Journal of Chemical Ecology, Vol. 31, No.

4, April 2005 ( #2005)

DOI: 10.1007/s10886-005-3544-x

COMPARISON OF ANTIFUNGAL AND ANTIOXIDANT

ACTIVITIES OF Acacia mangium AND A. auriculiformis
HEARTWOOD EXTRACTS

RIE MIHARA,1 KAREN M. BARRY,2,3 CAROLINE L. MOHAMMED,2,3,4

and TOHRU MITSUNAGA1,*
1
Faculty of Bioresources, Department of Environment Science and Engineering,
Mie University, 1515 Kamihama, Tsu 514-8507, Japan
2
CRC for Sustainable Production Forestry, Private Bag 12, Hobart Tasmania,
Australia 7001
3
School of Agricultural Science, University of Tasmania, Private Bag 12, Hobart Tasmania,
Australia 7001
4
CSIRO Forestry and Forest Products, Private Bag 12, Hobart Tasmania, Australia 7001

(Received July 27, 2004; accepted December 9, 2004)

Abstract—The effect of heartwood extracts from Acacia mangium (heartrot-

susceptible) and A. auriculiformis (heartrot-resistant) was examined on the
growth of wood rotting fungi with in vitro assays. A. auriculiformis heartwood
extracts had higher antifungal activity than A. mangium. The compounds
3,40 ,7,8-tetrahydroxyflavanone and teracacidin (the most abundant flavonoids
in both species) showed antifungal activity. A. auriculiformis contained higher
levels of these flavonoids (3.5- and 43-fold higher, respectively) than A.
mangium. This suggests that higher levels of these compounds may contribute
to heartrot resistance. Furthermore, both flavonoids had strong 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging activity and laccase inhibition. This
suggests that the antifungal mechanism of these compounds may involve
inhibition of fungal growth by quenching of free radicals produced by the
extracellular fungal enzyme laccase.

Key WordsVAcacia mangium, A. auriculiformis, heartrot, heartwood ex-

tracts, flavanone, Phellinus noxius, P. badius, antifungal activity, antioxidant,
laccase inhibition.

* To whom correspondence should be addressed. E-mail: mitunaga@cc.gifu-u.ac.jp

789
0098-0331/05/0400-0789/0 # 2005 Springer Science + Business Media, Inc.
790 MIHARA ET AL.

INTRODUCTION

Polyphenolic compounds are common in higher plants (Haslam, 1988) and have
properties that afford plant tissues protection from the external environment
including antimicrobial (Scalbert, 1991; Field and Lettinga, 1992; Mila et al.,
1996), antifeedant (Butler, 1992), and antioxidant properties (Hagerman et al.,
1998; Eyles et al., 2004). In woody tree species, polyphenolic compounds ac-
cumulate in bark, leaves, and heartwood, while lesser amounts are found in
sapwood (Hillis, 1987). Extracts from bark and heartwood of many woody tree
species have strong biological activities, such as enzyme inhibition (Mitsunaga
et al., 1997; Shimizu et al., 1998; Juntheikki and Julkunen-Titto, 2000), anti-
oxidant activity (Chang et al., 2001; Willför et al., 2003), and antifungal activity
(Kishino et al., 1995). Durability of wood (resistance to fungal decay) is often
attributed to extractive content (Hart and Hillis, 1974; Scalbert, 1992; Harju
et al., 2002).
Acacia mangium is a fast-growing hardwood planted widely in many areas
of Asia and is susceptible to heartrot (Lee et al., 1988; Mahmud et al., 1993; Ito
and Nanis, 1994; Barry et al., 2004). Heartrot is caused by fungal infections of
heartwood via dead or broken branches (Mahmud et al., 1993; Ito and Nanis,
1994). A. auriculiformis, a closely related species, and hybrids of A. auriculi-
formis
A. mangium are rarely affected by heartrot (Ito and Nanis, 1997). One
explanation for the difference in susceptibility is an increased incidence of
infections associated with the higher proportion of infection courts provided by
large-diameter branches that occur more frequently in A. mangium than in A.
auriculiformis (Ito and Nanis, 1997). Heartwood extractives may also influence
susceptibility to fungal infection and require further investigation in these
Acacia spp. (Barry et al., in press).
Extracts from A. mangium heartwood have been investigated (Tachi et al.,
1989; Lange and Hashim, 2001; Barry et al., in press). 3,40 ,7,8-Tetrahydroxy-
flavanone (1) (Figure 1) and 40 ,7,8-trihydroxyfavanone (2) are the main fla-
vonoid compounds in healthy A. mangium heartwood, but their levels are
markedly reduced in decayed heartwood (Barry et al., in press). This decrease is

FIG. 1. Structures of compounds 1, 2, and teracacidin.

COMPARISON OF ANTIFUNGAL AND ANTIOXIDANT ACTIVITIES 791

probably caused by enzymatic degradation of phenolic compounds associated

with heartrot fungi (Barry et al., in press). While the identities of fungi causing
A. mangium heartrot are not well known, Phellinus noxius has been isolated
from early stages of decay in A. mangium (Lee and Zakaria, 1993; Lee and
Yahya, 1999). P. badius has been isolated from the heartrot of Acacia spp.
(Quraishi and Ahmad, 1973; Stalpers, 1978)
In this study, we compare the antifungal properties of heartwood extracts
from A. mangium and A. auriculiformis against P. noxius and P. badius using in
vitro bioassays. Possible antifungal mechanisms (extracts acting as antioxidants
or laccase inhibitors) are investigated.

METHODS AND MATERIALS

Wood Materials. Six-year-old trees of A. mangium and A. auriculiformis

were harvested from an experimental plantation in northern Queensland,
Australia, in April 2003. Two trees from each species were analyzed. For bio-
assays, extracts from one tree of each species were used. Each tree was felled
and then a billet was cut from a height of 3 m above ground level. The billets
were dried at 40-C for 2 wk. The heartwood of each tree was separated from
the bark and sapwood, and milled using a Wiley Mill (Thomas Wiley Mill
Model 4; Arthur H. Thomas Company, USA). After drying at 40-C for 1 wk,
the ground heartwood samples (600 g) were extracted (two times) for 2 d each
with 5 l of methanol. Extracts were maintained in the dark at room temperature
on a shaker. The methanolic extracts were filtered through Whatman No. 5 filter
paper (UK). Solvents were evaporated to dryness on a rotary evaporator at 40-C.
The total extracts of A. mangium and A. auriculiformis wood were abbreviated
as M-T (yield = 2.9 and 4.0% based on air dried wood, w/w) and A-T (9.3 and
8.0%), respectively.
Successive Extraction. Extractions were carried out as previously reported
(Barry et al., in press). Methanol extracts (13 g) of A. mangium and A. auriculi-
formis were dissolved in 10 ml of methanol and extracted with 1 l of diethyl
ether, ethyl acetate, and n-butanol, successively. Each extraction was conducted
at room temperature for 2 hr. Residues were collected by centrifugation (1400
g,
10 min) and supernatants were evaporated at 40-C. Three fractions and the re-
sidue were obtained and coded as M-A (yield = 0.94% based on air dried wood,
w/w), M-B (0.43%), M-C (0.37%), and M-D (0.35%) for A. mangium, and A-A
(4.54%), A-B (1.04%), A-C (0.82%), and A-D (0.03%) for A. auriculiformis.
Isolation of Flavonoids. 3,40 ,7,8-Tetrahydroxyflavanone (1), 40 ,7,8-Trihy-
droxyflavanone (2), and teracacidin were isolated as previously described
(Barry et al., in press). M-A (2 g) was dissolved in n-hexane/acetone (1:1, v/v),
792 MIHARA ET AL.

applied to silica gel 60 for column chromatography (Merck, Australia), and

eluted with n-hexane/acetone (7:3 then 1:1) and methanol. The n-hexane/
acetone (1:1) fraction was further purified by preparative thin layer chromatog-
raphy (TLC) to isolate compounds 1 and 2. Preparative TLC was completed
using silica gel 60 plates (Merck, Germany) with methanol/dichloromethane
(8:92, v/v).
Teracacidin was isolated from the A. auriculiformis methanol extract be-
cause it occurred in much higher amounts than in A. mangium. Fraction A-A (3 g)
was dissolved in 300 ml of ethyl acetate and extracted with water (3
200 ml)
in a separatory funnel. The water-soluble fraction was purified by preparative
HPLC using a reverse phase column (Develosil ODS-10, 20 mm i.d.
250 mm;
Nomura Chemical Co. Ltd., Japan) monitored at 280 nm. The solvent system
used was as follows: a linear gradient elution for 40 min from 15% to 60%
solvent B (methanol) in solvent A (0.01% TFA in H2O) at a flow rate of 10 ml/
min. The elution time of teracacidin was 18 min.
Antifungal Bioassays. The liquid medium bioassay was adapted from Hart
and Hillis (1974). P. noxius (Melbourne C2558) and P. badius (Melbourne
C3437) were subcultured on malt agar plates (1% malt, 1.5% agar) for 2 wk at
20-C and 36-C, respectively. A 2-ml aliquot of 13% aqueous ethanol solution
containing a fixed amount of A. mangium and A. auriculiformis methanol ex-
tracts was added to 48 ml of 1% malt liquid media to obtain final concentrations
of 0.1, 1.0, and 10.0 mg/ml extract. The malt liquid medium was inoculated
with a 5-mm-diam plug of fungal culture and incubated at 20-C for 2 wk with
shaking. After incubation, the cultures were filtered (Whatman filter paper No.
2, UK), the mycelia washed with methanol, dried at 50-C for 2 d, and the fungal
weights measured. Three replicates were prepared for each treatment, and the
results were averaged.
The agar dilution bioassay was adapted from Yoshimoto et al. (1984). A
1-ml aliquot of 20% aqueous ethanol solution containing a fixed amount of
methanol extract and each fraction of A. mangium and A. auriculiformis was
added to 9 ml of malt agar solution to obtain a final concentration of 0.1%,
0.3%, and 0.5% (w/v) extract. These malt agar solutions (2 ml) were transferred
in sterile test tubes to make agar pieces 10 cm in length. The agar was inocu-
lated by placing a 5-mm-diam plug of fungal culture on the edge of the agar
(Figure 2) and incubated at 28-C. Mycelia growth was measured sequentially
over a period of 30 d. Three replicates were undertaken for each treatment, and
the results were averaged. The same method was used for bioassay of the pure
compounds that were tested at a concentration of 8 and 15 mM.
Quantitative HPLC Analysis. Quantification of compounds 1, 2, and tera-
cacidin was achieved by HPLC (CLASS M10A; Shimadzu Co. Ltd., Japan)
analysis of methanol extracts of A. mangium and A. auriculiformis using a De-
velosil ODS HG-5 reversed phase column (4.6 mm i.d.
250 mm; Nomura
COMPARISON OF ANTIFUNGAL AND ANTIOXIDANT ACTIVITIES 793

FIG. 2. Agar dilution bioassay method.

Chemical Co. Ltd., Japan) and monitoring at 280 nm. The solvent system used
was as follows: a linear gradient for 45 min from 5% to 90% solvent B
(methanol) in solvent A (0.01% TFA in H2O) at a flow rate of 1 ml/min. The
amount of each compound was calculated with a calibration curve of each pure
compound based on the relationship of the HPLC chromatogram peak area and
concentration of the compounds.
Evaluation of 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) Radical Scavenging
Activity. The DPPH radical scavenging activity was measured as previously
reported (Mihara et al., 2004). A test sample (ethanol solution, 2 ml) was added
to 0.5 mM DPPH in ethanol (1 ml) mixed with a 50 mM 2-monopholinoeth-
anesulfonic acid buffer (pH 7.4, 2 ml), and the mixture was shaken vigorously.
After 15 min incubation at 25-C, the relative decrease in absorbance of the test
sample mixtures at 517 nm was measured on a Jasco V-520 UV/Vis spectro-
photometer (JASCO Co. Ltd., Japan). The DPPH radical scavenging activity
was expressed as the relative activity compared to the 0.5 mM butylhydroxy-
toluen (BHT). Quercetin and gallic acid were tested as positive controls of
natural antioxidants (Amić et al., 2003; Eyles et al., 2004). The experiment was
performed in duplicate, and the following formula was used to calculate results:

DPPH radical scavenging activity ð%Þ

¼ fðethanol aloneÞ  ðtest sampleÞg=fðethanol aloneÞ  ðBHTÞg
100

Laccase Inhibitory Activity. This assay was conducted by using syringal-

dazine as a substrate (Leonowicz and Grzywnowicz, 1981). Laccase (EC
1.10.3.2) was purchased from Sigma-Aldrich Japan Co. Ltd. Each extract was
dissolved in 10% aqueous dimethyl sulfoxide (DMSO) to give 0.3 mg/ml of
sample solution. A 0.05-ml aliquot of sample solution was mixed with 0.9 ml of
0.1 M phosphate buffer (pH 6.5) and 0.067 ml of 0.5 mM syringaldazine
ethanol solution. A 0.1-ml aliquot of laccase solution (288 U/ml) was added to
the above mixture. After incubation at 30-C for 30 min, the absorbance at 525
nm was measured. For the control, 10% aqueous DMSO was used instead of the
794 MIHARA ET AL.

sample solution. The experiment was performed in duplicate, and the following
formula was used to calculate activity:

Laccase inhibition ð%Þ ¼ fðcontrolÞ  ðtest sampleÞg=control
100

Enzymatic Reaction Kinetics. A 0.1-ml aliquot of syringaldazine ethanol

solution (0.13Y0.5 mM) was mixed with 0.74 ml of 0.1 M phosphate buffer (pH
6.5) and 0.06 ml of 0.1 mM teracacidin or 10% aqueous DMSO. A 0.1-ml aliquot
of commercial laccase solution (288 U/ml) was added to the above mixture and
incubated at 30-C. Absorbance at 525 nm was measured over 20 min. Reaction
rates were calculated from the difference between absorbance at 15 and 20 min
(absorbance increased linearly between 10 and 25 min).
Statistical Analysis. All statistical analyses were completed with SAS for
Windows Version 8 (SAS Institute, 1999). For the liquid bioassay, a three-way
ANOVA was conducted on log-transformed data with fixed factors of extract
type (A. mangium or A. auriculiformis), extract concentration, and fungus type
(P. badius or P. noxius). For the agar dilution bioassay testing different frac-
tions, a two-way ANOVA was conducted on log-transformed data for fixed
factors extract type and concentration. The ANOVA was conducted on the data
from the final measurement at 30 d.

RESULTS

Comparison of Antifungal Activity. A. auriculiformis methanol extracts had

strong antifungal effects on the growth of P. noxius and P. badius. A. mangium
extracts showed no effect on P. noxius growth and little inhibition of P. badius
compared to A. auriculiformis (Figure 3). ANOVA of the whole bioassay
showed high significance (P < 0.001), and all main factors were significant,
including fungal species and extract type (P < 0.001) and extract concentration
(P < 0.001). Second-order interactions were significant between extract type

concentration (P < 0.001) and fungal species
extract concentration (P =
0.039).
The largest difference between the two Acacia spp. extracts was at the
highest concentration (10 mg/ml) on the growth of P. badius (Figure 3).
A. auriculiformis extract inhibited fungal growth, while A. mangium extract did
not. The inhibitory effect of A. auriculiformis extracts on growth of both
P. noxius and P. badius increased in a concentration-dependent manner.
An antifungal bioassay of fractions obtained by successive extraction was
conducted with an agar dilution method using P. badius only because P. noxius
growth appeared to be inhibited by ethanol (Figure 3). Fraction A-D was not
tested due to low yield (56 mg). Only the results from the highest concentration
COMPARISON OF ANTIFUNGAL AND ANTIOXIDANT ACTIVITIES 795

FIG. 3. Antifungal activities of methanol extracts from A. auriculiformis (filled diamond)

and A. mangium (open square) heartwood against (i) P. noxius and (ii) P. badius. Error
bars indicate TSEM of three replicates. Fungal growth rates represented as percentage
of fungal growth of water control. Fungal growth of ethanol control for P. noxius and
P. badius was 59.3% (T9.5) and 94.7% (T18.8), respectively.

(0.5%) are presented (Figure 4). The test was statistically significant based on
the two-factor fixed ANOVA (P < 0.001), and extract type (P < 0.001) con-
tributed to this more than concentration (P < 0.001). However, the interaction
between the factors was significant (P = 0.007). A. auriculiformis methanol
extract (A-T) and fractions A-A, A-B inhibited growth of P. badius. Only
the diethyl ether fraction of A. mangium (M-A) was inhibitory to P. badius
(Figure 4). Other fractions of A. mangium and A-C accelerated fungal growth.
HPLC analyses confirmed the presence of compounds 1, 2, and teracacidin in
fractions A-A, A-B, and M-A, while A-C, M-B, M-C, and M-D showed amal-
gamation of broad peaks.
The same assay was conducted using compound 1 and teracacidin at two
different concentrations. Compound 2 was not tested due to low yield. Both
compound 1 and teracacidin were inhibitory against P. badius at a concentration
of 15 mM, but not at 8 mM (Figure 5). Compound 1 showed slightly stronger
activity than teracacidin at similar concentrations. A. auriculiformis heartwood
contained 3.5-fold of compound 1, 1.5-fold of compound 2, and 43-fold of tera-
cacidin per unit weight of dry wood compared to A. mangium (Table 1).
Antioxidant and Laccase Inhibitory Activity. The DPPH radical scaveng-
ing activities of A. mangium and A. auriculiformis extracts were determined
(Table 2). DPPH radical scavenging activity was defined as the effective con-
centration of extract fraction needed to decrease the initial DPPH radical by 50%
(EC50). The EC50 value of A-T was ca. 3-fold higher than that of M-T. Frac-
tions A-A, A-B, and M-A also had strong antioxidant activity. Furthermore,
796 MIHARA ET AL.

FIG. 4. Antifungal activities of successive extracts from (i) A. mangium and (ii) A.
auriculiformis heartwood methanol extracts against P. badius. The concentration of
extracts was 0.5% per agar medium. Error bars indicate TSEM of three replicates. M-T and
A-T correspond to methanol extracts of A. mangium and A. auriculiformis heartwood,
respectively. M-A-C and A-A-C correspond to fractions obtained from A. mangium and
A. auriculiformis, respectively, by successive extractions with diethyl ether, ethyl acetate,
and n-butanol. M-D and A-D correspond to the extract residues.
COMPARISON OF ANTIFUNGAL AND ANTIOXIDANT ACTIVITIES 797

FIG. 5. Antifungal activities of compound 1 and teracacidin against P. badius. Error bars
indicate TSEM of three replicates.

compounds 1, 2, and teracacidin (the main compounds of A-A) had strong

antioxidant activity.
Compounds 1, 2, and teracacidin all had strongly inhibitory laccase activity
(Table 2). The rank order of laccase inhibition by these compounds was the
same as the DPPH radical scavenging activity (compound 2 > compound 1 >
teracacidin). Fractions that were strongly antioxidant (M-A, M-B, A-T, A-A,
and A-B) all inhibited laccase activity. LineweaverYBurk plots of teracacidin
activity toward syringaldazine as a substrate is shown in Figure 6. The results
suggest that, for teracacidin, the type of laccase inhibition is uncompetitive.
Relationships between antifungal, antioxidant, and laccase inhibitory
activity of each extract and fraction were explored using plots and calculating

TABLE 1. CONTENTS OF COMPOUNDS 1, 2, AND TERACACIDIN IN Acacia SPECIES

HEARTWOOD

A. mangium A. auriculiformis
a
% Per extract (w/w) % Per wood % Per extract % Per wood

Compound 1 4.52 0.15 6.25 0.53

Compound 2 1.23 0.04 0.68 0.06
Teracacidin 4.85 0.17 83.94 7.35
a
The mean of extracts from two individual trees.
798 MIHARA ET AL.

TABLE 2. ANTIOXIDANT AND LACCASE INHIBITORY ACTIVITIES OF HEARTWOOD

EXTRACTS FROM Acacia SPECIES

EC50 value of DPPH radical scavenging a

Sample (mg/ml) (nmol/ml) Laccase inhibition (%) b

M-T c 24.7 Y 36.5

M-A 9.5 Y 57.3
M-B 13.4 Y 78.4
M-C 32.6 Y 10.1
M-D 61.9 Y n.d.
A-T 6.4 Y 92.4
A-A 5.9 Y 92.0
A-B 7.5 Y 91.0
A-C 22.1 Y 14.9
A-D >80 Y 11.9
Compound 1 5.1 17.7 85.5
Compound 2 4.1 15.0 87.7
Teracacidin 6.0 20.5 73.3
Gallic acid d 1.7 10.0 95.8
Quercetin 2.3 27.6 98.2
a
Effective concentration of antioxidant necessary to decrease the initial DPPH radical by 50%.
b
Final concentration of each extract and fraction was 15 mg/ml. For compounds 1, 2, teracacidin,
gallic acid, and quercetin, that was 33.5 mM.
c
See Figure 4.
d
Positive control for DPPH radical scavenging assay.

FIG. 6. LineweaverYBurk plot of laccase and syringaldazine in the presence (filled

diamond) or absence (open diamond) of teracacidin.
COMPARISON OF ANTIFUNGAL AND ANTIOXIDANT ACTIVITIES 799

FIG. 7. Relationship between (i) antifungal and antioxidant activities, (ii) antifungal
activity and laccase inhibition, and (iii) laccase inhibition and antioxidant activity of
Acacia spp. extracts.
800 MIHARA ET AL.

correlation coefficients between each factor (Figure 7). Significant correlations

(R2 = 0.75Y0.85) were obtained between all three factors.

DISCUSSION

A. auriculiformis heartwood extracts have stronger antifungal properties

than A. mangium against P. noxius and P. badius. A. mangium heartwood ex-
tracts generally did not inhibit the mycelial growth of P. noxius and P. badius
(Figure 3). Previous studies have shown that methanolic extracts of A. mangium
contained ca. half the amount of total phenols compared to A. auriculiformis
(Barry et al., in press). This may explain why A. auriculiformis extract (A-T)
was inhibitory to fungal growth while A. mangium (M-T) was not, and also
implies that the heartrot susceptibility of A. mangium may be related to lower
quantities of active compounds in the extracts.
In the antifungal assays of each fraction obtained by successive extraction,
the diethyl ether fractions (M-A and A-A) of both Acacia spp. showed antifungal
activity (Figure 4). Compounds 1, 2, and teracacidin were present in highest
concentrations in M-A and A-A. Compound 1 and teracacidin inhibited the
growth of P. badius at 15 mM, but at 8 mM mycelial growth was accelerated
compared to the controls (Figure 5). This indicates that a twofold increase in the
concentration of these compounds can result in significant biological con-
sequences for fungal growth. Quantitative analysis by HPLC revealed that A.
auriculiformis contained 0.53% (w/w) of compound 1 and 7.3% of teracacidin
in dry wood (Table 1). This is a substantially higher concentration compared to
the in vitro biologically effective concentration of 15 mM (ca. 0.5% per agar
medium, w/w). A. mangium contained only 0.15% (w/w) of compound 1 and
0.17% of teracacidin in dry wood (Table 1) and as the concentration of 8 mM
(ca. 0.3% per agar medium, w/w) did not show in vitro antifungal effects, it can
be postulated that A. mangium heartwood does not contain a sufficient
concentration of antifungal compounds to inhibit P. badius. These results sug-
gest that the higher amount of antifungal compounds in A. auriculiformis con-
tribute to its heartrot resistance.
The n-butanol fractions (M-C and A-C) and M-D stimulated P. badius
growth (Figure 4). The acid butanol reaction and high BSA adsorption ability of
these fractions, as reported previously (Barry et al., in press), suggests that they
consist of proanthocyanidins. In cultures containing proanthocyanidins, growth
of several wood-rotting basidiomycete fungi also was accelerated (Mitsunaga
et al., 1999). Therefore, the acceleration of P. badius growth could be induced
by proanthocyanidins of A. mangium. Decolorization of samples was observed
in agar medium containing M-D or A-C. It is possible that the proanthocya-
nidins in these fractions were decomposed by enzymes produced by P. badius
COMPARISON OF ANTIFUNGAL AND ANTIOXIDANT ACTIVITIES 801

and then the products utilized as a nutrient source. Hashida et al. (2001) have
shown that another white rot fungus, Lentinus tigrinus, decomposed proantho-
cyanidin from the bark of A. mearnsii. Our previous study reported that A.
mangium had twofold higher proanthocyanidins per heartwood extracts
compared to A. auriculiformis (Barry et al., in press). This may be another
possible reason why heartwood extracts of A. mangium showed lower inhibitory
effects against Phellinus spp. than that of A. auriculiformis (Figures 3 and 4).
The fractions (M-A, A-T, A-A, and A-B) that showed antifungal activity
against P. badius also had strong antioxidant activity (Table 2). Because
compounds 1, 2, and teracacidin showed high DPPH radical scavenging ability,
it is suggested that the antioxidant properties of A-A and A-T can be attributed
to these compounds. A-T contained 1.4-fold more of compound 1 and 17-fold
more of teracacidin compared to M-T (Table 1).
The radical scavenging ability of several flavonoids could be due to the
hydroxylation pattern of the B ring (Amić et al., 2003). The pyrogallol and
catechol pattern of the B ring may impart radical scavenging activity. However,
compounds 1, 2, and teracacidin have a hydroxyl group at the C-40 position of
the B ring (Figure 1). These compounds have a pyrogallolic hydroxylation
pattern in the A ring that may be responsible for their antioxidant properties.
Teracacidin showed lower antioxidant activity than compound 1 or 2, which may
be due to the absence of a 4-carbonyl group (Figure 1). This group could also
play a role in the antioxidant behavior of flavonoids (Amić et al., 2003).
P. noxius and P. badius produce laccase (R. Mihara, unpublished data),
which is typical of white rot fungi. Phenol oxidases such as laccase and
polyphenol oxidase (PPO) oxidize phenolics by radical reactions (Guillén et al.,
2000). The kinetics study of laccase with teracacidin as an inhibitor suggests
that teracacidin might not act directly on the active site of laccase, but instead
inhibit the radical reaction after laccase forms a complex with syringaldazine
(Figure 6). Laccase activity is directly proportional to mycelial growth of a white
rot fungus, Agaricus bisporus (Wood, 1979). Therefore, the antifungal fractions
of A. auriculiformis and A. mangium may inhibit fungal growth as a result of
quenching the radicals produced by the extracellular enzymes of P. noxius and
P. badius. This hypothesis is further supported since the antifungal fractions that
inhibited laccase activity also showed high DPPH radical scavenging activity
(Table 2; Figure 7). The tannin fractions (M-C, A-C, and A-D) of Acacia spp.,
however, did not inhibit laccase activity. The structures of proanthocyanidins
in these Acacia spp. extracts are unknown (Barry et al., in press) and further
investigation is required.
While many previous studies have reported the antifungal and antimicro-
bial activities of polyphenols, the precise mechanisms involved with such
activities are not clear. Enzyme inhibition by the astringent character of tannin
and inhibition of the electron transport system on mitochondria have been
802 MIHARA ET AL.

investigated (Scalbert, 1991). In this study, proanthocyanidin fractions (A-C,

M-C, and M-D) did not show antifungal and laccase inhibitory activities. This
suggests that the antifungal properties of Acacia spp. extracts were not due to
the astringent character of proanthocyanidins (tannins). However, antifungal
activity, DPPH radical scavenging activity, and laccase inhibition were all
correlated with each other (Figure 7).
Mechanisms of wood degradation by wood-rotting fungi have been des-
cribed. Backa et al. (1993) hypothesized that hydroxyl radicals were produced
from the fungal degradation of wood. These hydroxyl radicals were produced
by an extracellular, low molecular weight substance and as the rate of wood
degradation increased so did the amounts of hydroxyl radicals (Tanaka et al.,
1999). Therefore, the antifungal property of A. auriculiformis heartwood may
be attributed to the scavenging of hydroxyl radicals as well as to laccase
inhibition.
We focused attention on heartwood extracts of A. mangium and A.
auriculiformis and found significant differences between the antifungal proper-
ties of both species that relates to the incidence of heartrot observed in these
species during field studies. We tested extracts from two individual trees of both
species; however, more trees should be sampled. It would be of interest to further
investigate the antifungal property of hybrids of A. mangium and A. auriculi-
formis, of which some have been shown to be resistant to heartrot (Ito, 2002).

AcknowledgmentsVThis research was partly funded by an ACIAR grant, project number FST/
00/123. R. Mihara acknowledges the Japanese Academic Frontiers Student Exchange Promotion
Program which funded a research visit to the University of Tasmania.

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