Inhibitory effects of gossypol, gossypolone, and apogossypolone on a collection of economically important filamentous fungi

Article abstract of DOI:10.1021/jf2044394

Racemic gossypol and its related derivatives gossypolone and apogossypolone demonstrated significant growth inhibition against a diverse collection of filamentous fungi that included Aspergillus flavus, Aspergillus parasiticus, Aspergillus alliaceus, Aspergillus fumigatus, Fusarium graminearum, Fusarium moniliforme, Penicillium chrysogenum, Penicillium corylophilum, and Stachybotrys atra. The compounds were tested in a Czapek agar medium at a concentration of 100 μg/mL. Racemic gossypol and apogossypolone inhibited growth by up to 95%, whereas gossypolone effected 100% growth inhibition in all fungal isolates tested except A. flavus. Growth inhibition was variable during the observed time period for all tested fungi capable of growth in these treatment conditions. Gossypolone demonstrated significant aflatoxin biosynthesis inhibition in A. flavus AF13 (B₁, 76% inhibition). Apogossypolone was the most potent aflatoxin inhibitor, showing greater than 90% inhibition against A. flavus and greater than 65% inhibition against A. parasiticus (B₁, 67%; G ₁, 68%). Gossypol was an ineffectual inhibitor of aflatoxin biosynthesis in both A. flavus and A. parasiticus. Both gossypol and apogossypolone demonstrated significant inhibition of ochratoxin A production (47%; 91%, respectively) in cultures of A. alliaceus.

Full text of DOI:10.1021/jf2044394

Article
pubs.acs.org/JAFC
Inhibitory Effects of Gossypol, Gossypolone, and Apogossypolone on
a Collection of Economically Important Filamentous Fungi
Jay E. Mellon,*,† Carlos A. Zelaya,‡ Michael K. Dowd,† Shannon B. Beltz,† and Maren A. Klich†
†Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1100 Robert E. Lee Boulevard,
New Orleans, Louisiana 70124, United States
‡Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148, United States
ABSTRACT: Racemic gossypol and its related derivatives gossypolone and apogossypolone demonstrated significant growth
inhibition against a diverse collection of filamentous fungi that included Aspergillus flavus, Aspergillus parasiticus, Aspergillus
alliaceus, Aspergillus fumigatus, Fusarium graminearum, Fusarium moniliforme, Penicillium chrysogenum, Penicillium corylophilum,
and Stachybotrys atra. The compounds were tested in a Czapek agar medium at a concentration of 100 μg/mL. Racemic gossypol
and apogossypolone inhibited growth by up to 95%, whereas gossypolone effected 100% growth inhibition in all fungal isolates
tested except A. flavus. Growth inhibition was variable during the observed time period for all tested fungi capable of growth in
these treatment conditions. Gossypolone demonstrated significant aflatoxin biosynthesis inhibition in A. flavus AF13 (B1, 76%
inhibition). Apogossypolone was the most potent aflatoxin inhibitor, showing greater than 90% inhibition against A. flavus and
greater than 65% inhibition against A. parasiticus (B1, 67%; G1, 68%). Gossypol was an ineffectual inhibitor of aflatoxin
biosynthesis in both A. flavus and A. parasiticus. Both gossypol and apogossypolone demonstrated significant inhibition of
ochratoxin A production (47%; 91%, respectively) in cultures of A. alliaceus.
KEYWORDS: filamentous fungi, mycotoxins, gossypol derivatives, inhibitory activities
A. flavus is a ubiquitous saprophytic fungus commonly found
in tropical and subtropical climes.14 This organism is also an
opportunistic pathogen of a number of oilseed crops (e.g.,
cotton, maize, peanuts, tree nuts) and has agronomic
significance due to its production of the potent carcinogenic
INTRODUCTION
■
Gossypol, an optically active disesquiterpene (C30) produced
by the cotton plant (Gossypium hirsutum), is principally located
in lysigenous glands found throughout aerial tissues, including
cottonseed, and along the external surfaces of nonglandular
root tissue. Cottonseed kernels contain, on average, about 1.3%
gossypol by weight.1 Gossypol is considered an antinutrient
component of cottonseed and cottonseed meal,2 which limits
its use as an animal feed and practicably precludes its use as a
human protein source. It is known to exhibit a wide range of
bioactivity that includes anticancer, antimicrobial, and antiviral
effects.3 Gossypolone is a gossypol derivative formed by
oxidation with ferric chloride.4 Though less actively studied,
gossypolone has been reported to exhibit some anticancer
effects, although in general this activity is reduced in
comparison with gossypol.5−7 Apogossypolone is a related
derivative that is formed by conversion of gossypol to
apogossypol followed by oxidation to apogossypolone.8,9 This
compound has recently been reported to have stronger activity
against some cancer cells and is of particular interest because it
binds to and interferes with Bcl proteins that are associated
with disrupting apoptosis mechanisms in mammalian cancer
cells.9,10
Gossypol contributes to plant defenses through anti-insect
activity,11 and it may be involved in other plant defense
functions that include fungal inhibition. The (−)-enantiomer of
gossypol is four times as active as the (+)-enantiomer in
inhibition of conidial germination, mycelial growth, and
conidiophore development12 in Aspergillus flavus. In more
recent work, racemic gossypol, optical gossypol, and a number
of related gossypol derivatives (Figure 1) were found to inhibit
the growth of A. flavus.13
mycotoxin aflatoxin B1.15
Our initial investigations regarding the effects of a series of
gossypol derivatives revealed that both gossypolone and
apogossypolone were more effective than gossypol as growth
inhibitors against an A. flavus strain.13 To further assess the
range of biological activity of these compounds, gossypol,
gossypolone, and apogossypolone were evaluated for inhibition
against a collection of economically significant filamentous
fungi. In addition to Aspergilli that produce aflatoxins, other
important fungal species selected for this study include the
following: Aspergillus fumigatus, a major causative agent of
human aspergillosis; Aspergillus alliaceus, which produces
ochratoxin; Fusarium graminearum, a wheat pathogen; Fusarium
moniliforme, a causal agent of ear rot in maize; Penicillium
chrysogenum, a human pathogen (lung); Penicillium corylophi-
lum, an animal pathogen, and Stachybotrys atra, which has
become a fungal problem on cellulosic substrates under
favorable conditions following flooding events in housing
structures. Results of this investigation are presented here.
Received: October 31, 2011
Revised: February 9, 2012
Accepted: February 13, 2012
Published: February 13, 2012
© 2012 American Chemical Society
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Figure 1. Synthesis of gossypolone and apogossypolone from gossypol.
Terpenoid Compound Preparation/Purification. Gossypol−
acetic acid (1:1) was isolated from cottonseed soapstock as described
previously.16 Racemic gossypol was purified by dissolving the solvate
in diethyl ether and washing the ether phase with equal volumes of
water three times. The ether was then evaporated under a stream of
dry nitrogen, and the product was stored under vacuum for several
days to remove residual ether. Proton NMR spectroscopy indicated
that the product contained only trace levels of ether and acetic acid.
Gossypolone was prepared from gossypol acetic acid by mild oxidation
as described by Haas and Shirley4 (Figure 1). Apogossypolone was
prepared by the basic procedures of Adams and Butterbaugh8 and
Zhan et al.,9 first eliminating the formyl groups in concentrated base,
then oxidizing the inner benzyl rings to yield apogossypolone (Figure
1). Product yields were comparable to prior reports, and the products
were essentially pure by HPLC. The products had the expected NMR,
UV−vis, and mass spectrometric properties.
MATERIALS AND METHODS
■
Biological Materials. Isolates were selected from the Southern
Regional Research Center Permanent Culture Collection on the basis
Table 1. Filamentous Fungi Included in Survey of Terpenoid
Compound Effects
SRRC
no.
other
isolate name
source
infected
cottonseed
designations
toxin type
1000-F A. flavus
aflatoxins B1,
B2
1532
A. flavus AF13 field soil sample, ATCC
aflatoxins B1,
B2
Arizona
96044
143-A A. parasiticus
Uganda peanuts
ATCC
201461
aflatoxins B1,
B2, G1, G2
Fungal Incubations. Standard Czapek Dox medium17 containing
0.01 g/L ZnSO4·7H2O, 0.005 g/L CuSO4·5 H2O, and 2% (w/w) agar
was utilized as the fungal growth medium. The medium was adjusted
to pH 6.0 before heat sterilization, followed by equilibration to 60 °C.
Terpenoid compounds were dissolved in acetone as a carrier solvent
and were dispersed in the fungal medium to yield a test concentration
of 100 μg/mL. Control plates were also treated with an equivalent
amount of acetone (i.e., 5% v/v). The media were introduced into
sterile, disposable Petri plates (9 cm, 25 mL/plate) and allowed to
solidify. Plates were placed in a dark fume hood for 24 h to allow
acetone to dissipate. Following acetone evaporation, plates were stored
at 5 °C in the dark until fungal inoculation. Plates were single-point
inoculated (2 μL) in the center and incubated in the dark at 25 °C for
up to 25 days. A plate system was chosen because (1) homogeneous
distribution of the test compound in the medium could be achieved
and (2) physical surface/air interface system simulated field conditions
better than a liquid system does.
7-A
A. alliaceus
dead blister
beetle
NRRL 315 ochratoxin A
2582
1052
A. fumigatus
human sinus
tissue
F.
air sample from
cotton field
graminearum
1083
2275
F. moniliforme horse feed
fumonisin B1
P.
pasture grass,
New Zealand
ATCC
48673
corylophilum
1397
273
P. chrysogenum human lung
tissue
S. atra
NRRL 2186
of pathogenicity and toxigenicity. Table 1 shows identification
information for the fungal isolates selected for this investigation;
they were screened for purity and toxin production prior to the start of
the experimental work. Fungal inocula were constructed from mature
cultures at concentrations of 106 spores per mL in sterile inoculation
medium (0.0005% Triton X-100; 0.2% agar), as determined by a
hemocytometer.
Biomass Estimation. Beginning at 72 h postinoculation, plates
were read every two days for colony growth progression. Measure-
ments were continued until control colonies filled their plates, at which
time the entire series for that fungal strain was terminated. A cutoff
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Table 2. Colony Areas of Fungal Species Grown in the Presence of Various Gossypol Derivatives at 100 μg/mL
a
Concentrations
c
mean colony areas of plates at terminal reading (cm2)
b
d
fungus
growth period
control
gossypol
gossypolone
apogossypolone
LSD
A. flavus 1000-F
A. flavus AF13
17
15
17
15
17
5
53.0 11.3 a
50.2 1.2 a
55.9 1.4 a
51.8 1.0 a
48.1 1.7 a
43.8 1.4 a
40.7 0.7 a
18.1 0.2 a
28.7 3.4 a
29.1 1.1 a
8.02 2.77 b
6.81 0.63 c
25.5 1.6 c
15.4 1.3 b
14.2 0.9 b
0.64 0.2 c
4.9 0.5 b
13.9 5.3 b
11.4 1.0 b
12.1 1.6 b
34.0 1.9 b
3.41 1.1 c
9.56 0.65 c
1.76 0.48 b
1.01 0.14 c
1.56 0.1 c
3.18 0.5 c
16.9 0.8 c
7.72
1.84
2.02
1.41
1.66
1.07
0.63
0.45
2.64
1.10
8.56 2.26 c
A. parasiticus 143-A
A. alliaceus 7-A
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
A. fumigatus 2582
F. graminearum 1042
F. moniliforme 1083
P. corylophilum 2275
P. chrysogenum 1397
S. atra 263
7
e
25
13.1 0.5 b
17.9 1.4 b
e
25
e
25
14.9 0.7 b
a
b
Six replicates at each condition; variances expressed as standard deviations. Days required for fungal colony to almost fill control plate (i.e., one
c
observation period prior to plates being filled). Means within a row not followed by a common letter are significantly different based on LSD
comparisons at P ≤ 0.05. Value shown is based on average number of observations per treatment. As the data was unbalanced for a few isolates, the
actual LSD used for comparing means may have differed slightly. Incubation period of 25 days; controls did not completely fill medium plates.
d
e
Figure 2. Variation in growth inhibition exhibited by gossypol related compounds in tested filamentous fungi during incubation periods. All
terpenoid compounds were tested at a concentration of 100 μg/mL. Gossypolone completely inhibited growth of all tested fungi except A. flavus;
◇
●
gossypolone data is shown only for A. flavus isolates. Symbol key: gossypol, ( ); gossypolone, ( ); apogossypolone, (×). Error bars represent
estimated standard deviations calculated by a propagation of error analysis.19
time of 25 days was given for colonies that failed to fill the plate. Each
data point consisted of an average of two colony diameter
measurements taken at 90° to each other. Colony areas were
calculated from diameter measurements; inhibition was taken as the
ratio of the treatment and control areas expressed as a percentage.
Mycotoxin Analysis. Plates from each A. flavus/Aspergillus
parasiticus treatment were extracted for aflatoxin analysis. The entire
contents from each plate were macerated, placed in 75 mL of 65%
acetone (v/v, aqueous), and shaken in an orbital shaker (25 °C, 125
rpm) for at least 1 h. Solids were removed by filtration through
qualitative filter paper (Whatman #4). Methylene chloride, 25 mL, was
added to the aqueous acetone filtrate. The biphasic mixture was
shaken; phases were allowed to separate, and the organic (lower)
phase was collected. Water was removed with anhydrous sodium
sulfate, and the solvent was evaporated under ambient conditions.
Each sample was resuspended in 5 mL of methylene chloride and
transferred to a 1 dram vial. Contents were again allowed to dry by
evaporation and resolvated in a small aliquot of acetone (volume
depends on toxin level). Four microliters of each sample was spotted
on silica gel G thin layer plates, which were developed in diethyl ether/
methanol/water (96:3:1) mobile phase. Aflatoxins B1 and G1 were
quantitated directly on thin layer plates by fluorescence densitometry
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(Shimadzu 9301PC), comparing Rf values to standards (Sigma
Chemical Co., St. Louis, MO).
some growth in the presence of this compound appeared to
lose a degree of potency over time (Figure 2A,B).
Ochratoxin A was extracted from A. alliaceus cultures as described
above. Ochratoxin A (OTA) analysis was performed according to the
procedure described by Bonvehi.18 Analyses were performed with a
Waters 2695 HPLC combined with a Waters 2475 fluorescence
detector; postcolumn derivatization was done with a photochemical
reactor for enhanced detection (PHRED) (Aura Industries Inc., New
York, NY) system. Detection of ochratoxin A was performed with an
excitation wavelength of 333 nm and an emission wavelength of 460
nm. Sample extracts (10 μL; n = 3) were analyzed by injection onto a
reverse phase column. The column used was a 150 mm × 3.9 mm i.d.,
5 μm, Nova-Pak C18, with a 20 mm × 3.9 mm i.d. guard column of the
same material (Waters, Milford, MA). Column temperature was
maintained at 38 °C. Elution flow rate was 0.8 mL/min with a mobile
phase solvent consisting of acetonitrile/water/acetic acid (51:47:2, v/
v/v). Retention time for ochratoxin A was about 6 min. A calibration
curve with high linearity (r2 = 0.998) was constructed for ochratoxin A
from a series of diluted standards (Sigma Chemical Co.).
Calculations and Statistics. The design of the experiment was
completely randomized with six replicates per treatment for each
isolate. At the completion of the growth cycle, three of the replicate
plates for the A. alliaceus, A. flavus, and A. parasiticus isolates were then
randomly chosen for toxin analysis. Least significant difference
comparisons at a significance level of P ≤ 0.05 were performed on
fungal colony areas and on total toxin levels. Standard deviations for %
inhibition were estimated from an analysis of propagation of error
based on the control and treatment colony areas and their standard
deviations.19
Aflatoxin analysis of the A. flavus/A. parasiticus cultures
following incubation termination revealed apparent inhibition
of aflatoxin biosynthesis by all of the tested compounds (Table
3). However, when the data were normalized to account for
Table 3. Effect of Gossypol-Related Compounds on
Mycotoxin Production in A. flavus, A. parasiticus, and A.
a
alliaceus
toxin
b
toxin level
normalized toxin inhibn
c
treatment
control
type
SD, μg
level
(%)
A. flavus 1000-F
27.3 0.94
9.64 2.29
7.55 2.21
0.36 0.12
3.13
B1
B1
B1
B1
n/a
n/a
0
gossypol
63.8
28.7
1.66
gossypolone
apogossypolone
0
94
d
LSD
A. flavus AF13
30.4 1.32
2.87 0.73
1.25 0.82
0.21 0.1
1.62
control
B1
B1
B1
B1
n/a
n/a
30.6
76
gossypol
21.1
7.31
0.83
gossypolone
apogossypolone
LSD
97
A. parasiticus 143-A
34.1 1.87
13.8 1.62
6.83 0.56
2.92
control
B1
B1
B1
n/a
n/a
gossypol
apogossypolone
LSD
30.3
11.2
11.1
67.2
RESULTS
■
A fairly wide range of growth rates was displayed by the fungi
under the growth conditions of the experiment (Table 2). The
Fusarium isolates displayed the fastest growth rates, with the
control plates being fully covered in 7 to 9 days. The Aspergillus
isolates all showed relatively moderate growth rates, covering
the plates in 15 to 19 days. The Penicillium and Stachybotrys
isolates grew at significantly slower rates and had not fully
covered the plates after 25 days of growth.
A. parasiticus 143-A
136 10.2
102 17.4
26.4 0.7
23.3
control
G1
G1
G1
n/a
224
43.4
n/a
0
gossypol
apogossypolone
LSD
68.1
A. alliaceus 7-A
928 83
control
OTA
OTA
OTA
n/a
494
82.4
n/a
Table 2 shows that the three compounds of interest
displayed variable growth inhibition against the fungal isolates.
Racemic gossypol demonstrated a wide range of inhibition. It
showed relatively poor activity against the slower growing
Penicillium species, while it was moderately effective against F.
graminearum and A. flavus. Apogossypolone was a more
effective growth inhibitor, demonstrating good activity in all
tested fungi except A. parasiticus and S. atra. Gossypolone was
the most effective growth inhibitor tested, resulting in full
inhibition in all tested fungi except A. flavus.
Analysis of the growth inhibition activity displayed during the
course of the fungal incubations revealed a range of temporal
trends. In some cases, relative inhibition remained somewhat
nonvariable over the course of the incubation. For example, the
measured gossypol growth inhibition of A. fumigatus was 70%
at both 3 and 17 days, with little variation in the intermediate
time points (Figure 2E). Likewise, gossypol growth inhibition
of A. alliaceus varied from 76% at 3 days to 70% at 15 days
(Figure 2D). In other cases, inhibition potency appeared to
increase over the incubation period, e.g., gossypol inhibition of
A. flavus 1000-F varied from about 48% at 3 days to 85% at 17
days (Figure 2A). Significant decreases in apparent inhibition
over time were also observed. This trend was observed for
gossypol inhibition of the Penicillium species (Figure 2H,I) and
for apogossypolone inhibition of A. parasiticus and S. atra
(Figure 2C,J). While almost all fungi studied were completely
inhibited by gossypolone, both A. flavus isolates that exhibited
gossypol
apogossypolone
LSD
147 14
46.8
91.1
5.42 1.21
96.6
a
b
c
Data is reported as average weight of mycotoxin per plate (n = 3).
B1 = aflatoxin B1; G1 = aflatoxin G1; OTA = ochratoxin A.
Mycotoxin data was transformed to normalize area differences
d
between control and treated plates (n/a = not applicable). LSD of
treatments at P ≤ 0.05 for each isolate/toxin combination.
colony area differences between the control and compound-
treated cultures, it became apparent that gossypol exhibited
little aflatoxin inhibitory activity. In contrast, both gossypolone
and apogossypolone appeared to inhibit aflatoxin production in
Aspergillus species. Apogossypolone was the most effective
agent with respect to aflatoxin inhibition, reducing toxin levels
by 67−68% for A. parasiticus and 94−97% for A. flavus.
Gossypolone also demonstrated significant aflatoxin inhibition
activity in the A. flavus AF13 cultures (76% reduction in toxin)
but not in the 1000-F cultures. Both gossypol and
apogossypolone demonstrated significant inhibitory activity
against ochratoxin A production in A. alliaceus cultures (Table
3).
DISCUSSION
■
The considerable range in growth rates for the fungi used in
this investigation was not unexpected. A. flavus, A. parasiticus,
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and A. fumigatus isolates are adapted to more rapid growth at
higher temperatures (30−37 °C).20 The Fusarium isolates are
adapted to optimal growth near 25 °C.21 The Stachybotrys atra
isolate is known to be a slow grower under most conditions.
The Czapek Dox medium used in the study also may have
contributed to the different rates of growth, as the fungi would
likely have different abilities to cope with the limited available
resources.
Racemic gossypol displayed a large variability in its ability to
inhibit growth in the fungi included in this investigation. It was
particularly effective (>80% inhibition) against Fusarium
species, but much less effective (<30% inhibition) against the
Penicillium species. This observation may be related to a
differential effect of gossypol penetration through fungal cell
walls, depending on the lipid composition of the fungal
plasmalemma in question.22 Overall, apogossypolone and
gossypolone were more effective growth inhibitors, with
gossypolone causing total inhibition in all tested fungi except
A. flavus.
The variable inhibitions observed among species, and the
changes in potency over time within a species, suggest that the
compounds likely interact with the fungi through a variety of
mechanisms. The literature of gossypol-affected metabolic
enzymes is large and the current study of apogossypolone as
a Bcl inhibitor also points to metabolic inhibition. In addition,
the relatively biplanar structure of gossypol (i.e., essentially
planar naphthalene rings with hydrophobic and hydrophilic
sides oriented approximately perpendicular to each other)
suggests that the molecule can at least partially intercalate into
membranes and interfere with transport functions.22 Other
effects are also possible. Gossypol has been shown to be an
uncoupler (inhibitor) of oxidative phosphorylation in mamma-
lian mitochondrial membranes, presumably through perturba-
tion of normal membrane function.23 In addition, gossypol
perturbation of membranes may negatively affect the function
of membrane proteins (e.g., P450 oxidases), contributing to
some observed biological activities. For example, spore
germination inhibition is distinct from mycelial growth
inhibition. Gossypol is a known inhibitor of A. flavus conidial
germination12 and also appears to inhibit viral replication.3 The
almost complete inhibition of fungal growth by gossypolone
suggests that the compound may act as a general spore
germination inhibitor.
The mechanism of action for the inhibitory effects of
gossypol and its derivatives is not well understood.
Gossypolone differs structurally from gossypol in that its
quinoid rings are more flexible (and able to pucker modestly
into flattened boat-type conformations) compared with the
essentially planar naphthalene rings of gossypol. In addition, the
presence of the quinoid oxygen atom disrupts the side-to-side
naphthalene ring polarity present within the gossypol molecule
(Figure 1). The added flexibility might allow for stronger
interactions with active sites, which may contribute to the
improved inhibition of the compound compared with gossypol.
Apogossypolone also would have the added flexibility of the
backbone structure but differs in the loss of the formyl moieties.
The aldehyde groups of gossypol are well-known to form
Schiff’s base derivatives with amines, including the terminal and
lysine side chain amines of proteins. These groups allow
gossypol to react in a nonspecific manner with amines of
various moieties, including cellular proteins. This effect has
been assumed to reduce the effective intracellular concentration
of the compound and lower its activity. If this were the only
important factor, however, then apogossypolone would be
expected to be more effective than gossypolone as a growth
inhibitor. That it is not suggests that the aldehyde moieties play
a more direct role in the inhibition, possibly allowing for
gossypolone−Schiff’s base interactions with proteins that
contribute to critical functions.
Biological activity and animal toxicity effects of gossypol are
well-known to be variable and subject to change with particular
conditions used during study, which has made understanding
the compound’s various metabolic effects, or exploiting of the
compound’s range of activity, challenging. Nevertheless, the
large variation in fungal growth inhibition observed over
incubation time was unexpected. That some cultures were able
to reduce the inhibitory effects over time suggests that the
organism was able to adjust metabolically (e.g., by increasing an
affected enzyme level) to correct for critical function, or was
able to sequester or modify the compounds to reduce their
effect. In some cases, fungal inhibition was relatively constant,
suggesting that these organisms may have been less able to
adjust or accommodate the inhibitory processes. Some fungi
showed increased inhibition over time, suggesting that slowly
increasing intracellular concentrations resulting from rate
limiting penetration of the compounds through mycelial walls
may have been a factor in the observed effects. In the case of
reversals of growth inhibition (Figure 2A,B), reversibility of
terpenoid binding to sites of action is consistent with this
observation. Undoubtedly, all of these factors contribute to the
net effect, with the apparent variation resulting from small
differences in the capabilities of the various fungal species.
It is possible that gossypolone and apogossypolone show
biological activities similar to those of plant-derived antimicro-
bial phytoalexins. A possible mechanism of action for this class
of antimicrobial substances would be interference with
membrane transport characteristics in cellular metabolism.24
Thus, perhaps gossypolone and apogossypolone are disrupting
the ability of the fungus to import needed carbon and nitrogen
resources, limiting cell wall biosynthesis. This hypothesis is
consistent with the ability of gossypol to interfere with
transporter mechanisms. Gossypol is a known inhibitor of
hexose transporter mechanisms in mammalian membranes.25,26
The mechanism of aflatoxin inhibitory activity expressed by
apogossypolone is currently unknown. There is good evidence
to support the model that later stages of aflatoxin biosynthesis
occur in vesicles termed “aflatoxisomes”.27 Further, the export
of aflatoxin outside of the mycelium appears to occur through
exocytosis by fusion of these vesicles with the fungal
plasmalemma membrane.28 Possibly, apogossypolone exerts
its antiaflatoxigenic effects either by interfering with the
formation of aflatoxisomes or by interfering with transport
phenomena of aflatoxin substrates through vesicle membranes.
In short, these gossypol-related compounds are probably
exerting their inhibitory effects at multiple cellular locations
through a variety of mechanisms.
Gossypol is generally understood to be a plant defensive
compound that helps reduce insect predation. However, the
compound is also found along the exterior root bark of cotton
plants where insect damage is less likely; hence, it is possible
that gossypol has additional biological roles to reduce or
impede infection of root tissue. Nematode infection of cotton
roots has not been found to elicit gossypol or its related
compounds strongly,29 but methyl jasmonate strongly elicits
the production of gossypol in cotton hairy root tissue
cultures.30 The finding that gossypol has growth inhibitory
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Journal of Agricultural and Food Chemistry
Article
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activity against a collection of fungal plant pathogens is
consistent with this hypothesis.
Although gossypol and its derivatives have demonstrated a
wide range of bioactivity, it has yet to find a commercial
purpose that would justify the cost of its recovery from cotton
plant tissues. This application deficit may be due to the lack of
sufficient activity to compete with current products and
unwanted side effects, i.e., insufficient therapeutic index, or
complicating physical properties, e.g., instability to light and
nonspecific reactivity with cellular components. The com-
pounds evaluated in this report have not been well studied for
antifungal activity. Given the ability of gossypol to inhibit fungal
growth, the compound might be useful as a seed coating to
impede fungal contamination of germinating seedlings. These
compounds, as a class, tend to be sensitive to UV radiation, and
this type of application might overcome this complication.
Since gossypolone and apogossypolone exhibit some improved
inhibitory activity compared with gossypol, testing these
compounds for this purpose might also be warranted. Proof
of the efficacy of these agents for this application, however, will
require further investigation.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: (504)286-4358. Fax: (504) 286-4419. E-mail: Jay.
Mellon@ars.usda.gov.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The assistance of C. H. Carter-Wientjes in the acquisition of
ochratoxin A data and of D. Boykin in the programming of the
statistical analyses is greatly appreciated. Mention of trade
names or commercial products in this paper is solely for the
purpose of providing specific information and does not imply
recommendation or endorsement by the U.S. Department of
Agriculture.
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P. T. T. Gossypol effects on the structure and dynamics of
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119−125.
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