Holz Roh Werkst (2007) 65: 23–28 DOI 10.1007/s00107-006-0120-1
ORIGINALARBEITEN · ORIGINALS
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 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. 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 Dichlormethan- und Ethanolfraktionen vorhandenen Verbindungen sowie biologischer Pr¨ufungen k¨onnen die Unterschiede in der Dauerhaftigkeit von Eperua falcata und Eperua grandiflora aufgezeigt werden. Der Hauptunterschied zwischen den beiden Arten besteht in der sauren Fraktion des Diterpenoid-Extraktstoffes, der sich bei Pr¨ufungen unter 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:
[email protected] 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 Bˆat. 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 u¨ ber die erstmalige Isolierung von Cativins¨aure aus Eperua falcata 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 extractives; total extractives may be more efficient than the individual 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 environmental 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 hydraulic 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 important role in durability observed in a standing tree (Klingstr¨om 1969, Medina and De Santis 1981). In our attempt to understand 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
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the natural compounds playing a role in durability when they have been exposed to white-rot fungus Coriolus versicolor. Plates agar tests assays have been completed by mini wood block tests. These tests allow to approach the behaviour of wood by taking into account the wood/extractives interaction.
2 Experimental 2.1 General NMR spectra were obtained with an autospect WG spectrophotometer. Mass spectra were obtained by GC/MS on a Buker DRX500 instrument. Centrifugal thin layer chromatography (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 falcata and one Eperua grandiflora from French Guiana (Paracou 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.
Fig. 1 Fractionation of Eperua falcata and Eperua grandiflora dichloromethane extract Abb. 1 Fraktionierung des Dichlormethanextrakts von Eperua falcata und Eperua grandiflora
Eperua falcata. Six pure compounds were found for Eperua grandiflora: compound 3: for C20 H34 O2 , (eperuic acid, mixture of the isomer, M = 306), compound 4 and 5: C20 H32 O2 , (Z and E-copalic acid, M = 304), compound 6: C20 H32 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, 13 C and 1 HNMR 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
2.3 Extraction The sawdust (40 g, moisture content 8%) was sequentially extracted in a Soxhlet for eight hours with dichloromethane (CH2 Cl2 , 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, respectively. 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 (Na2 SO4 ) 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 CH2 Cl2 , dried (Na2 SO4 ) and the solvent removed giving the crude acid (Fig. 1 and Table 1). The acidic subfractions from both species were chromatographed 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 compounds: compound 1: C20 H34 O2 (eperuic acid, M = 306) and compound 2: C20 H34 O2 , (cativic acid, M = 306) for
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Fungus used in this study was Coriolus versicolor (L.ex Fr) Qu´elet, strain CTB 863 A. All antifungal tests were performed three times and the data were averaged. All fractions (dichloromethane fraction and ethanolic fraction) and subfractions (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). A 0.5 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).
Table 1 Yields (%, in relation to the sawdust before extraction) of extracts 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 Dichloromethane fraction Acid subfraction Neutral subfraction Ethanol fraction
Eperua falcata 4.50% (0.3) 1.28% (0.2) 3.12% (0.3) 28.50% (1.4)
Eperua grandiflora 3.80% (0.4) 0.22% (0.1) 0.32% (0.1) 14.46% (0.4)
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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 inhibition) 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 × 5 mm3 , LRT) of Eperua falcata and Eperua grandiflora 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,
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Table 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 of diterpenoid mixture 1.7% 1.3% 0.1% Control
% Inhibition (AI) 100 45 20
Concentration of acid subfraction 1.1% 0.2% 0.07%
% Inhibition (AI) 100 50 27
Table 3 Inhibition of the growth (AI) of Coriolus versicolor by Eperua 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 of diterpenoid mixture 5.4% 1.3% 0.1% Control
% Inhibition (AI) 46 18 2
Concentration of ethanolic fraction 4.3% 2.1% 0.8% 0
% Inhibition (AI) 75 57 49
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 extraction, 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 percentage 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, cultivated 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 logging of the specimens. 30 controls of Fagus sylvatica wood samples having the same sizes were used to validate the virulence 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 measure of their moisture content and then dried (24 hours at room temperature, then at 103 ◦ C over night) and then weighed again. The mass loss due to decay was calculated as the difference between dry mass of each wood block before and
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Concentration of neutral subfraction 3.1% 1.5% 0.3% 0
% Inhibition (GI) 10 5 0
Concentration of ethanolic fraction 4.3% 2.1% 0.8%
% Inhibition (AI) 82 69 54
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 heartwood of both species removed alcohol, resin acids and terpenoids compounds (Blake and Jones 1963). Ethanol extracts include monoflavonoids and polyflavonoids compounds (Villeneuve and Vergnet 1988). The ethanolic fraction 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 compared 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 Eperua 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 index against white rot fungus (Coriolus versicolor) of various 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 versicolor. 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
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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 compounds in Eperua grandiflora Fig. 2. This is the first report of cativic acid occurring in Eperua falcata. The relative configuration 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 13 C NMR 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 enantiomeric form 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 difference in chemical composition. The ethanolic fraction from both species showed slight fungal inhibition compared to the dichloromethane fraction from Eperua falcata. 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 compounds on the growth of many fungi in culture is well documented (Smith et al. 1989). They play a role in defending trees against pathogens because they can inhibit fungal invasion 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 extracts 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 grandiflora depends on the quantitative and qualitative extractive composition of the wood, but the tests do not take into account 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. Figures 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-
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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 extraction 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 = Dichlormethan)
tractives in the wood blocks, particularly polyphenolic compounds. The extraction is less efficient with wood blocks than with sawdust. The yield of extractives obtained with successive 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 compounds 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 extraction, 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
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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 exposure 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 composition 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 falcata 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. The compounds identified in the acidic subfraction in Eperua falcata 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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