Chemical compounds from Eperua falcata and Eperua grandiflora heartwood and their biological activities against wood destroying fungus (Coriolus versicolor)

Abstract

The chemical analysis of the compounds present in dichloromethane and ethanolic fractions as well
as bioassays enable to understand the differences in the durability of Eperua falcata
and Eperua grandiflora. The main distinction between these two species
is the acidic subfraction of diterpenoid extract, which is antifungal in Eperua
falcata when tested under in-vitro conditions. This study also shows that ethanolic fraction
plays an important role in the mechanism of natural durability. Furthermore, it reports the first isolation
of cativic acid in Eperua falcata wood.

Full text

DOI 10.1007/s00107-006-0120-1
ORIGINALARBEITEN ·ORIGINALS
Holz Roh Werkst (2007) 65: 23–28
Chemical compounds from Eperua falcata and Eperua grandiflora
heartwood and their biological activities against wood destroying
fungus (Coriolus versicolor)
Nadine Amusant · Christian Moretti · Bernard Richard · Elise Prost ·
Jean Marc Nuzillard · Marie France Th´
evenon
Published online: 3 August 2006
©Springer-Verlag 2006
Abstract The chemical analysis of the compounds present
in dichloromethane and ethanolic fractions as well as bioas-
says enable to understand the differences in the durability of
Eperua falcata and Eperua grandiflora. The main distinc-
tion between these two species is the acidic subfraction of
diterpenoid extract, which is antifungal in Eperua falcata
when tested under in-vitro conditions. This study also shows
that ethanolic fraction plays an important role in the mech-
anism of natural durability. Furthermore, it reports the first
isolation of cativic acid in Eperua falcata wood.
Chemische Inhaltsstoffe aus dem Kernholz von Eperua
falcata und Eperua grandiflora und ihre Wirkung gegen
Holz zerst¨
orende Pilze (Coriolus versicolor)
Zusammenfassung Anhand chemischer Analyse der in Di-
chlormethan- und Ethanolfraktionen vorhandenen Verbin-
dungen sowie biologischer Pr¨
ufungen k¨
onnen die Unter-
schiede in der Dauerhaftigkeit von Eperua falcata und Epe-
rua grandiflora aufgezeigt werden. Der Hauptunterschied
zwischen den beiden Arten besteht in der sauren Fraktion
des Diterpenoid-Extraktstoffes, der sich bei Pr¨
ufungen un-
ter in-vitro Bedingungen bei Eperua falcata als pilzwidrig
N. Amusant (u)·M.F. Th
´
evenon
73 rue JF Breton, TA 10/16,
34398 Montpellier Cedex 5, France
e-mail: nadine.amusant@cirad.fr
C. Moretti
IRD, Unit´
e S 84,
BP 165, 97323 Cayenne Guyane Franc¸aise, France
B. Richard · E. Prost · J. M. Nuzillard
Laboratoire de Pharmacognosie, UMR 6013, CPCBAI Bˆ
at. 18,
BP 1039, 51687 Reims Cedex 2, France
erwies. Anhand dieser Arbeit konnte auch gezeigt werden,
dass die Ethanolfraktion eine wichtige Rolle bez¨
uglich der
nat¨
urlichen Dauerhaftigkeit spielt. Dar ¨
uber hinaus wird ¨
uber
die erstmalige Isolierung von Cativins¨
aure aus Eperua fal-
cata Holz berichtet.
1 Introduction
Hawley et al. (1924) were the first to demonstrate antifungal
activity from the presence of small amounts of extractives
that are toxic to fungi and other wood-attacking organisms.
Heartwood durability is ascribed to highly antifungal extrac-
tives; total extractives may be more efficient than the in-
dividual components due to synergism (Hart 1981, Schultz
et al. 1995). Several authors investigated the relationships
between the wood properties and extractives (Lesley et al.
1989, Schultz et al. 1990, Chang et al. 1999). From the envi-
ronmental perspective, finding natural constituents in highly
durable wood species and elucidating the mechanisms of
their action is one of the best approaches to achieve wood
protection while preserving the heavy construction and hy-
draulic works (class 1 of durability; very durable), timber
from Eperua grandiflora does not last in damp conditions
(class 2 or 3 of durability; moderately durable to). However,
there has been little research investigating the relationships
between the wood properties of Eperua spp. and extractives.
Indeed, one could suspect that acid resin plays an import-
ant role in durability observed in a standing tree (Klingstr ¨
om
1969, Medina and De Santis 1981). In our attempt to un-
derstand why these two very close tropical species, Eperua
falcata (very durable) and Eperua grandiflora (durable),
show different decay resistance, extracts from heartwood
have been tested for their antifungal activity. We report here
about the extraction method, and chemical identification of
13

24 Holz Roh Werkst (2007) 65: 23–28
the natural compounds playing a role in durability when
they have been exposed to white-rot fungus Coriolus ver-
sicolor. Plates agar tests assays have been completed by
mini wood block tests. These tests allow to approach the be-
haviour of wood by taking into accountthe wood/extractives
interaction.
2 Experimental
2.1 General
NMR spectra were obtained with an autospect WG spec-
trophotometer. Mass spectra were obtained by GC/MS on
a Buker DRX500 instrument. Centrifugal thin layer chro-
matography (CTLC) was performed using a chromatotron
manufactured by Harisson research (Palo alto, California).
The glass plate is covered by a 2 mm thick layer of Silica
Gel (Merck Kieselgel 60PF254 gipshaltig).
2.2 Materials
Sampling consisted of two trees including one Eperua fal-
cata and one Eperua grandiflora from French Guiana (Para-
cou forest) harvested in April 2000. Outer heartwood chips
(next to the sapwood and situated 3 m from the base) were
prepared from a green cut tree, conditioned (65% HR –
20 ◦C) for several weeks and then milled to a 60-mesh sieve
size.
2.3 Extraction
The sawdust (40 g, moisture content 8%) was sequentially
extracted in a Soxhlet for eight hours with dichloromethane
(CH2Cl2, 200 ml-technical grade) and ethanol (EtOH,
200 ml-technical grade) in increasing order of polarity. The
solvents were evaporated under vacuum at 40 ◦C, respec-
tively. The extraction yield was estimated by weighing
the residual product and expressed as % of dry sawdust.
The extractions were carried out in triplicate. The reported
values are average values with standard deviations.For each
species, a part of the extract (0.200 g) was shaken with KOH
(10%), dried (Na2SO4) and the solvent removed (organic
phase) leaving the neutral subfraction. Acidifications of the
KOH extract (aqueous phase) gave the acidic subfraction
which was extracted with CH2Cl2, dried (Na2SO4)andthe
solvent removed giving the crude acid (Fig. 1 and Table 1).
The acidic subfractions from both species were chro-
matographed on a 2 mm Silicagel plate using CHCl3-EtOH
(95 :5 v/v) as eluent. The observation of the spot under UV
and the Rf comparison allowed to obtain two pure com-
pounds: compound 1: C20H34 O2(eperuic acid, M = 306)
and compound 2: C20H34O2, (cativic acid, M = 306) for
Fig. 1 Fractionation of Eperua falcata and Eperua grandiflora
dichloromethane extract
Abb. 1 Fraktionierung des Dichlormethanextrakts von Eperua fal-
cata und Eperua grandiflora
Eperua falcata. Six pure compounds were found for Epe-
rua grandiflora:compound 3: for C20H34O2, (eperuic acid,
mixture of the isomer, M = 306), compound 4 and 5:
C20H32 O2, (Z and E-copalic acid, M = 304), compound 6:
C20H32 O2, (7- oxalabd-8-en-15-oic acid, M = 320) and
compound 7: (7 -oxalabda-8, 13-E-dien-15-oic acid, M =
318). The isolated constituents were identified by, COSY,
TOCSY, ROESY, HSQC and HMBC, 13Cand1HNMR in
(CDCl3) and EI-MS analysis and the structures are shown in
Fig. 2. The compound 8 is not yet identified.
2.4 Determination of antifungal activity on agar
Fungus used in this study was Coriolus versicolor (L.ex Fr)
Qu´
elet, strain CTB 863 A. All antifungal tests were per-
formed three times and the data were averaged. All fractions
(dichloromethane fraction and ethanolic fraction) and sub-
fractions (acidic and neutral subfractions) were added to
sterilised malt agar medium (40 :20 g/l distilled water) to
give three concentrations of extractives (Tables 2 and 3).
A0.5 cm diameter plug of C. versicolor mycelium was
transferred into the centre of the Petri dishes and the testing
plates were incubated at 27 ◦C–70% HR (relative humidity).
Tab le 1 Yields (%, in relation to the sawdust before extraction) of ex-
tracts from Eperua falcata and Eperua grandiflora (standard deviation
in brackets)
Tabelle 1 Ausbeute der Extraktstoffe (in % des Ausgangsmaterials)
von Eperua falcata und Eperua grandiflora (Standardabweichung in
Klammern)
Fractions Eperua falcata Eperua grandiflora
Dichloromethane fraction 4.50% (0.3) 3.80% (0.4)
Acid subfraction 1.28% (0.2) 0.22% (0.1)
Neutral subfraction 3.12% (0.3) 0.32% (0.1)
Ethanol fraction 28.50% (1.4) 14.46% (0.4)
13

Holz Roh Werkst (2007) 65: 23–28 25
Fig. 2 Configuration of compounds from the acidic fraction of Eperua falcata (1) Eperuic acid, (2) cativic acid and of Eperua grandiflora (4
and 5) Z and E-copalic acid; (6) 7-oxalabda-8-en-15-oic acid; (7) 7-oxalabda-8,13-Edien- 15-oic acid
Abb. 2 Konfiguration der verschiedenen Bestandteile der sauren Fraktion von Eperua falcata (1) Eperus¨
aure, (2) Cativins¨
aure und von Eperua
grandiflora (4 und 5) Z und E-Kopals¨
aure; (6) 7-Oxalabda-8-en-15-s¨
aure; (7) 7- Oxalabda-8,13-E-dien-15-s¨
aure
When the mycelium reached the edge of the control plate
(without adding extractives), the antifungal index (AI) was
calculated:
AI (%) = [1-(radial growth on the test medium / radial
growth on the control medium)] ×100. The growth index
varies from 0 when there is no fungal inhibition to 100
when there is no fungal growth (i.e., total fungal inhibi-
tion) and gives information about the in-vitro activity of
extractives.
2.5 Determination of antifungal activity
by wood-block tests on agar
Extraction of the wood blocks Wood block specimens (10×
10 ×5mm
3,LRT)ofEperua falcata and Eperua grandi-
flora were machined from a single flat-sawn board from
the outer heartwood. The thickness (5 mm) was selected
to ensure good penetration by extracting solvents. They
were conditioned (20 ◦C – 65% RH) until constant mass,
13

26 Holz Roh Werkst (2007) 65: 23–28
Tab le 2 Inhibition of the growth (AI) of Coriolus versicolor by Eperua falcata fraction with different concentrations (g extract/100 ml medium)
Tabelle 2 Hemmung des Wachstums (AI) von Coriolus versicolor in Eperua falcata Fraktionen unterschiedlicher Konzentrationen
(g Extraktstoff/100 ml N¨
ahrstoff)
Concentration % Inhibition Concentration % Inhibition Concentration % Inhibition Concentration % Inhibition
of diterpenoid (AI) of acid (AI) of neutral (GI) of ethanolic (AI)
mixture subfraction subfraction fraction
1.7% 100 1.1% 100 3.1% 10 4.3% 82
1.3% 45 0.2% 50 1.5% 5 2.1% 69
0.1% 20 0.07% 27 0.3% 0 0.8% 54
Control 0
Tab le 3 Inhibition of the growth (AI) of Coriolus versicolor by Epe-
rua grandiflora fraction with different concentrations (g extract/100 ml
medium)
Tabelle 3 Hemmung des Wachstums (AI) von Coriolus versicolor
in Eperua grandiflora Fraktionen unterschiedlicher Konzentrationen
(g Extraktstoff/100 ml N¨
ahrstoff)
Concentration % Inhibition Concentration % Inhibition
of diterpenoid (AI) of ethanolic (AI)
mixture fraction
5.4% 46 4.3% 75
1.3% 18 2.1% 57
0.1% 2 0.8% 49
Control 0
then dried at 103 ◦C. The wood blocks were extracted with
a Soxhlet extraction apparatus in groups of 10 for eight
hours with dichloromethane and ethanol alone and with
dichloromethane and ethanol successively. Following ex-
traction, all blocks were allowed to dry at room conditions
(20 ◦C – 65% RH) to constant mass, then dried at 103◦C.
The amount of extraneous material removed by each solvent
was calculated as the difference between the original and the
extracted wood of each wood block and expressed as a per-
centage of the original (between dry mass). The average
mass losses due to extraction were calculated for the wood
blocks extracted by each solvent. Preparation of the decay
test Decay resistance was determined by a modified version
of the European standard EN 350-1 and EN 113 procedures.
The wood blocks were dried at 103 ◦C until constant mass
to determine dry mass, sterilised (autoclave) and exposed to
actively growing, pure culture of Coriolus versicolor, culti-
vated in Petri dishes. Before inoculation, a sterile perforated
polycarbonate barrier was placed on the malt-agar medium
(40 :20 g/l distilled water) surface to prevent water log-
ging of the specimens. 30 controls of Fagus sylvatica wood
samples having the same sizes were used to validate the vir-
ulence of the fungus. Petri dishes were incubated for eight
weeks at 25 ◦C – 70% RH. At the end of exposure the test
blocks were cleaned of mycelium, weighed to give a meas-
ure of their moisture content and then dried (24 hours at
room temperature, then at 103 ◦C over night) and then wei-
ghed again. The mass loss due to decay was calculatedas the
difference between dry mass of each wood block before and
after incubation and expressed as a percentage of dry mass
loss. The average mass loss due to decay in each group of
ten replicate wood blocks was calculated. Comparison with
non-extracted wood blocks allowed to evaluate the loss of
durability after extraction and gives information about the
activity of the extractives.
3 Results and discussion
The mean and standard deviation of the yields of extract
from Eperua falcata and Eperua grandiflora heartwood are
listed in Table 1. These data provide a direct measure of
the amount of extractives removed by each solvent in the
experiment. The dichloromethane extraction of the heart-
wood of both species removed alcohol, resin acids and
terpenoids compounds (Blake and Jones 1963). Ethanol
extracts include monoflavonoids and polyflavonoids com-
pounds (Villeneuve and Vergnet 1988). The ethanolic frac-
tion from Eperua falcata contains more extractives than
Eperua grandiflora,and the observation is the same with the
dichlormethane fraction. The content of acidic and neutral
subfractions of Eperua grandiflora is particularly low com-
pared to the dichloromethane fraction. These results can be
explained by the formation of a persistent emulsion witch
prevents a good separation between both phases. For Epe-
rua falcata, the main part of the dichloromethane fraction is
composed of the neutral subfraction. The acidic subfraction
from Eperua falcata was analysed for structural component.
There is no doubt that extractives are the most significant
factor influencing the durability of wood. The antifungal in-
dex against white rot fungus(Coriolus versicolor)ofvarious
extracts from Eperua falcata and Eperua grandiflora are
presented in Tables 2 and 3. The dichloromethane fraction
from E. falcata has a strong activity against Coriolus versi-
color. The degree of inhibition was positively related to the
concentration of the fraction. The antifungal activity of the
dichloromethane fraction is due to the acidic subfraction.
The acidic diterpenoid isolated from the acidic subfraction
is known to have strong inhibitory effects on the growth of
fungi (Bauch et al. 1977, Gref 1987). At high concentration,
the dichloromethane fraction from E. grandiflora affects the
13

Holz Roh Werkst (2007) 65: 23–28 27
growth of the mycelium, but the action is limited (AI = 46).
Because of this low value, no test was carried out with the
acidic subfraction. After isolation and purification by CTLC
as well as the chemical analysis (1H-NMR, spectrum mass
...) of the acidic fraction from both species, we observed
that the composition of both acidic fractions is different:
there are two compounds in Eperua falcata and six com-
pounds in Eperua grandiflora Fig. 2. This is the first report
of cativic acid occurring in Eperua falcata. The relative con-
figuration of the asymmetric centres at C-5, C-9 and C-10
were ascertained by analysis of the ROESY spectrum of
compound 1, and deduced to be identical in compound 2 for
biogenetic reason. From the previously published 13CNMR
spectrum of eperuic acid (Dey and Wolf 1978) the absolute
configuration at C-13 is determined as 13-(S) and deduced
to be identical in compound 2. Previous works (Bajmer et al.
1968) showed that the decalin system could exist either like
in compound 1 or in its enantiomericform like in labdanolic
acid, but always with a 13-(S) centre in the side-chain.
The differences of behaviour against fungus between
the acidic fractions from both species are due to a dif-
ference in chemical composition. The ethanolic fraction
from both species showed slight fungal inhibition com-
pared to the dichloromethane fraction from Eperua fal-
cata. The ethanolic fraction mainly contains polyphenolic
compounds, which are known to contribute to protect the
trunk against pathogenic and wood-rotting micro-organisms
(Wang 1983). The inhibitory effect of polyphenolic com-
pounds on the growth of many fungi in culture is well doc-
umented (Smith et al. 1989). They play a role in defending
trees against pathogens because they can inhibit fungal in-
vasion by complexing with extracellular enzymes and with
proteins in the fungal cell wall. In our study reduction of
growth of mycelium is observed in presence of ethanolic ex-
tracts from both species. For high concentration (more than
5%) the assay is not appropriate for this type of compounds
since the medium does not easily harden as polyphenolic
compound complex with compounds in the medium like
agar (Zucker 1983). Thus, the agar plate tests give a part
of the response on the activity of extractives: the difference
of durability between Eperua falcata and Eperua grandi-
flora depends on the quantitative and qualitative extractive
composition of the wood, but the tests do not take into ac-
count the relation between wood, extractives and fungus
(Celimene et al. 1999).
To examine the impact of extractives on mechanism of
durability, an evaluation of the mass loss of wood blocks after
dichloromethane and ethanol extraction was carried out. Fig-
ures 3 and 4 show the mean mass loss after extraction of wood
blocks and the mean mass loss after the decay exposure. As
shown in Fig. 3, the mean mass losses of the extractives after
extraction are lower than the yields of extraction obtained
directly with the sawdust. This means that there are still ex-
Fig. 3 Mean of mass loss of the wood blocks after extraction with
different sequence of solvents in %, (E = extraction, dichlo =
dichloromethane)
Abb. 3 Mittlerer Masseverlust der Holzpr¨
ufk¨
orper nach Extraktion
mit unterschiedlichen L¨
osungsmitteln, in % (E= Extraktion, dichlo =
Dichlormethan)
Fig. 4 Mean of mass loss (in %) of wood blocks as a function of ex-
traction after exposure to Coriolus versicolor. (Control = wood blocks
not extracted, E = extraction, dichlo = dichloromethane)
Abb. 4 Mittlerer Masseverlust (in %) der Holzpr¨
ufk¨
orper nach Befall
mit Coriolus versicolor in Abh¨
angigkeit von der Extraktion (Control
= nicht extrahierte Holzpr¨
ufk¨
orper, E = Extraktion, dichlo = Dichlor-
methan)
tractives in the wood blocks, particularly polyphenolic com-
pounds. The extraction is less efficient with woodblocks than
with sawdust. The yield of extractives obtained with succes-
sive extraction (dichloromethane and ethanol) is lower than
the sum of the yield of extract obtained with each solvent.
This result can be explained by the fact that some of the com-
pounds extracted by the first solvent block the vessels and
prevent the extraction of the second set of compounds. This
result could have an effect on the behaviour of wood block
during the exposure to the fungus. The control Beech wood
blocks exposed to Coriolus versicolor gave a mass loss of
20% and validated the virulence of the fungus. Without ex-
traction, the mean mass loss of the wood blocks exposed to
the fungus is 2.6% for both species (Fig. 4). No significant
difference of mass loss was observed between these species.
Compared to the standardised tests it might be possible that
eight weeks are not sufficient to distinguish the durability of
both species. The loss of extractives leads to an increase of
mass loss of the wood-blocks exposed to Coriolus versicolor,
meaning that there is a loss of natural durability. It seems that
extractives from Eperua falcata are more active than those of
13

28 Holz Roh Werkst (2007) 65: 23–28
Eperua grandiflora because the mass losses observed with E.
falcata after the contamination of the extracted wood blocks
are higher. It is likely that the mass loss after fungal expo-
sure would be higher if the extraction time had been longer.
This difference of behaviour can be partially attributed to the
quantitative and qualitative differences between the compo-
sition of extractives from both species. This test underlines
the role of the polyphenolic compounds, which seems to be
more active than terpenoid compounds. This result confirms
the hypothesis proposed above, the agar plate test was not
adapted to evaluating the antifungal effect of the ethanolic
fraction. The dichloromethane fraction and ethanolic fraction
act on the mechanism of wood resistance against fungus.
4 Conclusion
Eperua falcata is classified as an excellent durability species
while Eperua grandiflora is durable to moderately durable.
Our study showed that the high durability of Eperua fal-
cata depends on the presence of compounds extracted with
dichloromethane and ethanolic solvents. The quantitative
contents of dichloromethane and ethanolic extracts are higher
in Eperua falcata than in Eperua grandiflora.Thecom-
pounds identified in the acidic subfraction in Eperua fal-
cata include 2 compounds while 6 compounds in Eperua
grandiflora. However the agar plate test is not sufficient or
adapted to evaluating the antifungal activity of extractives.
The wood block method used allows observing the role of the
polyphenolic compounds. The difference of durability with
Eperua grandiflora seems to be related to the difference in
diterpenoid acid composition and polyphenolic compounds
composition.
Acknowledgement The authors are extremely grateful to CNES
(Centre National d’Etudes Spatiales), which supported the study and
the PhD. Our thanks also go to Guillaume Marti (IRD) and Meriem
Fournier (Engref).
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