Synthesis of oxylipin mimics and their antifungal activity against the citrus postharvest pathogens

Article abstract of DOI:10.3390/molecules21020254

Nine oxylipin mimics were designed and synthesized starting from D-mannose. Their antifungal activity against three citrus postharvest pathogens was evaluated by spore germination assay. The results indicated that all the compounds significantly inhibited the growth of Penicillium digitatum, Penicillium italicum and Aspergillus Niger. The compound (3Z,6Z,8S,9R,10R)-octadeca-3,6-diene-8,9,10-triol (3) exhibited excellent inhibitory effect on both Penicillium digitatum (IC₅₀ = 34 ppm) and Penicillium italicum (IC₅₀ = 94 ppm). Their in vivo antifungal activities against citrus postharvest blue mold were tested with fruit inoculated with the pathogen Penicillium italicum. The compound (3R,4S)-methyl 3,4-dihydroxy-5-octyltetrahydrofuran-2-carboxylate (9) demonstrated significant efficacy by reducing the disease severity to 60%. The antifungal mechanism of these oxylipin mimics was postulated in which both inhibition of pathogenic mycelium and stimuli of the host oxylipin-mediated defense response played important roles.

Full text of DOI:10.3390/molecules21020254

molecules
Article
Synthesis of Oxylipin Mimics and Their Antifungal
Activity against the Citrus Postharvest Pathogens
Jimei Ma, Yupeng Li, Hangwei Chen, Zhen Zeng, Zi-Long Li and Hong Jiang *
Department of Chemistry, College of Sciences, Huazhong Agricultural University, Wuhan 430070, China;
majimei@mail.hzau.edu.cn (J.M.); liyupeng@webmail.hzau.edu.cn (Y.L.); chw234234@hotmail.com (H.C.);
zengzhen@mail.hzau.edu.cn (Z.Z.); lizilong@mail.hzau.edu.cn (Z.-L.L.)
*
Correspondence: Jianghong0066@126.com; Tel.: +86-27-8728-4018
Academic Editors: Jean Jacques Vanden Eynde and Derek J. McPhee
Received: 29 September 2015 ; Accepted: 13 January 2016 ; Published: 22 February 2016
Abstract: Nine oxylipin mimics were designed and synthesized starting from D-mannose. Their
antifungal activity against three citrus postharvest pathogens was evaluated by spore germination
assay. The results indicated that all the compounds significantly inhibited the growth of Penicillium
digitatum, Penicillium italicum and Aspergillus niger. The compound (3Z,6Z,8S,9R,10R)-octadeca-3,6-
diene-8,9,10-triol (3) exhibited excellent inhibitory effect on both Penicillium digitatum (IC50 = 34 ppm)
and Penicillium italicum (IC50 = 94 ppm). Their in vivo antifungal activities against citrus postharvest
blue mold were tested with fruit inoculated with the pathogen Penicillium italicum. The compound
(3R,4S)-methyl 3,4-dihydroxy-5-octyltetrahydrofuran-2-carboxylate (9) demonstrated significant
efficacy by reducing the disease severity to 60%. The antifungal mechanism of these oxylipin
mimics was postulated in which both inhibition of pathogenic mycelium and stimuli of the host
oxylipin-mediated defense response played important roles.
Keywords: oxylipin; synthesis; antifungal activity; citrus; pathogen
1
. Introduction
The biologically active compounds oxidized from polyunsaturated fatty acids in aerobic
organisms are collectively termed oxylipins, which play an important role in a variety of functions
including growth, aging, development, and defense responses to environmental stimuli [ ].
Generally, oxylipins are enzymatically or spontaneously synthesized de novo in response to mechanical
injury, pathogen attack and other environmental inputs [ ]. The chemical structures of oxylipins
1–4
5–7
are diverse including hydroxyl, aldehyde, epoxy, ketol and divinyl-ether derivatives. It has been
reported that quite a few oxylipins and their metabolisms play important roles in defense against
microbial pathogens [
against Rhizoctonia solani and Pythium ultimum [10]. The compound 12-oxo-10,15(Z)-phytodienoic acid
strongly inhibited mycelial growth and spore germination of eukaryotic microbes [ ]. Jasmonic acid
could suppress the reproductive development and secondary metabolism of Aspergillus specie [11 12].
8,9]. For example, linolenic acid and linoleic acid exhibited antifungal activity
8
,
Free fatty acid methyl ester fractions isolated from Linum usitatissimum L. seeds exhibited antifungal
activity against toxigenic Apsergillus [13,14].
Citrus fruit is one of the most abundant agricultural economic crops in the world due to its taste
and nutritive properties. However, decay during postharvest storage always brings significant loss
in the economy [15]. The most common culprits are green and blue mold [16–19]. At present, the
application of germicides is a principal method to control postharvest decay. The safety and endurance
of these compounds are mainly considered, besides the preservative effect. Oxylipin, which is a natural
plant metabolite and generally low in toxicity, was reported to show notable antifungal activity. In this
Molecules 2016, 21, 254; doi:10.3390/molecules21020254
www.mdpi.com/journal/molecules

Molecules 2016, 21, 254
2 of 11
paper, a series of oxylipin mimics were designed and synthesized (Scheme 1). Their antifungal effects
on P. digitatum, P. italicum and Aspergillus niger in Petri dishes were also investigated.
Scheme 1. The structures of designed oxylipin mimics.
2
. Results and Discussion
2
.1. Synthetic Chemistry
In this work, we designed a few oxylipin mimics which were mainly unsaturated alkane with a
multi-hydroxyl group forming an aliphatic carbon chain ranging from 15 to 21. Furthermore, a few
mimics with a tetrahydrofuran ring were also synthesized to investigate the influence of different
structures on antifungal activities [2,20].
The compounds
groups. The synthetic route of compounds
,3:5,6-di-O-isopropylidene-D-mannose (10), prepared according to the literature [21], was reacted with
1
–9
were synthesized from D-mannose, which provided inherent multi-hydroxyl
1–9
was shown in Scheme 2. The compound
2
the corresponding Grignard reagent to give compound 11. Oxidation of compound 11 with periodic
acid [22] afforded compound 12. The Wittig/Horner-Wadsworth-Emmons reaction of compound 12
and subsequent deprotection resulted in the desired oxylipin mimics
products were characterized by H-NMR and HRMS.
1–9
[23
,
24]. All the synthesized
1
Scheme 2. The synthetic route to compounds
1–9. 11a, 12a, 13a, 14a, 15a: R = nC H ; 11b, 12b, 13b,
11 23
1
4b, 15b: R = nC H ; 11c, 12c, 13c, 14c, 15c: R = nC H .
10
21
8
17
2
.2. Evaluation of in Vitro Antifungal Activity
All the synthesized compounds were screened to investigate their antifungal activities against
P. digitatum, P. italicum and A. niger on Petri dishes using the agar dilution method. Linolenic acid

Molecules 2016, 21, 254
3 of 11
was reported to inhibit a few fungi [10,25] and was thus used as the reference compound. IC , the
50
concentration inhibiting 50% of fungal activity, was calculated and shown in Table 1. All the tested
compounds showed significant antifungal activities against P. digitatumi and diverse activities against
P. italicum and A. niger. Compound
the IC50 value of 23 ppm. Compound
IC50 = 94 ppm). Compound 9 inhibited the growth of A. niger with the IC50 value of 202 ppm.
2
strongly reduced the conidia germination of P. digitatumi with
3
demonstrated substantial antifungal activity against P. italicum
(
Table 1. IC50 values of the tested compounds against three citrus postharvest pathogens.
IC50 for Penicillium
digitatumi (ppm)
IC50 for Penicillium
italicum (ppm)
IC50 for Aspergillus
niger (ppm)
Compound
1
2
3
4
5
6
7
8
35.54 ˘ 1.75
22.73 ˘ 3.43
33.64 ˘ 1.85
47.59 ˘ 0.13
31.45 ˘ 3.26
28.43 ˘ 0.82
201.65 ˘ 6.48
59.73 ˘ 4.31
123.69 ˘ 8.79
57.96 ˘ 2.16
133.12 ˘ 2.27
114.01 ˘ 7.63
94.41 ˘ 7.62
256.22 ˘ 18.93
385.56 ˘ 12.74
311.04 ˘ 5.71
243.64 ˘ 11.31
301.01 ˘ 3.54
263.81 ˘ 8.84
257.99 ˘ 9.32
333.15 ˘ 13.62
202.40 ˘ 2.99
272.14 ˘ 5.47
156.97 ˘ 10.13
191.37 ˘ 8.14
182.33 ˘ 13.50
272.83 ˘ 18.73
208.85 ˘ 12.28
212.60 ˘ 11.08
234.21 ˘ 10.43
9
Linolenic acid
Compounds 1–9 showed antifungal activities against three pathogens. It is reasonable to speculate
that compounds possessing many hydroxylic groups are able to interact with fungal cytoderm
composed mainly from chitin. This results in increased cytoderm permeability [26]. If considering
structural features, triols
1–6 containing long aliphatic chains showed better antifungal activities
than compounds , which were characterized by cyclic structure and a lower number of hydroxyl
7–9
groups. It seems that the length of the chain for similar structures did not show an apparent difference.
Overall, oxylipin mimic treatment could result in the inhibition of germination or spore death.
2
.3. Evaluation of in Vivo Antifungal Activity
To further explore the antifungal activities of these oxylipin mimics, the test to control citrus
postharvest blue mold on fruit inoculated with the pathogens was conducted. Results summarized in
Table 2 showed that all the compounds reduced the severity of blue mold to some extent as compared
to the control after 10 days. The most effective, compound
reduced the disease severity to 82.5%. However, compound
3
, in spore germination assay simply
9
exhibited the best antifungal activity
in vivo. Compound
9 reduced the disease severity to 60%, which is more effective than that of linolenic
acid (65%).
Table 2. The disease index after 10 days on citrus treated with tested compounds.
Compound
Disease Index (%)
Control
97.5
80.0
75.0
82.5
90.0
77.5
65.0
62.5
65.0
60.0
65.0
1
2
3
4
5
6
7
8
9
Linolenic acid

Molecules 2016, 21, 254
4 of 11
It is noteworthy that the antifungal activities of the tested compounds against P. italicum on
citrus and in vitro Petri dish are inconsistent. Compounds , which displayed moderate inhibitory
7–9
effect on P. italicum in a Petri dish, showed better a inhibitory effect in vivo than those compounds
without an ester group. It is possible that the ester group could be a contributing factor which could
be converted to acid by hydrolase. It is well known that the antifungal activity of oxylipin is not
simply due to the inhibition of the growth of mycelia or killing fungi spores directly. Oxylipin may
act as a signal-transmitting substance to activate the immunomodulation properties of the host to
induce a systemic defense response [
6]. The reactive oxygen species [27] and related enzymes [1,28]
such as lipoxygenase, -dioxygenase and allene oxide synthase could also be influenced by oxylipin.
α
Whether the compounds in this work demonstrate a significant effect through similar mechanisms is
worth investigating in the future.
3
. Experimental Section
3
.1. General
All reagents were commercially available and used without further purification. Solvents were
1
dried prior to use. All the reactions were performed under argon atmosphere. The H-NMR spectra
were collected at room temperature on 400 MHz Bruker AM spectrometers (Bruker Corporation,
1
Madison, WI, USA). The residual solvent signals were taken as the reference (7.26 ppm for H-NMR
and 77.0 ppm for 13C-NMR in CDCl ). Chemical shift (
δ) is reported in ppm, coupling constants (J)
3
are given in Hz. The following abbreviations classify the multiplicity: s = singlet, brs = broad singlet,
d = doublet, t = triplet, m = multiplet or unresolved. HR-MS (ESI) spectra were recorded on Waters
Q-Tof premier
™
(Waters Corporation, Milford, MA, USA) and Bruker MicroTOF II mass spectrometer
1
(Bruker Corporation). The H-NMR and HRMS spectra were attached in the Supplementary Materials.
3
.2. Synthesis of Compounds 1–9
(2R,4S,5R)-1,2:4,5-Diisopropylideneundecyl-1,2,3,4,5,6-hexaol (11a). To a suspension of magnesium
turnings (3.65 g, 0.15 mol) in 100 mL of anhydrous ether, 1-bromoundecane (4.7 g, 0.02 mol) was
added. The reaction was initiated by addition of 0.1 mL dibromoethane. Then 1-bromoundecane
(
18.8 g, 0.08 mol) was added dropwise to keep gently refluxing. After 3 h, the mixture was cooled
˝
to room temperature and then
0
2
´10 C, which was followed by addition of compound 10 (2.6 g,
.01 mol). After completion of addition, the mixture was warmed to room temperature and stirred for
h. Ice water was added to quench the reaction and the resulting suspension was filtered through
Celite. The filtrate was concentrated and purified by column chromatography on silica gel (ethyl
1
acetate/petroleum ether = 1:5) to give compound 11a as a colorless oil (3.54 g, 85%). H-NMR (400 MHz,
CDCl3) δ: 0.87 (t, J = 6.8 Hz, 3H, CH ), 1.23–1.53 (m containing 4 s, 32H, 4CH , 10CH ), 3.59–3.62 (m,
3 3 2
1
4
H, CH), 3.77–3.88 (m, 1H, CH), 3.97–4.14 (m, 4H, 2CH, CH ), 4.34–4.41 (m, 1H, CH); HRMS (ESI) m/z
2
+
39.3036 (calcd. for C H O Na [M + Na] , 439.3036).
24
44
6
(
1
4
3R,4S)-3,4-Isopropylidene-5-undecyltetrahydrofuran-2,3,4-triol (12a). A mixture of periodic acid (3.84 g,
6.95 mmol) and compound 11a (2.35 g, 5.65 mmol) in 200 mL of anhydrous ether was stirred for
h at room temperature. The suspension was filtered to remove the extra periodic acid. The filtrate
was washed with saturated NaHCO solution to neutral and then washed with brine. The organic
3
phase was dried over Na SO , filtered and concentrated. The residue was purified by column
2
4
chromatography on silica gel (ethyl acetate/petroleum ether = 1:10) to give compound 12a as a
1
colorless oil (1.55 g, 87%). H-NMR (400 MHz, CDCl )
δ
: 0.86 (t, J = 6.8 Hz, 3H, CH ), 1.24–1.46 (m
3
3
containing 2 s, 24H, 2CH , 9CH ), 1.68 (dd, J = 14.7, 7.2 Hz, 2H, CH ), 3.31(s, 1H, OH), 4.10 (dt, J = 6.8,
3
2
2
3
.6 Hz, 1H, CH), 4.56–4.57 (m, 1H, CH), 4.60–4.64 (m, 1H, CH), 5.32 (d, J = 1.6 Hz, 1H, CH); HRMS
+
(ESI) m/z 337.2355 (calcd. for C H O Na [M + Na] , 337.2354).
18
34
6

Molecules 2016, 21, 254
5 of 11
(3Z,6Z,8S,9R)-8,9-Isopropylidene-heneicosa-3, 6-diene-8, 9, 10-triol (13a). n-Butyl lithium (9.88 mL,
1
1
.6 M) was added dropwise to a solution of (Z)-hex-3-ene triphenylphosphonium iodine (5.81 g,
˝
2.3 mmol) in 70 mL of THF at
´
30 C. After 0.5 h, a solution of compound 12a (1.10 g, 3.5 mmol)
˝
in 5 mL of THF was added to the mixture, which was then allowed to warm to 0 C. The reaction
was continued for another 6 h. The resulting mixture was filtered. The filtrate was dried over
Na SO , filtered and concentrated. The residue was purified by column chromatography on silica
2
4
gel (ethyl acetate/petroleum ether = 1:20) to give compound 13a as a colorless oil (0.65 g, 49%).
1
H-NMR (400 MHz, CDCl3) δ: 0.88 (t, J = 6.8 Hz, 3H, CH ), 0.97 (t, J = 7.4 Hz, 3H, CH ), 1.26–1.51 (m
3 3
containing 2 s, 26H, 2CH , 10CH ), 2.02–2.10 (m, 2H, CH ), 2.80–2.86 (m, 2H, CH ), 3.56–3.59 (m 1H,
3
2
2
2
CH), 3.97–4.00 (m, 1H, CH), 4.98 (dd, J = 8.6, 6.8 Hz, 1H, CH), 5.24–5.74 (m, 4H, 2CH=CH); HRMS
+
(
ESI) m/z 403.3191 (calcd. for C H O Na [M + Na] , 403.3188).
24
44
3
(3Z,6Z,8S,9R,10R)-Henicosa-3,6-diene-8,9,10-triol (1). Compound 13a (0.46 g, 1.2 mmol) was added
to a solution of concentrated HCl (11 mL) in water/methanol (75 mL, v/v = 1:9). The mixture was
stirred for 3 h at room temperature and then neutralized with NaOH solution. The resulting mixture
was concentrated to remove methanol. The residue was diluted with EtOAc and washed with water.
The organic phase was dried over Na SO , filtered and concentrated. The residue was purified by
2
4
column chromatography on silica gel (ethyl acetate/petroleum ether = 1:2) to give compound
1
as a
1
colorless oil (0.32 g, 78%). H-NMR (400 MHz, CDCl )
δ
: 0.87 (t, J = 6.8 Hz, 3H, CH ), 0.97 (t, J = 7.5 Hz
,
3
3
3
H, CH ), 1.25–1.51 (m, 20H, 10CH ), 2.02–2.08 (m, 2H, CH ), 2.15 (brs, 3H, 3OH), 2.79–2.89 (m, 2H,
3 2 2
CH ), 3.28–3.48 (m, 1H, CH), 3.64–3.85 (m, 1H, CH), 4.22–4.67 (m, 1H, CH), 5.27–5.83 (m, 4H, CH=CH);
2
+
HRMS (ESI) m/z 363.2875 (calcd. for C H O Na [M + Na] , 363.2875).
21
40
3
(
3Z,6Z,8S,9R,10R)-Icosa-3,6-diene-8,9,10-triol (2). To a suspension of magnesium turnings (4.86 g, 0.2 mol)
in 200 mL of anhydrous ether, 1-bromodecane (8.85 g, 0.04 mol) was added. The reaction was initiated
by addition of 0.1 mL dibromoethane. Then 1-bromodecane (35.39 g, 0.16 mol) was added dropwise to
˝
keep gently refluxing. After 3 h, the mixture was cooled to room temperature and then
´10 C, which
was followed by addition of compound 10 (5.21 g, 0.02 mol). After completion of addition, the mixture
was warmed to room temperature and stirred for 2 h. Ice water was added to quench the reaction
and the resulting suspension was filtered through Celite. The filtrate was concentrated and purified
by column chromatography on silica gel (ethyl acetate/petroleum ether = 1:5) to give compound
1
1
1b as a colorless oil (7.08 g, 88%). A mixture of periodic acid (11.97 g, 52.5 mmol) and compound
1b (7.04 g, 17.5 mmol) in 250 mL of anhydrous ether was stirred for 4 h at room temperature.
The suspension was filtered to remove the extra periodic acid. The filtrate was washed with saturated
NaHCO solution to neutral and then washed with brine. The organic phase was dried over Na SO ,
3
2
4
filtered and concentrated. The residue was purified by column chromatography on silica gel (ethyl
acetate/petroleum ether = 1:10) to give compound 12b as a colorless oil (4.16 g, 79%). n-Butyl lithium
(
(
11.8 mL, 1.6 M) was added dropwise to a solution of (Z)-hex-3-ene triphenylphosphonium iodine
˝
7.44 g, 15.75 mmol) in 80 mL of THF at
´
30 C. After 0.5 h, a solution of compound 12b (1.35 g,
˝
4
.5 mmol) in 5 mL of THF was added to the mixture, which was then allowed to warm to 0 C.
The reaction was continued for another 6 h. The resulting mixture was filtered. The filtrate was
dried over Na SO , filtered and concentrated. The residue was purified by column chromatography
2
4
on silica gel (ethyl acetate/petroleum ether = 1:20) to give compound 13b as a colorless oil (0.76 g,
6%). Compound 13b (0.75 g, 2.05 mmol) was added to a solution of concentrated HCl (8.5 mL)
4
in water/methanol (100 mL, v/v = 1:9). The mixture was stirred for 3 h at room temperature and
then neutralized with NaOH solution. The resulting mixture was concentrated to remove methanol.
The residue was diluted with EtOAc and washed with water. The organic phase was dried over
Na SO , filtered and concentrated. The residue was purified by column chromatography on silica gel
2
4
1
(
(
2
ethyl acetate/petroleum ether = 1:2) to give compound
400 MHz, CDCl3) : 0.88 (t, J = 6.4 Hz, 3H, CH ), 0.97 (t, J = 7.5 Hz, 3H, CH ), 1.24–1.73 (m, 18H, 9CH ),
.00–2.10 (m containing brs, 3H, CH , OH), 2.44 (brs, 1H, OH), 2.63 (brs, 1H, OH), 2.77–2.91 (m, 2H,
2 as a colorless oil (0.48 g, 72%). H-NMR
δ
3
3
2
2

Molecules 2016, 21, 254
6 of 11
CH ), 3.39–3.46 (m, 1H, CH), 3.66–3.83 (m, 1H, CH), 4.22–4.30 (m, 0.5H, CH), 4.64–4.66 (m, 0.5H, CH),
2
+
5
.28–5.83 (m, 4H, 2CH=CH); HRMS (ESI) m/z 349.2713 (calcd. for C H O Na [M + Na] , 349.2706).
20
38
3
(
3Z,6Z,8S,9R,10R)-Octadeca-3,6-diene-8,9,10-triol (
3
). To a suspension of magnesium turnings (4.86 g,
0.2 mol) in 200 mL of anhydrous ether, 1-bromoctane (7.72 g, 0.04 mol) was added. The reaction
was initiated by addition of 0.1 mL dibromoethane. Then 1-bromodecane (30.9 g, 0.16 mol) was
added dropwise to keep gently refluxing. After 3 h, the mixture was cooled to room temperature and
˝
then
´
10 C, which was followed by addition of compound 10 (5.2 g, 0.02 mol). After completion
of addition, the mixture was warmed to room temperature and stirred for 2 h. Ice water was
added to quench the reaction and the resulting suspension was filtered through Celite. The filtrate
was concentrated and purified by column chromatography on silica gel (ethyl acetate/petroleum
ether = 1:5) to give compound 11c as a colorless oil (5.91 g, 79%). A mixture of periodic acid (10.77 g,
4
4
7.25 mmol) and compound 11c (5.90 g, 15.75 mmol) in 250 mL of anhydrous ether was stirred for
h at room temperature. The suspension was filtered to remove the extra periodic acid. The filtrate
was washed with saturated NaHCO solution to neutral and then washed with brine. The organic
3
phase was dried over Na SO , filtered and concentrated. The residue was purified by column
2
4
chromatography on silica gel (ethyl acetate/petroleum ether = 1:10) to give compound 12c as a
colorless oil (3.36 g, 78%). n-Butyl lithium (10.5 mL, 1.6 M) was added dropwise to a solution of
˝
(
Z)-hex-3-ene triphenylphosphonium iodine (6.61 g, 14 mmol) in 80 mL of THF at
´
30 C. After 0.5 h,
a solution of compound 12c (1.1 g, 4 mmol) in 5 mL of THF was added to the mixture, which was then
allowed to warm to 0 C. The reaction was continued for another 6 h. The resulting mixture was filtered.
The filtrate was dried over Na SO , filtered and concentrated. The residue was purified by column
˝
2
4
chromatography on silica gel (ethyl acetate/petroleum ether = 1:20) to give compound 13c as a colorless
oil (0.48 g, 36%). Compound 13c (0.47 g, 1.4 mmol) was added to a solution of concentrated HCl (6 mL)
in water/methanol (100 mL, v/v = 1:9). The mixture was stirred for 3 h at room temperature and
then neutralized with NaOH solution. The resulting mixture was concentrated to remove methanol.
The residue was diluted with EtOAc and washed with water. The organic phase was dried over Na SO ,
2
4
filtered and concentrated. The residue was purified by column chromatography on silica gel (ethyl
acetate/petroleum ether = 1:2) to give compound
as a colorless oil (0.39 g, 75%). 1H-NMR (400 MHz
CDCl3) : 0.88 (t, J = 6.8 Hz, 3H, CH ), 0.96(t, J = 7.5 Hz, 3H, CH ), 1.26–1.72(m, 14H, CH ), 2.02–2.06(m,
H, CH ), 2.20 (brs, 2H, 2OH), 2.60(brs, 1H, OH), 2.77–2.86 (m, 2H, CH ), 3.44–3.48 (m, 1H, CH), 3.65(s,
3
,
δ
3
3
2
2
2
2
1
5
H, CH), 4.23 (s, 1H, CH), 5.30–5.37 (m, 1H, =CH), 5.43–5.49(m, 1H, =CH), 5.58–5.64 (m, 1H, =CH),
+
.77–5.84(m, 1H, =CH); HRMS (ESI) m/z 321.2405 (calcd. for C H O Na [M + Na] , 321.2400).
18
34
3
(
5S,6R,7R,Z)-Octadeca-1,3-diene-5,6,7-triol (
4
). n-Butyl lithium (8.5 mL, 1.6 M) was added dropwise to
˝
a solution of allyl triphenylphosphonium bromide (4.02 g, 10.5 mmol) in 60 mL of THF at
After 0.5 h, a solution of compound 12a (0.94 g, 3.0 mmol) in 5 mL of THF was added to the mixture,
which was then allowed to warm to 20 C. The reaction was continued for another 6 h. The resulting
mixture was filtered. The filtrate was dried over Na SO , filtered and concentrated. The residue
´10 C.
˝
2
4
was purified by column chromatography on silica gel (ethyl acetate/petroleum ether = 1:20) to give
compound 14a as a colorless oil (0.39 g, 40%). Compound 14a (0.37 g, 1.1 mmol) was added to a
solution of concentrated HCl (10 mL) in water/methanol (70 mL, v/v = 1:9). The mixture was stirred
for 3 h at room temperature and then neutralized with NaOH solution. The resulting mixture was
concentrated to remove methanol. The residue was diluted with EtOAc and washed with water.
The organic phase was dried over Na SO , filtered and concentrated. The residue was purified
2
4
by column chromatography on silica gel (ethyl acetate/petroleum ether = 1:3) to give compound
1
4
as a colorless oil (0.24 g, 74%). H-NMR (400 MHz, CDCl3)
δ: 0.88 (t, J = 6.8 Hz, 3H, CH3),
1.01–1.73 (m, 20H, 10CH ), 3.34–3.37 (m, 1H, CH), 3.54–3.97 (m, 2H, 2CH), 5.14–5.34 (m, 2H, =CH ),
2 2
5
.57–5.82 (m, 1H, =CH), 6.22–6.72 (m, 2H, 2CH=); HRMS (ESI) m/z 321.2404 (calcd. for C H O Na
18
34
3
+
[M + Na] , 321.2400).

Molecules 2016, 21, 254
7 of 11
(
5S,6R,7R,Z)-Heptadeca-1,3-diene-5,6,7-triol (
5
). n-Butyl lithium (11.8 mL, 1.6 M) was added dropwise to
˝
a solution of allyl triphenylphosphonium bromide (6.77 g, 15.75 mmol) in 80 mL of THF at
After 0.5 h, a solution of compound 12b (1.35 g, 4.5 mmol) in 5 mL of THF was added to the mixture,
which was then allowed to warm to 20 C. The reaction was continued for another 6 h. The resulting
mixture was filtered. The filtrate was dried over Na SO , filtered and concentrated. The residue
´10 C.
˝
2
4
was purified by column chromatography on silica gel (ethyl acetate/petroleum ether = 1:20) to give
compound 14b as a colorless oil (0.65 g, 45%). Compound 14b (0.65 g, 2 mmol) was added to a
solution of concentrated HCl (8 mL) in water/methanol (50 mL, v/v = 1:9). The mixture was stirred
for 3 h at room temperature and then neutralized with NaOH solution. The resulting mixture was
concentrated to remove methanol. The residue was diluted with EtOAc and washed with water.
The organic phase was dried over Na SO , filtered and concentrated. The residue was purified by
2
4
column chromatography on silica gel (ethyl acetate/petroleum ether = 1:3) to give compound
5
as a
1
colorless oil (0.38 g, 68%). H-NMR (400 MHz, CDCl )
δ
: 0.87 (t, J = 6.8 Hz, 3H), 1.25–1.62 (m, 18H),
3
2
.27–2.85 (brs, 1H, OH), 3.04–3.18 (brs, 1H, OH), 3.83–3.89 (m, 2H, 2CH), 3.99–4.00 (m, 1H, CH), 4.04 (s,
1
3
H, OH), 4.74–4.89 (m, 2H, =CH ), 5.22–5.55 (m, 2H, 2=CH), 6.15–6.66 (m, 1H, =CH); HRMS (ESI) m/z
2
+
07.2250 (calcd. for C H O Na [M + Na] , 307.2244).
17
32
3
(
5S,6R,7R,Z)-Pentadeca-1,3-diene-5,6,7-triol (
6
). n-Butyl lithium (10.5 mL, 1.6 M) was added dropwise
˝
to a solution of allyl triphenylphosphonium bromide (6.02 g, 14 mmol) in 80 mL of THF at
After 0.5 h, a solution of compound 12c (1.1 g, 4 mmol) in 5 mL of THF was added to the mixture,
which was then allowed to warm to 20 C. The reaction was continued for another 6 h. The resulting
mixture was filtered. The filtrate was dried over Na SO , filtered and concentrated. The residue
´10 C.
˝
2
4
was purified by column chromatography on silica gel (ethyl acetate/petroleum ether = 1:20) to give
compound 14c as a colorless oil (0.49 g, 41%). Compound 14c (0.48 g, 1.6 mmol) was added to a
solution of concentrated HCl (6.7 mL) in water/methanol (40 mL, v/v = 1:9). The mixture was stirred
for 3 h at room temperature and then neutralized with NaOH solution. The resulting mixture was
concentrated to remove methanol. The residue was diluted with EtOAc and washed with water.
The organic phase was dried over Na SO , filtered and concentrated. The residue was purified by
2
4
column chromatography on silica gel (ethyl acetate/petroleum ether = 1:3) to give compound
6 as
1
a colorless oil (0.31 g, 76%). H NMR (400 MHz, CDCl )
δ
: 0.89 (t, J = 6.4 Hz, 3H, CH ), 1.25–1.59
3
3
(
m, 14H, 7CH ), 2.98 (brs, 3H, 3OH), 3.33–3.36 (m, 0.5H, CH), 3.41–3.42 (m, 0.5H, CH), 3.50–3.53 (m,
2
0
.5H, CH), 3.62–3.65 (m, 0.5H, CH), 3.77–3.86 (m, 0.5H, CH), 3.94–4.03 (m, 0.5H, CH), 4.31–4.78 (m, 1H,
=
CH ), 5.12–5.33 (m, 1.5H, =CH , =CH), 5.49–5.58 (m, 0.5H, =CH), 5.71–5.85 (m, 0.5H, =CH), 6.13–6.24
2 2
(
m, 0.5H, =CH), 6.29–6.37 (m, 0.5H, =CH), 6.56–6.70 (m, 0.5H, =CH); HRMS (ESI) m/z 279.1937 (calcd.
+
for C H O Na [M + Na] , 279.1931).
15
28
3
(3R,4S)-Methyl 3,4-dihydroxy-5-undecyl-tetrahydrofuran-2-acetate (7). n-Butyl lithium (5.5 mL, 1.6 M) was
added dropwise to a solution of triethyl phosphonoacetate (1.93 g, 8.6 mmol) in 70 mL of THF at
˝
´
30 C. After 0.5 h, a solution of compound 12a (1.89 g, 6.0 mmol) in 5 mL of THF was added to the
mixture, which was then allowed to warm to room temperature. The reaction mixture was stirred
overnight and then filtered. The filtrate was dried over Na SO , filtered and concentrated. The residue
2
4
was purified by column chromatography on silica gel (ethyl acetate/petroleum ether = 1:20) to give
compound 15a as a colorless oil (1.02 g, 44%). Compound 15a (1.0 g, 2.6 mmol) was added to a
solution of concentrated HCl (20 mL) in water/methanol (150 mL, v/v = 1:9). The mixture was stirred
for 3 h at room temperature and then neutralized with NaOH solution. The resulting mixture was
concentrated to remove methanol. The residue was diluted with EtOAc and washed with water.
The organic phase was dried over Na SO , filtered and concentrated. The residue was purified by
2
4
column chromatography on silica gel (ethyl acetate/petroleum ether = 1:3) to give compound
7
as a
1
colorless oil (0.51 g, 69%). H-NMR (400 MHz, CDCl )
δ
: 0.86 (t, J = 6.8 Hz, 3H, CH ), 1.24–1.72 (m,
3
3
2
0H, 10CH ), 2.58–2.86 (m, 2H, CH ), 3.60–3.90 (m containing s, 5H, CH, OCH ), 3.96–4.02 (m, 1H,
2
2
3
+
CH), 4.79–4.98 (m, 1H, CH); HRMS (ESI) m/z 353.2287 (calcd. for C H O Na [M + Na] , 353.2298).
18
34
5

Molecules 2016, 21, 254
8 of 11
(3R,4S)-Methyl 5-decyl-3,4-dihydroxytetrahydrofuran-2-acetate (
8). n-Butyl lithium (4.1 mL, 1.6 M) was
added dropwise to a solution of triethyl phosphonoacetate (1.45 g, 6.45 mmol) in 70 mL of THF at
˝
´
30 C. After 0.5 h, a solution of compound 12b (1.3 g, 4.3 mmol) in 5 mL of THF was added to the
mixture, which was then allowed to warm to room temperature. The reaction mixture was stirred
overnight and then filtered. The filtrate was dried over Na SO , filtered and concentrated. The residue
2
4
was purified by column chromatography on silica gel (ethyl acetate/petroleum ether = 1:20) to give
compound 15b as a colorless oil (0.76 g, 48%). Compound 15b (0.74 g, 2 mmol) was added to a
solution of concentrated HCl (8 mL) in water/methanol (50 mL, v/v = 1:9). The mixture was stirred
for 3 h at room temperature and then neutralized with NaOH solution. The resulting mixture was
concentrated to remove methanol. The residue was diluted with EtOAc and washed with water.
The organic phase was dried over Na SO , filtered and concentrated. The residue was purified by
2
4
column chromatography on silica gel (ethyl acetate/petroleum ether = 1:3) to give compound
8
as a
1
colorless oil (0.47 g, 75%). H-NMR (400 MHz, CDCl )
δ
: 0.86 (t, J = 0.90 Hz, 3H, CH ), 1.23–1.55 (m,
3
3
1
3
8H, 9CH ), 2.61 (dd, J = 16.2, 7.4 Hz, 1H, CH ), 2.75 (dd, J = 16.2, 5.9 Hz, 1H, CH ), 3.78 (s, 3H, OCH ),
2
2
2
3
13
.71–3.75 (m, 1H, CH), 3.78–3.85 (m, 2H, 2CH), 3.95–4.00 (m, 1H, CH); C NMR (100 MHz, CDCl3)
δ:
1
4.1 (CH ), 22.6 (CH ), 25.5 (CH ), 29.3 (CH ), 29.5 (2CH ), 29.6 (3CH ), 31.9 (CH ), 38.3 (CH ), 52.0
3
2
2
2
2
2
2
2
(
OCH ), 74.8 (CH), 74.9 (CH), 78.4 (CH), 84.3 (CH),172.5 (C=O); HRMS (ESI) m/z 339.2144 (calcd. for
3
+
C H O Na [M + Na] , 339.2142).
17
32
5
(
3R,4S)-Methyl 3,4-dihydroxy-5-octyltetrahydrofuran-2-acetate (
9
). n-Butyl lithium (4.3 mL, 1.6 M) was
˝
added dropwise to a solution of triethyl phosphonoacetate (1.28 g, 5.7 mmol) in 80 mL of THF at
After 0.5 h, a solution of compound 12c (1.04 g, 3.8 mmol) in 5 mL of THF was added to the mixture,
which was then allowed to warm to 20 C. The reaction was continued for another 6 h. The resulting
mixture was filtered. The filtrate was dried over Na SO , filtered and concentrated. The residue
´10 C.
˝
2
4
was purified by column chromatography on silica gel (ethyl acetate/petroleum ether = 1:20) to give
compound 15c as a colorless oil (0.53 g, 41%). Compound 15c (0.52 g, 1.5 mmol) was added to a
solution of concentrated HCl (6 mL) in water/methanol (40 mL, v/v = 1:9). The mixture was stirred
for 3 h at room temperature and then neutralized with NaOH solution. The resulting mixture was
concentrated to remove methanol. The residue was diluted with EtOAc and washed with water.
The organic phase was dried over Na SO , filtered and concentrated. The residue was purified by
2
4
column chromatography on silica gel (ethyl acetate/petroleum ether = 1:3) to give compound
9
as a
1
colorless oil (0.32 g, 74%). H-NMR (400 MHz, CDCl )
δ
: 0.85 (t, J = 7.2 Hz, 3H, CH ), 1.23–1.73 (m,
3
3
1
4H, 7 CH ), 2.58–2.85 (m, 2H, CH ), 3.57–4.01 (m containing s, 6H, CH , 3CH), 4.55–4.99 (m, 1H, CH);
2 2 3
+
HRMS (ESI) m/z 289.2007 (calcd. for C H O [M + H] , 289.2010).
15
29
5
3
.3. Antifungal Biology Assay
3
.3.1. Strain
The P. digitatum, P. italicum and A. niger strain was obtained from the College of Horticulture and
Forestry Sciences, Huazhong Agricultural University. The strain was incubated with potato dextrose
agar (PDA). The P. italicum conidial suspension was prepared by diluting with 0.1% Tween-80 sterile
6
´1
water. After three days incubation, the concentration was adjusted to 1
with sterile distilled water using a hemocytometer.
ˆ
10 conidia mL by diluting
3
.3.2. In Vitro Antifungal Activity [29]
The mycelium was inoculated by a hole punch uniformly in 70 mm sterile plastic Petri dishes. Afte
a pre-study, the tested compounds were diluted with PDA to 15, 25, 50, 75, 150 ppm for P. digitatum and
0, 100, 200, 300, 400 ppm for P. italicum and A. niger detection. Control plates contained culture medium
5
˝
without tested compounds. All the Petri dishes were incubated in biochemical incubator at 28 C.
Colony diameters (D) were measured after fourdays for P. digitatum, seven days for P. italicum and A.

Molecules 2016, 21, 254
9 of 11
niger. Three measurements were taken on each plates and in triplicate for each concentration. The mean
value was used for statistical analysis. Inhibitory rate% = [(Dcontrol ´ Dtest)/D ] ˆ 100%.
control
Plot was set with inhibitory rate as Y axial and concentration as X axial. IC , the half maximal
50
concentration to inhibit 50% of fungal activity, was calculated from the plot.
3
.3.3. In Vivo Antifungal Activity
Citrus reticulate used for experiments were collected from orchards in Wuhan, China. After being
stored for one week at room temperature, tested fruits were selected for uniformity of size and maturity
without any physical injury or infection. All fruits were washed with tap water and dried with tissue
carefully before use. A uniform wound (3 mm deep and 4 mm wide) was made at the equator of each
orange with a sterile needle. Then, 20 min later, 20
each wound. After 2 h, 20 L 300 ppm oxylipin mimics were inoculated and 20
control. The tested fruits were stored in artificial climatic box at 28 C with the relative humidity of
0%. The disease incidence and lesion diameters were recorded for 10 days. Analysis of 10 fruits, in
µL P. italicum conidial suspension was injected into
µ
µL sterile water for
˝
9
triplicate for each compound was done.
The disease index for fruit was observed by assessing the extent of total disease symptoms on
each fruit surface using the following scale:
‚
‚
‚
‚
‚
0: lesion diameter = 0 mm (no visible disease symptoms);
1: 1 mm ď lesion diameter ď 10 mm;
2: 10 mm < lesion diameter ď 20 mm;
3: 20 mm < lesion diameter ď 30 mm;
4: lesion diameter > 30 mm.
ř
The disease index was calculated using the formula [30]: (disease scale
ˆ
number of fruit in
each class)/(number of total fruit
vernier caliper using the cross method, and the unit was milimeter.
ˆ
highest disease scale)
ˆ
100. Lesion diameter was measured by a
3
.4. Statistical Analysis
The analysis was carried out using Statistical Package of Social Sciences (SPSS 17.0, SPSS Inc.
Chicago, IL, USA).
4
. Conclusions
In conclusion, a series of oxylipin mimics were designed and synthesized. The in vitro bioassay
indicated that all these oxylipin mimics showed significant inhibitory effect to the growth of P. digitatum,
P. italicum and A. niger. The in vivo experiment on citrus-inoculated P. italicum. demonstrated that the
most effective compound could reduce the disease severity to 60%. However, the mechanism for the
antifungal activity of these oxylipin mimics needs further investigation.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
2
1/2/254/s1.
Acknowledgments: The financial supports (Program No. 2014QC007, 2012BQ029) provided by the Fundamental
Research Funds for the Central Universities, China and the National Natural Science Foundation of China
(21402056) are gratefully acknowledged.
Author Contributions: Jimei Ma, Zi-Long Li and Hong Jiang conceived and designed the experiments; Yupeng Li
and Zhen Zeng performed the experiments; Yupeng Li and Hangwei Chen analyzed the data; Jimei Ma wrote
the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Griffiths, G. Biosynthesis and analysis of plant oxylipins. Free Radic. Res. 2015, 49, 565–582. [CrossRef]
PubMed]
[

Molecules 2016, 21, 254
10 of 11
2
.
Fiorentino, A.; D’Abrosca, B.; DellaGreca, M.; Izzo, A.; Natale, A.; Pascarella, M.T.; Pacifico, S.; Zarrelli, A.;
Monaco, P. Chemical characterization of new oxylipins from Cestrum parqui, and their effects on seed
germination and early seedling growth. Chem. Biodivers. 2008, 5, 1780–1791. [CrossRef] [PubMed]
Mosblech, A.; Feussner, I.; Heilmann, I. Oxylipins: Structurally diverse metabolites from fatty acid oxidation.
Plant Physiol. Biochem. 2009, 47, 511–517. [CrossRef] [PubMed]
Chehab, E.W.; Perea, J.V.; Gopalan, B.; Theg, S.; Dehesh, K. Oxylipin pathway in rice and arabidopsis. J. Integr.
Plant Biol. 2007, 49, 43–51. [CrossRef]
Savchenko, T.V.; Zastrijnaja, O.M.; Klimov, V.V. Oxylipins and plant abiotic stress resistance. Biochemistry
3.
4.
5.
6.
7.
8.
2
014, 79, 362–375. [CrossRef] [PubMed]
Howe, G.A.; Schilmiller, A.L. Oxylipin metabolism in response to stress. Curr. Opin. Plant Biol. 2002, 5,
30–236. [CrossRef]
2
Böttcher, C.; Pollmann, S. Plant oxylipins: Plant responses to 12-oxo-phytodienoic acid are governed by its
specific structural and functional properties. FEBS J. 2009, 276, 4693–4704. [CrossRef] [PubMed]
Prost, I.; Dhondt, S.; Rothe, G.; Vicente, J.; Rodriguez, M.J.; Kift, N.; Carbonne, F.; Griffiths, G.;
Esquerré-Tugayé, M.-T.; Rosahl, S.; et al. Evaluation of the antimicrobial activities of plant oxylipins supports
their involvement in defense against pathogens. Plant Physiol. 2005, 139, 1902–1913. [CrossRef] [PubMed]
Shimada, T.L.; Hara-Nishimura, I. Leaf oil bodies are subcellular factories producing antifungal oxylipins.
Curr. Opin. Plant Biol. 2015, 25, 145–150. [CrossRef] [PubMed]
9
1
1
1
.
0. Walters, D.; Raynor, L.; Mitchell, A.; Walker, R.; Walker, K. Antifungal activities of four fatty acids against
plant pathogenic fungi. Mycopathologia 2004, 157, 87–90. [PubMed]
1. Christensen, S.A.; Kolomiets, M.V. The lipid language of plant–fungal interactions. Fungal Genet. Biol. 2011
8, 4–14. [CrossRef] [PubMed]
2. Santino, A.; Taurino, M.; de Domenico, S.; Bonsegna, S.; Poltronieri, P.; Pastor, V.; Flors, V. Jasmonate
,
4
signaling in plant development and defense response to multiple (a)biotic stresses. Plant Cell Rep. 2013, 32,
1
085–1098. [CrossRef] [PubMed]
1
1
1
1
3. Abdelillah, A.; Houcine, B.; Halima, D.; sari Meriel, C.; Imane, Z.; Eddine, S.D.; Abdallah, M.; sari Daoudi, C.
Evaluation of antifungal activity of free fatty acids methyl esters fraction isolated from Algerian Linum
usitatissimum L. seeds against toxigenic Aspergillus. Asian Pac. J. Trop. Biomed. 2013, 3, 443–448. [CrossRef]
4. Chandrasekaran, M.; Senthilkumar, A.; Venkatesalu, V. Antibacterial and antifungal efficacy of fatty acid
methyl esters from the leaves of Sesuvium portulacastrum L. Eur. Rev. Med. Pharmacol. Sci. 2011, 15, 775–780.
[PubMed]
5. Lorente, D.; Escandell-Montero, P.; Cubero, S.; Gómez-Sanchis, J.; Blasco, J. Visible–NIR reflectance
spectroscopy and manifold learning methods applied to the detection of fungal infections on citrus fruit.
J. Food Eng. 2015, 163, 17–24. [CrossRef]
6. Chafer, M.; Sanchez-Gonzalez, L.; Gonzalez-Martinez, C.; Chiralt, A. Fungal decay and shelf Life of oranges
coated with chitosan and bergamot, thyme, and tea tree essential oils. J. Food Sci. 2012, 77, E182–E187.
[CrossRef] [PubMed]
1
1
7. Moscoso-Ramírez, P.; Palou, L. Preventive and curative activity of postharvest potassium silicate treatments
to control green and blue molds on orange fruit. Eur. J. Plant Pathol. 2014, 138, 721–732. [CrossRef]
8. Droby, S.; Eick, A.; Macarisin, D.; Cohen, L.; Rafael, G.; Stange, R.; McColum, G.; Dudai, N.; Nasser, A.;
Wisniewski, M.; et al. Role of citrus volatiles in host recognition, germination and growth of Penicillium
digitatum and Penicillium italicum. Postharvest Biol. Technol. 2008, 49, 386–396. [CrossRef]
1
2
2
2
9. Moscoso-Ramírez, P.; Montesinos-Herrero, C.; Palou, L. Antifungal activity of sodium propylparaben alone
or in combination with low doses of imazalil against Penicillium decay on citrus fruit. Eur. J. Plant Pathol.
2
014, 140, 145–157. [CrossRef]
0. Marques, J.P.R.; Amorim, L.; Silva-Junior, G.J.; Spósito, M.B.; Appezzato-da Gloria, B. Structural and
biochemical characteristics of citrus flowers associated with defence against a fungal pathogen. AoB Plants
2
015, 7. [CrossRef] [PubMed]
1. Zeng, J.; Vedachalam, S.; Xiang, S.; Liu, X.-W. Direct C-glycosylation of organotrifluoroborates with glycosyl
fluorides and its application to the total synthesis of (+)-Varitriol. Org. Lett. 2011, 13, 42–45. [CrossRef]
[PubMed]
2. Wu, W.L.; Wu, Y.L. Stereoselective synthesis of methyl (11S,12S,13S)-(9Z,15Z)-11-hydroxy-12,13-epoxy
octadecadienoate from D-mannose. Tetrahedron 1993, 49, 4665–4670. [CrossRef]

Molecules 2016, 21, 254
11 of 11
2
3. Michieletti, M.; Bracci, A.; Compostella, F.; de Libero, G.; Mori, L.; Fallarini, S.; Lombardi, G.; Panza, L.
Synthesis of -galactosyl ceramide (KRN7000) and analogues thereof via a common precursor and their
α
preliminary biological assessment. J. Org. Chem. 2008, 73, 9192–9195. [CrossRef] [PubMed]
4. Ohrui, H.; Jones, G.H.; Moffatt, J.G.; Maddox, M.L.; Christensen, A.T.; Byram, S.K. C-Glycosyl nucleosides. V.
Some unexpected observations on the relative stabilities of compounds containing fused five-membered
rings with epimerizable substituenes. J. Am. Chem. Soc. 1975, 97, 4602–4613.
2
2
2
2
5. Yan, S.; Liang, Y.; Zhang, J.; Chen, Z.; Liu, C.-M. Autoxidated linolenic acid inhibits aflatoxin biosynthesis in
Aspergillus flavus via oxylipin species. Fungal Genet. Biol. 2015, 81, 229–237. [CrossRef] [PubMed]
6. Upchurch, R.G. Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress.
Biotechnol. Lett. 2008, 30, 967–977. [CrossRef] [PubMed]
7. Fu, L.-H.; Hu, K.-D.; Hu, L.-Y.; Li, Y.-H.; Hu, L.-B.; Yan, H.; Liu, Y.-S.; Zhang, H. An antifungal role of
hydrogen sulfide on the postharvest pathogens Aspergillus niger and Penicillium italicum. PLoS ONE 2014, 9.
[
CrossRef] [PubMed]
8. Liavonchanka, A.; Feussner, I. Lipoxygenases: Occurrence, functions and catalysis. J. Plant Physiol. 2006, 163,
48–357. [CrossRef] [PubMed]
9. Si, W. The Study on the Biological Characters of Postharvest Pathogen Diplodia natalensis in Ponkan Fruit
and Their Control. Master’s Thesis, Huazhong Agricultural University, Wuhan, China, 2011.
2
2
3
3
0. Zhu, Y.; Yu, J.; Brecht, J.K.; Jiang, T.; Zheng, X. Pre-harvest application of oxalic acid increases quality and
resistance to Penicillium expansum in kiwifruit during postharvest storage. Food Chem. 2016, 190, 537–543.
[CrossRef] [PubMed]
Sample Availability: Samples of the compounds 7 and 8 are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
(