal of Agricultural and Food Chemistry
199
Kakapo, Karamea) were positively correlated to factor 2 and negatively correlated to factor 1.
200
Four cultivars (Unique, Opal Star, Triumph, Waingaro) were negatively correlated to both
201
factors.
202
We then sought to distinguish the compounds that were positively and negatively
203
correlated with bioactivity. The multivariate PLSR analysis determined those compounds to
204
the right side of the mid-line as being positively correlated to factor 1, while those
205
compounds to the top side of the mid-line were positively correlated to factor 2 (Figure 3B).
206
The data analysis illustrated there were 17 compounds negatively correlated to both factors 1
207
and 2, while there were 26 compounds positively correlated to both factors. To define the
208
compounds that were significantly correlated with bioactivity, we conducted further
209
statistical analyses using a Martens uncertainty test based on the first two factors in the PLSR
210
analysis. We detected 22 compounds that were significantly correlated with bioactivity (18
211
positively correlated, four negatively correlated), of which nine matched with >85% SI to
212
compounds in the NIST11 database (Table 1). Using the minimum criterion of 85% similarity
213
to the NIST11 database for identification, we tentatively annotated nine significantly
214
positively correlated compounds based on β-coefficiency: compound 26 (β-phellandrene),
215
compound 141 (flavone), compound 4 (4-cyclopentene-1,3-dione), compound 105 (ledene),
216
compound 113 (unknown), compound 95 (β-caryophyllene), compound 46 (N-methyl-N-
217
nitroso-2-propanamine), compound 20 (2H-pyran-2,6(3H)-dione) and compound 47
218
(unknown). In order of correlation based on β-coefficiency, the four significantly negatively
219
correlated compounds were compounds 52, 61, 131 and 140, none of which could be
220
annotated based on ≥85% similarity with fragmentation patterns in the NIST11 database
221
(Table S1). The results of these statistical correlations with two factors were validated using
222
cross-validation in the PLSR model. The model generates a validation rate for each factor,
223
wherein validation for the model factors was high; for the first factor (r2 = 25.1%) and this
10 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
224
was further increased with the second factor (r2 = 35.1%), results that are consistent with
225
previous reports of two factors that reliably correlated biological activity and metabolic
226
profile.26
227
Of the nine significantly positively correlated compounds tentatively identified on the
228
basis of >85% similarity to the NIST database, three were commercially available (flavone,
229
4-cyclopentene-1,3-dione, and β-caryophyllene). We obtained the GC-MS fragmentation
230
patterns and retention times of the authentic standards for these three compounds and
231
compared these profiles with the profiles of the tentatively identified compounds detected in
232
the feijoa cultivars (Table 1A). In each case the fragmentation patterns and retention times
233
were identical, confirming the identity of these three compounds.
234
Unique Compounds in Novel Feijoa Cultivars. To follow up on our identification
235
of the FFF5 and FFF6 cultivars as having the greatest bioactivity (Figure 2; Figure 3A), we
236
investigated compounds with >85% similarity to database matches that were identified only
237
in these cultivars. We therefore examined the GC-MS analyses of these cultivars and detected
238
(E)-3-hexenyl butyrate and two unknown compounds unique to the FFF5 cultivar (Table 1B),
239
while there were no unique compounds detected in FFF6. We then compared the GC-MS
240
fragmentation patterns and retention times of our tentatively identified (E)-3-hexenyl butyrate
241
from the FFF5 cultivar with those of an authentic standards, which were the same as those of
242
the standards, confirming the identity of this compound as being present only in the novel
243
FFF5 feijoa cultivar.
244
Confirmation of Compound Bioactivity. To confirm the prediction of our
245
multivariate analyses that the definitively identified flavone, β-caryophyllene and 4-
246
cyclopentene-1-3-dione were positively correlated with growth inhibition of S. cerevisiae,
247
we measured growth inhibition of S. cerevisiae in the presence and absence of standards.
248
Relative to untreated cells, the IC50 values for the three compounds were 2.3 µM, 35.8 µM
11 ACS Paragon Plus Environment
Page 12 of 36
Page 13 of 36
Journal of Agricultural and Food Chemistry
249
and 1.3 µM for flavone, ß-caryophyllene, and 4-cyclopentene-1-3-dione, respectively (Table
250
2). The unique compound observed only in the FFF5 cultivar, (E)-3-hexenyl butyrate,
251
conferred growth inhibition via an IC50 of 22.7 µM (Table 2). These results validate our
252
multivariate analysis-based correlation strategy, showing that positively correlated
253
compounds predicted by the PLSR analysis are indeed bioactive in yeast cells.
254
Antifungal Activity of Feijoa Cultivars and Compounds Against Human
255
Pathogens. Candida species commonly cause fungal infections and fatalities in
256
immunocompromised hosts with more than 400,000 fatalities worldwide every year.3 To
257
determine whether the compounds we identified as being inhibitory to growth of the saprobic
258
yeast S. cerevisiae were more widely effective against human pathogenic fungal species, we
259
quantified growth of four Candida species (C. albicans, C. glabrata, C. parapsilosis, and C.
260
tropicalis) in the presence of three compounds that were positively correlated with bioactivity
261
(flavone, β-caryophyllene, 4-cyclopentene-1-3-dione) and one compound that was only
262
identified in the FFF5 cultivar ((E)-3-hexenyl butyrate). Relative to untreated cells, the IC50
263
values for the four compounds were 1.5-2.6 µM for flavone, 1.5-2.3 µM for 4-cyclopentene-
264
1-3-dione, 35.8-75.2 µM for β-caryophyllene, and 16.0-38.2 µM for (E)-3-hexenyl butyrate
265
(Table 2). Overall, the bioactivity of these four compounds against human pathogenic
266
Candida species was consistent with bioactivity against S. cerevisiae (Table 2), providing
267
further validation of our use of S. cerevisiae as a model fungus to study the antifungal
268
bioactivity of feijoa cultivars.
269
As these four compounds are merely a fraction of the 151 compounds that we
270
identified in the 16 feijoa cultivars, we sought to further understand the antifungal potency of
271
the total methanolic extracts derived from these cultivars, testing their abilities to inhibit the
272
growth of C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis. At 0.01% v/v, the 16
273
cultivar extracts significantly inhibited growth of the four Candida species by 12-87%
12 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
274
compared to untreated cells. Specifically, the FFF5 and FFF6 cultivar extracts inhibited
275
growth of the pathogenic Candida strains by 69-85% compared to untreated cells (Table 3),
276
consistent with these cultivar extracts being the most bioactive against S. cerevisiae (Figure
277
2; Table 3).
278
Antifungal Mechanism of Action of 4-cyclopentene-1-3-dione. Based on potent
279
antifungal activity (IC50 = 1-2 µM), 4-cyclopentene-1,3-dione has the potential to be a lead
280
for developement as a clinical antifungal drug once a mechanism of action is determined.
281
Haploinsufficiency (hypersensitivity) of a heterozygous diploid yeast strain indicates the
282
mutant gene is the drug target based on inhibition of the product of a single gene copy by the
283
drug.24 To determine if the target of 4-cyclopentene-1,3-dione is a known antifungal drug
284
target, we evaluated sensitivity of heterozygous mutants bearing deletions of known
285
antifungal drug targets. We observed that heterozygosity of the chitin synthase-1 and chitin
286
synthase-2 genes (chs1∆/CHS1 and chs2∆/CHS2) conferred significant hypersensitivity to 4-
287
cyclopentene-1,3-dione with 70% and 74% growth reductions, respectively, compared to
288
10% growth reduction seen in the diploid WT strain (BY4743) (Figure 4A). For comparison,
289
other antifungal drug targets were not hypersensitive to 4-cyclopentene-1,3-dione including
290
the cell wall 1,3-β-D-glucan synthase FKS1 gene (target of caspofungin), the ergosterol
291
biosynthesis ERG11 gene (target of ketoconazole), the sphingolipid synthesis LCB1 gene
292
(target of myriocin), ergosterol biosynthesis HMG1 gene (target of atorvastatin), the cytosine
293
deaminase FCY1 gene (resistance to flucytosine) and the RNAse subunits POP1 gene
294
(hypersensitive to nystatin) (Figure 4A). Interestingly, there was significant, albeit slight
295
(21%), inhibition of the hmg1∆/HMG1 strain by 4-cyclopentene-1,3-dione, though not to the
296
same extent as chs1∆/CHS1. The CHS1 and CHS2 genes are the target of polyoxin,27 and the
297
hypersensitivity of the chs1∆/CHS1 and chs2∆/CHS2 strains to 4-cyclopentene-1,3-dione
298
mirrored a 76% and 65% growth reduction in these strains by polyoxin (Figure 4B).
13 ACS Paragon Plus Environment
Page 14 of 36
Page 15 of 36
Journal of Agricultural and Food Chemistry
299
If a drug target is absent, cells will be resistant to that drug.24 Consistent with CHS1
300
and CHS2 being the target of 4-cyclopentene-1,3-dione, chs1∆ and chs2∆ haploid mutants
301
were resistant to both 4-cyclopentene-1,3-dione and polyoxin, which induced no growth
302
inhibition in chs1∆ and chs2∆ compared to 59% growth inhibition for each compound in the
303
WT strain (Figure 4C). To further characterize chitin synthesis inhibition, we examined the
304
abundance of chitin in the cell wall. Using a fluorescent stain specific for chitin (calcafluor
305
white),28 both 4-cyclopentene-1,3-dione and polyoxin B reduced abundance of chitin in the
306
septum (Figure 4D), a morphology that is consistent with our demonstrated inhibition of the
307
CHS1 and CHS2 gene products, which are the major regulators of chitin distribution in
308
septa.29 Together, these results indicate 4-cyclopentene-1,3-dione and polyoxin share a
309
common mechanism of action by targeting the major chitin synthesis genes CHS1 and CHS2
310
and inhibiting the synthesis of chitin, a major structural component of the fungal cell wall that
311
does not exist in mammalian cells and is critical to growth of all fungi. This specificity for
312
chitin was further supported with an absence of antibacterial activity of 4-cyclopentene-1,3-
313
dione against four bacterial species at 1.3 µM, the concentration that achieved IC50 antifungal
314
activity. Consequently, antibacterial activity of 4-cyclopentene-1,3-dione was reduced 22-70
315
fold relative to antifungal activity with IC50 values of 85±11, 60±1, 33±2 and 105±14 µM
316
against B. subtilis (Gram positive lab strain), E. coli (Gram negative lab strain), S. aureus
317
(pathogenic strain) and K. pneumoniae (pathogenic strain), respectively.
318 319
DISCUSSION
320
In this study, we investigated agronomically diverse feijoa cultivars to identify
321
antifungal compounds with pharmaceutical potential. These compounds were sourced from
322
feijoa peel waste to provide the first explicit attempt to add high value to the peels that are
323
currently discarded by the ton in the feijoa juicing industry. We investigated 16 feijoa
14 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
324
cultivars, both commercial and non-commercial within a feijoa breeding program, and in an
325
unbiased fashion correlated antifungal bioactivity with the observed metabolite content (151
326
volatile compounds) rather than in a traditional reductionist fashion of bioassay-guided
327
fractionation of a subset of these compounds. Using established methodology to link
328
metabolomic and bioassay results,25 our multivariate analysis demonstrated strong correlation
329
between antifungal bioactivity and 22 compounds present in methanolic extracts of feijoa
330
fruit peels. One of these compounds, 4-cyclopentene-1,3-dione, was potently antifungal
331
against Candida species that cause the most fungal infections in humans, including C.
332
albicans, via a fungal-specific mechanism targeting chitin in fungal cell walls. As no chitin
333
synthesis inhibitor has been clinically approved for use in humans,29 our results indicate 4-
334
cyclopentene-1,3-dione has such clinical potential, particularly since no cyclopentenedione
335
reported to date has demonstrated inhibition of pathogenic Candida species via a chitin
336
synthesis mechanism.
337
In addition to recovering compounds detected in previous studies such as flavone,30 β-
338
caryophyllene,30 3-octanone,30 trans-calamenene30 and globulol,31 we also detected
339
compounds that have not been reported from feijoa to date, although these are primarily
340
tentative annotations and not formal positive identifications. In one case of a positive
341
identification, we provide the first report of 4-cyclopentene-1,3-dione being present in feijoa.
342
We confirmed the compound identity via GC-MS analysis of an authentic standard, and
343
demonstrated potent antifungal activity via growth inhibition of multiple pathogenic Candida
344
species. The presence of 4-cyclopentene-1,3-dione in a plant species is not specific to feijoa
345
as this compound has previously been identified in walnut oil.32 Interestingly, the most potent
346
FFF5 cultivar contained relatively the most 4-cyclopentene-1,3-dione among the 16 cultivars,
347
raising the possibility that the antifungal potency of FFF5 is largely a consequence of 4-
348
cyclopentene-1,3-dione itself or enhanced 4-cyclopentene-1,3-dione bioactivity due to
15 ACS Paragon Plus Environment
Page 16 of 36
Page 17 of 36
Journal of Agricultural and Food Chemistry
349
synergy with other bioactive compounds (e.g., butyrate compounds can increase the
350
bioactivity of other volatile organic compounds33).
351
In regards to the potency of antifungal bioactivity, 4-cyclopentene-1,3-dione is
352
comparable with established antifungal drugs. Clinically used drugs from the polyene group
353
such as simvastatin and atorvastatin exhibit IC50 values of 3 µM and 10 µM, respectively,
354
against Candida albicans,34 while azole drugs used in the clinic such as fluconazole have IC50
355
values of ~3 µM against different Candida species.35 Our study determined that 4-
356
cyclopentene-1,3-dione exhibits IC50 values of 1.5-2.3 µM against four Candida species,
357
indicating that this compound is as potent as antifungal drugs used in the clinic today.
358
Although the antifungal activity of 4-cyclopentene-1,3-dione has not previously been
359
demonstrated, it is consistent with antifungal activity for structurally related coruscanone A.36
360
Coruscanone A and a desmethoxy derivative were each comparable to the clinically approved
361
antifungals fluconazole and amphotericin B with IC50 values of 0.5-3 µM against Candida
362
species . While the antifungal mechanism of coruscanone A is not yet resolved, our results
363
show that 4-cyclopentene-1,3-dione is antifungal via chitin synthesis inhibition.
364
As chitin is a major component of fungal cell walls that is missing in vertebrate
365
organisms, chitin biosynthesis is a specific and promising target to treat fungal infections
366
without inhibiting those same pathways in human hosts.3,29 Nikkomycin and polyoxin are two
367
established chitin synthase inhibitors that were isolated more than 40 years ago from cultures
368
of Streptomyces species.29 While both are pyrimidine nucleoside peptides, neither has been
369
clinically approved as a pharmaceutical drug, albeit polyoxin is an active ingredient in
370
commercial fungicides in the agricultural and horticultural industries.29 Both nikkomycin and
371
polyoxin inhibit CHS1 and CHS2, which is consistent with our results comparing 4-
372
cyclopentene-1,3-dione and polyoxin. Although these three compounds share a mechanism, it
373
is plausible that 4-cyclopentene-1,3-dione will exhibit different pharmacokinetics than
16 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 18 of 36
374
nikkomycin and polyoxin given their completely different structural backbones. For example,
375
the more lipophillic cyclopentenedione backbone will exhibit vastly different solubility than
376
pyrimidine nucleoside peptides, and also will be less susceptible to hydrolysis. Given the
377
Michael acceptor
378
cycopentenedione scaffold, covalent inhibition of targets is also plausible. In addition to
379
nikkomycin and polyoxin isolated from bacteria, there have been other structurally diverse
380
natural product chitin synthesis inhibitors with antifungal activity, notably five compounds
381
isolated from four fungal species and 14 compounds isolated from 10 plant species.29 With
382
our results, 4-cyclopentene-1,3-dione can be added to this list of natural chitin synthase
383
inhibitors.
and
acidic
1,3-diketo
functional
groups
within
the
compact
384
Cyclopentenedione compounds are a relatively new group of ~100 secondary
385
metabolites isolated primarily from plants as well as fungi and bacteria.37 It is plausible that
386
other cyclopentenedione compounds, in addition to our demonstration for 4-cyclopentene-
387
1,3-dione, are antifungal by a common mechanism of chitin synthesis inhibition. For
388
example, the cyclopentenediones linderone and methyllinderone exhibited antifungal activity
389
against plant pathogens via chitin synthase inhibition.38 However, these cyclopentenediones
390
did not inhibit CHS1 activity, which was the main reason these compounds did not
391
effectively kill Candida species (IC50 > 700 µM). In contrast, we demonstrated that 4-
392
cyclopentene-1,3-dione inhibited CHS1 and thus inhibited growth of four Candida species
393
with IC50 ~ 1-2 µM. More broadly than specificity for chitin synthase genes, the reports of
394
anti-cancer and anti-inflammatory bioactivities among cyclopentenedione compounds in
395
mammalian cells suggests that chitin synthesis inhibition is not the only bioactivity
396
mechanism in this family of compounds since mammalian cells do not contain chitin.37 A
397
similar phenomenon was evident in this study, with 4-cyclopentene-1,3-dione exhibiting low
17 ACS Paragon Plus Environment
Page 19 of 36
Journal of Agricultural and Food Chemistry
398
level antibacterial activity relative to the potent antifungal activity (consistent with bacteria
399
not containing chitin).
400
Recognizing the potential use of cyclopentenedione compounds as novel
401
pharmaceutical drugs, a library of compounds derived from a parental cyclopentenedione
402
compound was generated and chemically characterized but not characterized for any
403
biological activity.39 Our finding of potent antifungal activity of 4-cyclopentene-1,3-dione as
404
a chitin synthesis inhibitor clearly warrants further evaluation of structure-activity
405
relationships. Similar medicinal chemistry approaches have been used to enhance the anti-
406
inflammatory and anti-cancer activities of compounds with a cyclopentenedione backbone.37
407
As the cyclopentenedione backbone is highly reactive, it has a high potential as a lead for
408
commercial synthesis and testing of analogues to probe antifungal structure-activity
409
relationships.
410
In addition to the potential of 4-cyclopentene-1,3-dione as a pharmaceutical
411
antifungal with specificity for a target that does not exist in humans and animals, there is also
412
potential for this compound to be a specific fungicide as well as insecticide (the insect
413
exoskeletion contains chitin) in an agricultural setting. Regarding insects, a number of chitin
414
synthesis inhibitors are currently used worldwide to combat arthropod pests in agriculture and
415
forestry responsible for billions of dollars of damage.40 It will thus be interesting to evaluate
416
chitin-specific insecticide activity of 4-cyclopentene-1,3-dione, particularly since insecticide
417
activity of cylcopentenedione compounds is poorly understood with only one publication and
418
the few cyclopentenedione compounds with insecticide activity were not chitin synthesis
419
inhibitors.41 Specifically, it will be interesting to determine if 4-cyclopentene-1,3-dione
420
behaves in a similar fashion to the chitin synthesis inhibitor polyoxin in only exhibiting
421
insecticide activity when administered orally or injected into the insects.40 Regarding fungal
422
plant pathogens, the cyclopentenedione compounds chrysotrione A and B have exhibited
18 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
423
antifungal activity against Fusarium verticilloides, a major pathogen of corn.42 Notably,
424
chrysotrione A and B have not been shown to be antifungal through a chitin synthesis
425
inhibition mechanism such as that seen in this study for 4-cyclopentene-1,3-dione.
426
In conclusion, we conducted the first systematic evaluation of bioactivity across feijoa
427
cultivars and demonstrated there is enormous functional chemical diversity among feijoa
428
cultivars to exploit as a natural source of bioactive compounds from agricultural waste. We
429
used bioactivity-guided metabolite profiling of peels derived from 16 commercial and
430
experimental feijoa cultivars to correlate 151 volatile compounds with antifungal activity.
431
These compounds were identified via unbiased metabolite profiling of both novel, non-
432
commercial and commercially available feijoa cultivars, which collectively indicate that there
433
is extensive chemical diversity within closely related feijoa cultivars. All cultivars were
434
substantially antifungal at 0.01% (v/v) based on growth inhibition of Saccharomyces and
435
Candida species. The two experimental cultivars FFF5 and FFF6 that exhibited the most
436
antifungal activity contained the compound 4-cyclopentene-1,3-dione, which we showed to
437
be potently antifungal against Saccharomyces and Candida species. It will be necessary to
438
measure the abundance of 4-cyclopentene-1,3-dione in each feijoa peel to precisely determine
439
how much of this potent antifungal compound can be isolated from a given amount of feijoa
440
peel waste. Via our identification of the first cyclopentenedione compound to potently kill
441
Candida species via a fungal-specific mechanism of chitin synthesis inhibition, there is
442
potential of a high value antifungal pharmaceutical in a waste product in the feijoa juicing
443
industry, which warrants further investigation of volatile and non-volatile compounds for
444
more activities in addition to antifungal activity.
445
446
ASSOCIATED CONTENT
447
Supporting Information 19 ACS Paragon Plus Environment
Page 20 of 36
Page 21 of 36
Journal of Agricultural and Food Chemistry
448
Figure S1. GC-MS chromatogram of FFF6 feijoa cultivar.
449
Table S1. List of compounds detected in 16 feijoa cultivars, with experimental and literature
450
LRI values and similarity indices to NIST database.
451 452
AUTHOR INFORMATION
453
Corresponding Author
454
(A.B.M.) Phone: +64 4 463 5171. Email:
[email protected] 455 456
Funding
457
This work was supported by a Victoria University of Wellington Doctoral Scholarship
458
(M.M.).
459 460
Notes
461
There are no conflicts of interest to report.
462 463
ACKNOWLEDGMENTS
464
We express our gratitude to Jeffrey Sheridan for technical assistance in figure preparation and
465
to all the employees at Foretaste Feijoa Fruit Ltd.
466
467
REFERENCES
468
(1)
469
leads. Biochim. Biophys. Acta 2013, 1830, 3670-3695.
470
(2)
471
and combat the antibiotic resistome. Nat. Rev. Microbiol. 2017, 15, 422-434.
Cragg, G. M.; Newman, D. J. Natural products: a continuing source of novel drug
Crofts, T. S.; Gasparrini, A. J.; Dantas, G. Next-generation approaches to understand
20 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
472
(3)
Perfect, J. R. The antifungal pipeline: a reality check. Nat. Rev. Drug Discov. 2017,
473
16, 603-616.
474
(4)
475
Berlin Heidelberg, 2013.
476
(5)
477
R. Antibacterial activity in Actinidia chinensis, Feijoa sellowiana and Aberia caffra. Int. J.
478
Antimicrob. Agents 1997, 8, 199-203.
479
(6)
480
Minichiello, A.; Manzo, F.; Carafa, V.; Basile, A.; Rigano, D.; Sorbo, S.; Castaldo-
481
Cobianchi, R.; Schiavone, E. M.; Ferrara, F.; De Simone, M.; Vietri, M. T.; Cioffi, M.; Sica,
482
V.; Bresciani, F.; de Lera, A. R.; Altucci, L.; Molinari, A. M. Feijoa sellowiana derived
483
natural flavone exerts anti-cancer action displaying HDAC inhibitory activities. Int. J.
484
Biochem. Cell Biol. 2007, 39, 1902-1914.
485
(7)
486
Myrtaceae): A review. Food Chem. 2010, 121, 923-926.
487
(8)
488
towards selected probiotic and pathogenic bacteria. Benef. Microbes 2012, 3, 309-18.
489
(9)
490
C.; de Guzman, E.; Trower, T.; Perry, N. B. JAK2 and AMP-kinase inhibition in vitro by
491
food extracts, fractions and purified phytochemicals. Food Funct. 2015, 6, 305-12.
492
(10)
493
Extracts of feijoa inhibit toll-like receptor 2 signaling and activate autophagy implicating a
494
role in dietary control of IBD. PLoS ONE 2015, 10, e0130910.
495
(11)
496
Basile, A.; Cuomo, R. Acetonic extract from the Feijoa sellowiana Berg. fruit exerts
Razzaghi-Abyaneh, M.; Rai, M. Antifungal Metabolites from Plants. Springer-Verlag
Basile, A.; Vuotto M. L.; Violante, U.; Sorbo, S.; Martone, G.; Castaldo-Cobianchi,
Bontempo, P.; Mita, L.; Miceli, M.; Doto, A.; Nebbioso, A.; De Bellis, F.; Conte, M.;
Weston, R. J. Bioactive products from fruit of the feijoa (Feijoa sellowiana,
Hap, S.; Gutierrez, N. A. Functional properties of some New Zealand fruit extracts
Martin, H.; Burgess, E. J.; Smith, W. A.; McGhie, T. K.; Cooney, J. M.; Lunken, R.
Nasef, N. A.; Mehta, S.; Powell, P.; Marlow, G.; Wileman, T.; Ferguson, L. R.
Turco, F.; Palumbo, I.; Andreozzi, P.; Sarnelli, G.; De Ruberto, F.; Esposito, G.;
21 ACS Paragon Plus Environment
Page 22 of 36
Page 23 of 36
Journal of Agricultural and Food Chemistry
497
antioxidant properties and modulates disaccharidases activities in human intestinal epithelial
498
cells. Phytother. Res. 2016, 30, 1308-15.
499
(12)
500
Center Pacific Islands 2001, 34, 169-175.
501
(13)
502
properties of essential oil of Feijoa sellowiana O. Berg (pineapple guava). J. Pure Appl.
503
Microbiol. 2008, 2, 227-230.
504
(14)
505
properties of acetonic extract of Feijoa sellowiana fruits and its effect on Helicobacter pylori
506
growth. J. Med. Food 2010, 13, 189-195.
507
(15)
508
Souza, K. C. B.; Fuentefria, A. M. Reversal of fluconazole resistance induced by a
509
synergistic effect with Acca sellowiana in Candida glabrata strains. Pharm. Biol. 2016, 54,
510
2410-2419.
511
(16)
Shan, Y. Comprehensive Utilization of Citrus By-Products. Academic Press, 2016.
512
(17)
Lemmon, E. W.; McLinden, M. O.; Friend, D. G. NIST Chemistry WebBook, NIST
513
Standard Reference Database Number 69. National Institute of Standards and Technology,
514
Gaithersburg, Maryland 2015.
515
(18)
516
T. W.-M.; Fiehn, O.; Goodacre, R.; Griffin, J. L.; Hankemeier, T.; Hardy, N.; Harnly, J.;
517
Higashi, R.; Kopka, J.; Lane, A. N.; Lindon, J. C.; Marriott, P.; Nicholls, A. W.; Reily, M.
518
D.; Thaden, J. J.; Viant, M. R. Proposed minimum reporting standards for chemical analysis
519
Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI).
520
Metabolomics 2007, 3, 211-221.
Nakashima, H. Biological activity of Feijoa peel extracts. Kagoshima Univ. Res.
Saj, O. P.; Roy, R. K.; Savitha, S. V. Chemical composition and antimicrobial
Basile, A.; Conte, B.; Rigano, D.; Senatore, F.; Sorbo, S. Antibacterial and antifungal
Machado, G. R. M.; Pippi, B.; Dalla Lana, D. F.; Amaral, A. P. S.; Teixeira, M. L.;
Sumner, L. W.; Amberg, A.; Barrett, D.; Beale, M. H.; Beger, R.; Daykin, C. A.; Fan,
22 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
521
(19)
Ruggles, K. V.; Garbarino, J.; Liu, Y.; Moon, J.; Schneider, K.; Henneberry, A.;
522
Billheimer, J.; Millar, J. S.; Marchadier, D.; Valasek, M. A.; Joblin-Mills, A.; Gulati, S.;
523
Munkacsi, A. B.; Repa, J. J.; Rader, D.; Sturley, S. L. A functional, genome-wide evaluation
524
of liposensitive yeast identifies the “ARE2 required for viability”(ARV1) gene product as a
525
major component of eukaryotic fatty acid resistance. J. Biol. Chem. 2014, 289, 4417-4431.
526
(20)
527
Quantitative Raman spectroscopy for the analysis of carrot bioactives. J. Agric. Food Chem.
528
2013, 61, 2701-2708.
529
(21)
530
R.; Patterson, A. V.; Ackerley, D. F. Discovery and evaluation of Escherichia coli
531
nitroreductases that activate the anti-cancer prodrug CB1954. Biochem. Pharmacol. 2010, 79,
532
678-687.
533
(22)
534
Munro, C. A.; Rodrigues, A. G.; Pina‐Vaz, C. Determination of chitin content in fungal cell
535
wall: an alternative flow cytometric method. Cytometry A 2013, 83, 324-328.
536
(23)
537
Chua, G.; Sopko, R.; Brost, R. L.; Ho, C. H; Wang, J.; Ketela, T.; Brenner, C.; Brill, J. A.;
538
Fernandez, G. E.; Lorenz, T. C.; Payne, G. S.; Ishihara, S.; Ohya, Y.; Andrews, B.; Hughes,
539
T. R.; Frey, B. J.; Graham, T. R.; Andersen, R. J.; Boone, C. Exploring the mode-of-action of
540
bioactive compounds by chemical-genetic profiling in yeast. Cell 2006, 126, 611-625.
541
(24)
542
A.; Jitkova, Y.; Gronda, M.; Wu, Y.; Kim, M. K.; Cheung-Ong, K.; Torres, N. P.; Spear, E,
543
D.; Han, M. K.; Schlecht, U.; Suresh, S.; Duby, G.; Heisler, L. E.; Surendra, A.; Fung, E.;
544
Urbanus, M. L.; Gebbia, M.; Lissina, E.; Miranda, M.; Chiang, J. H.; Aparicio, A. M.;
545
Zeghouf, M.; Davis, R. W.; Cherfils, J.; Boutry, M.; Kaiser, C. A.; Cummins, C. L.; Trimble,
Killeen, D. P.; Sansom, C. E.; Lill, R. E.; Eason, J. R.; Gordon, K. C.; Perry, N. B.
Prosser, G. A.; Copp, J. N.; Syddall, S. P.; Williams, E. M.; Smaill, J. B.; Wilson, W.
Costa‐de‐Oliveira, S.; Silva, A. P.; Miranda, I. M.; Salvador, A.; Azevedo, M. M.;
Parsons, A. B.; Lopez, A.; Givoni, I. E.; Williams, D. E.; Gray, C. A.; Porter, J.;
Lee, A. Y.; Onge, R. P. S.; Proctor, M. J.; Wallace, I. M.; Nile, A. H.; Spagnuolo, P.
23 ACS Paragon Plus Environment
Page 24 of 36
Page 25 of 36
Journal of Agricultural and Food Chemistry
546
W. S.; Brown, G. W.; Schimmer, A. D.; Bankaitis, V. A.; Nislow, C.; Bader, G. D.; Giaever,
547
G. Mapping the cellular response to small molecules using chemogenomic fitness signatures.
548
Science 2014, 344, 208-211.
549
(25)
550
Metabolite profiling of sugarcane genotypes and identification of flavonoid glycosides and
551
phenolic acids. J. Agric. Food Chem. 2016, 64, 4198-4206.
552
(26)
553
hydrolysis with subsequent mild thermal oxidation of tallow on precursor formation and
554
sensory profiles of beef flavours assessed by partial least squares regression. Meat Sci. 2014,
555
96, 1191-1200.
556
(27)
557
cerevisiae by polyoxin D and nikkomycins. Antimicrob. Agents Chemother. 1991, 35, 170-
558
173.
559
(28)
560
Enzymol. 1991, 194, 732-735.
561
(29)
562
antifungal agents. Mini Rev. Med. Chem. 2013, 13, 222-236.
563
(30)
564
Chemical composition of the essential oil from Feijoa (Feijoa sellowiana Berg.) peel. J.
565
Essent. Oil Res. 2004, 16, 274-275.
566
(31)
567
Chem. 1989, 37, 734-736.
568
(32)
569
correction: a calibration method for handling instrumental drifts of gas chromatography-mass
570
spectrometry systems. J. Chromatogr. A 2006, 1116, 248-258. .
Coutinho, I. D.; Baker, J. M.; Ward, J. L.; Beale, M. H.; Creste, S.; Cavalheiro, A. J.
Song, S.; Tang, Q.; Hayat, K.; Karangwa, E.; Zhang, X.; Xiao, Z. Effect of enzymatic
Cabib, E. Differential inhibition of chitin synthetases 1 and 2 from Saccharomyces
Pringle, J. R. Staining of bud scars and other cell wall chitin with Calcofluor. Methods
Chaudhary, P. M.; Tupe, S. G.; Deshpande, M. V. Chitin synthase inhibitors as
Fernandez, X.; Loiseau, A.-M.; Poulain, S.; Lizzani-Cuvelier, L.; Monnier, Y.
Binder, R. G.; Flath, R. A. Volatile components of pineapple guava. J. Agric. Food
Deport, C.; Ratel, J.; Berdagué, J. L.; Engel, E. Comprehensive combinatory standard
24 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
571
(33)
Strobel, G. A.; Spang, S.; Kluck, K.; Hess, W. M.; Sears, J.; Livinghouse, T.
572
Synergism among volatile organic compounds resulting in increased antibiosis in Oidium sp.
573
FEMS Microbiol. Lett. 2008, 283, 140-145.
574
(34)
575
Candida species and Aspergillus fumigatus by statins. FEMS Microbiol. Lett. 2006, 262, 9-
576
13.
577
(35)
578
D0870 compared with those of other azoles against fluconazole-resistant Candida spp.
579
Antimicrob. Agents Chemother. 1995, 39, 868-871.
580
(36)
581
G.; Smillie, T. J.; Khan, I. A.; Walker, L. A.; Clark, A. M. Antifungal cyclopentenediones
582
from Piper coruscans. J. Am. Chem. Soc. 2004, 126, 6872-3.
583
(37)
584
biological activities of cyclopentenediones: A Review. Mini Rev. Med. Chem. 2014, 14, 322-
585
331.
586
(38)
587
D.; Kim, S. U. Inhibition of chitin synthase 2 and antifungal activity of lignans from the stem
588
bark of Lindera erythrocarpa. Planta Med. 2007, 73, 679-682.
589
(39)
590
Sykora, J.; Storch, J.; Vacek, J. A comprehensive LC/MS analysis of novel
591
cyclopentenedione library. J. Pharm. Biomed. Anal. 2016, 128, 342-351.
592
(40)
593
Insect Sci. 2013, 20, 121-138.
594
(41)
595
Screening of dialkoxybenzenes and disubstituted cyclopentene derivatives against the
Macreadie, I. G.; Johnson, G.; Schlosser, T.; Macreadie, P. I. Growth inhibition of
Wardle, H. M.; Law, D.; Moore, C. B.; Mason, C.; Denning, D. W. In vitro activity of
Li, X. C.; Ferreira, D.; Jacob, M. R.; Zhang, Q.; Khan, S. I.; ElSohly, H. N.; Nagle, D.
Sevcikova, Z.; Pour, M.; Novak, D.; Ulrichova, J.; Vacek, J. Chemical properties and
Hwang, E. I.; Lee, Y. M.; Lee, S. M.; Yeo, W. H.; Moon, J. S.; Kang, T. H.; Park, K.
Papouskova, B.; Bernard, M.; Ottenschlager, J.; Karban, J.; Velisek, P.; Hrbac, J.;
Merzendorfer, H. Chitin synthesis inhibitors: old molecules and new developments.
Akhtar, Y.; Isman, M. B.; Paduraru, P. M.; Nagabandi, S.; Nair, R.; Plettner, E.
25 ACS Paragon Plus Environment
Page 26 of 36
Page 27 of 36
Journal of Agricultural and Food Chemistry
596
cabbage looper, Trichoplusia ni, for the discovery of new feeding and oviposition deterrents.
597
J. Agric. Food Chem. 2007, 55, 10323-10330.
598
(42)
599
acylcyclopentenediones from fruiting bodies of Hygrophorus chrysodon. J. Nat. Prod. 2007,
600
70, 137-139.
Gilardoni, G.; Clericuzio, M.; Tosi, S.; Zanoni, G.; Vidari, G. Antifungal
601
26 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
602
Figure legends
603
604
Figure 1. Representative images of feijoa including feijoa tree (upper left), feijoa flowers
605
(upper right), feijoa fruit (lower left), and cross-section of feijoa fruit (lower right). Images
606
provided by Foretaste Feijoa Fruit Ltd., Takaka, New Zealand.
607
608
Figure 2. Bioactivity of methanolic extracts (0.01% v/v) of 16 feijoa cultivars based on
609
growth inhibition of S. cerevisiae. The growth percentage inhibition was calculated via
610
optical density (OD600) at mid-log in treated cells relative to untreated cells. Data shown as
611
mean ± S.D. **, p < 0.005, Student’s t-test comparing treated and untreated cells. ***, p