Insoluble ellagitannins in Castanea sativa and Quercus petraea woods

Phytochemistry,Vol. 30, No. 3, pp. 775-778, 1991

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INSOLUBLE ELLAGITANNINS IN CASTANEA SATIVA AND QUERCUS PETRAEA WOODS SHUYUN PENG, AUGUSTIN SCALBERT* and BERNARD MONTIES Laboratoire de Chimie Biologique (INRA), Centre de Biotechnologie Agro-industrielle, Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, France

(Received 10 July 1990) Key Word Index--Castanea sativa; Quercuspetraea; Fagaceae; wood; ageing; oxidation; ellagitannins; ellagic acid.

Abstract--Sapwood and heartwood samples of sweet chestnut (Castanea sativa) and sessile oak (Quercus petraea) were extracted with aqueous acetone. Ellagitannins were estimated in both extracts and extraction residues, by the Folin-Ciocalteu method and by determination of ellagic acid formed after acid degradation. Ellagitannins were found to accumulate in comparable concentrations (ca 10% DM) in the heartwoods of both species. The concentrations of soluble ellagitannins slowly decreased from the periphery to the centre of the heartwood as it ages. This decrease was balanced by an equivalent increase of insoluble ellagitannins, resulting probably from the slow oxidative polymerization or copolymerization with cell-wall components of soluble ellagitannins.

INTRODUCTION Many woods contain tannins [1, 21 which may be proanthocyanidins (condensed tannins) or hexahydroxydiphenoyl esters (ellagitannins). Condensed tannins accumulate in many softwoods and hardwoods. EUagitannins accumulate in some hardwoods, including some important commercial species belonging to Quercus, Castanea or Eucalyptus genera. Both quebracho (Schinopsis sp.) in Argentina and Paraguay, and chestnut (Castanea sativa Mill.) in France and Italy provide wood for the industrial extraction of tannins. Tannins may be soluble (extractives) or insoluble (bound to cell wall). The content of soluble tannins in woods varies largely according to the position of the sample in the tree [1]. It increases sharply at the border between sapwood and heartwood, and decreases from the periphery to the centre of heartwood. This decrease might be explained by a progressive insolubilization of tannins as the wood ages. The occurrence of insoluble condensed tannins is well known. In some plant tissues like leaves [3] or woods [4], they may represent up to 90% of the total tannins of the sample. These insoluble tannins may correspond to highM, fractions, which would be strongly adsorbed on the cell-wall matrix through weak interactions [5]. A more likely mechanism of insolubilization would involve the formation of covalent linkages while the plant tissues are still alive or while they are already dead. In a living plant cell, phenolic glycosides could be enzyme-incorporated in cell-wall polysaccharides during their biosynthesis [6, 7]; tannins or their precursors could also form covalent linkages with lignins during their dehydrogenative polymerization [8]. In dead tissues such as heartwoods or barks, tannins may be slowly non-enzymatically oxidized and would thus copolymerize with different cell-wall polymers [4, 9]. *Author to whom correspondence should be addressed.

The occurrence of insoluble ellagitannins is less well documented. However, their presence has been previously reported after detection of ellagic acid in alkalinetreated extraction residues [1], but no quantitative data have been provided, although some attempts have been made on Eucalyptus woods [10]. We report here quantitative estimation of insoluble ellagitannins in sweet chestnut (Castanea sativa Mill.) and sessile oak (Quercus petraea Liebl.) woods, Both woods are known to contain high amounts of ellagitannins [2], the main structures being castalagin, vescalagin [11-13] and their dimers [14]. Each of these molecules contains one hexahydroxydipbenoyl unit which gives eUagic acid upon acid degradation (Scheme 1). Different extraction procedures are compared in order to clarify the distinction between soluble and insoluble tannins. RESULTS

Different extraction solvents were compared in order to solubilize most of the extractable tannin (Fig. 1). Solvents can be ranked in the following order: water, aqueous methanol, dimethylsulphoxide and dimethylformamide, aqueous acetone. The proportion of water in the aqueous methanol or acetone, in the range considered, had no effect on the amount of tannin solubilized. Extractibility of tannin by water could be improved significantly by raising the temperature to 100 ° or by increasing the pH from pH 4 (measured pH of wood aqueous extrac0 to pH 10; a value close to that obtained with aqueous acetone was thus obtained; increasing the temperature from room temperature to 65 ° had a slight positive effect on aqueous methanol extraction, and a negligible effect on aqueous acetone extraction (data not shown); in any case, varying solvents, temperature or pH did not improve the extraction yields as compared to those obtained with aqueous acetone at room temperature.

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Fig. 2. Total soluble phenols in wood cross sections of sweet chestnut (A) and sessile oak (A) trunks; dotted line: sapwoodheartwood transition from sweet chestnut (1) and sessile oak (2).

The amounts of soluble ellagitannins were measured along chestnut and oak tree sections, using the Folin-Ciocalteu colorimetric method. Results are expressed in gallic acid equivalent (GAE), as a function of the average age of the sample (Fig. 2). Both species showed similar tannin contents, and comparable tannin content variations; the amount of soluble tannin increased sharply at the sapwood-heartwood boundary, and then decreased slowly during the next four to five decades. Losses during this last period were estimated to 23 and 25 mg GAE g - I wood for chestnut and oak respectively. Ellagitannin contents were also measured by degradation in alcohol-hydrochloric acid mixtures and estimation of the resulting ellagic acid by HPLC. Conditions of acid degradation was optimized with solutions of purified castalagin, vescalagin or raw wood extracts. Two solvents were compared: n-butanol, classically used for the acid degradation of condensed tannins, and methanol. The first was finally avoided as it gave poor yields of ellagic

acid and many secondary products. The effect of temperature was also studied: when the degradation was run at 95 °, the maximum yield was not reached after 400 min of reaction. At 140°, a maximum yield was reached at c a 4 0 rain followed by a decrease, due to the degradation of ellagic acid. The temperature was finally set at 120°. Under these conditions, a molar ratio approaching one was obtained by degradation of castalagin or vescalagin (Fig. 3). A reaction time of 160 min was finally chosen as some degradation of ellagic acid was observed under longer treatment of more complex samples such as chestnut wood extract. Results are expressed as castalagin equivalents (CE; Fig. 4). Data for the extracts are in good agreement with the ones presented above (Fig. 2). Losses during heartwood ageing, were estimated to 26 and 25 mg CE g - ~ wood for chestnut and oak respectively. These losses are balanced by an increase of ellagitannin content in the extraction residue as heartwood ages. These increases were estimated to be 22 and 9 mg CE g - t wood for

Insoluble eUagitannins in wood chestnut and oak respectively. As a consequence, the total amounts of ellagitannin in all heartwood samples remain fairly stable.

wood [18], is however not excluded. This insolubilization phenomenon is probably not specific for tannins, but may well affect any easily oxidized polyphenols. The amounts of insolubilized polyphenolic extractives is probably related to the ease with which they are oxidized in air. It is possibly significant that the proportion of insoluble ellagitannins after 50 years of ageing, does not exceed 41% in the samples analysed, whereas it commonly exceeds 90% for condensed tannins [4]. Indeed, it is known that purified ellagitannins are more stable when stored dry, than are condensed tannins which become coloured with time [19]. These oxidation reactions may not only occur within the living tree but also during storage or processing of wood: they are probably responsible for the decrease of tannin content during storage of oak wood [20], the decrease of (+)-catechin content during storage of western hemlock chips [21] and the loss of reactivity of condensed tannins during drying of Turkey oak [4]. The consequences of the existence of these insoluble ellagitannins are several. From an analytical point of view, insoluble tannins may be confused with lignins, when methods like Klason determination are used [10, 22, 23]. Lignins would thus be overestimated. Several wood properties will probably be modified, two examples of which are: woods rich in tannins from species like some oaks or chestnut are usually highly resistant to biodegradation [24, 25]. This property is possibly explained by the astringency of tannins which would lead to the inhibition of microbial enzymes or to complexation with substrates [26]. Oxidation and polymerization of tannins would probably modify considerably their astringent character and hence their toxicity [27], and would thus explain the commonly observed reduction of durability of central heartwood as compared to external heartwood [28, 29]. Another consequence may be related to the colour of woods rich in tannins, or pulp produced from these woods [30]. Indeed, oxidized and polymerized tannins have certainly retained part of their reactivity. They may contribute, as much as the soluble polyphenols, to the

DISCUSSION It is demonstrated here that the slow decrease of soluble tannin concentration, as the wood ages within the trunk, can be explained by progressive insolubilization. It is thus not due to a lesser amounts of tannins in young as compared to older trees. This insolubilization probably results from their slow oxidation, which leads to their polymerization [15] or copolymerization with cell-wall components [16]. The oxidation in wood is probably non-enzymatic as several boiled heartwoods consume oxygen [17]. A possible role of polyphenol oxidases, identified in oak heart-

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S. PENG et al.

778

discoloration of w o o d or of pulps p r o d u c e d u n d e r alkaline conditions. Last, insoluble t a n n i n s m i g h t also affect the mechanical properties of w o o d [31, 32].

EXPERIMENTAL General. Castalagin and vescalagin were purified from oak wood by combination of Sephadex LH-20 chromatography and reverse phase HPLC [33]. Ellagic acid and gallic acid were obtained from Fluka and 1-naphthol from Aldrich. Plant materials. Oak (Quercus petraea Liebl.) and chestnut (Castanea sativa Mill.) trees used for sampling were 90 and 50 years old respectively; samples were collected two months after the trees were felled; radial sections were cut at the base of the trunk for chestnut and at 6 m above ground for oak and airdried. Eight samples of different ages were made out of each section and ground in a Retsch mill SM 1 (particule size less than 60 mesh). Polyphenol extraction. Wood meal (100 mg) was extracted at room temp. by 5 ml H20, buffer or organic solvent in a screw cap tube under magnetic stirring for 160 min. It was checked that a plateau was reached at this time. Extractions were run in triplicates. The suspensions are left to sediment a few rain and a 0.1 ml aliquote diluted by 4.9ml H20 before estimation of polyphenols by the Folin-Ciocalteu method [2]. Results are expressed in gallic acid equivalents (GAE). Extracted wood meals were recovered by filtration and air-dried. Extracted wood meals and one extract out of the three triplicates were acid degraded. Acid degradation and ellagic acid estimation. Tannin (10 mg) in a teflon lined screw cap glass tube was dissolved in 5 ml MeOH-aq. HC1 6 M (9:1) containing 0.5 mg 1-naphthol as int. standard. 1-Naphthol was chosen as it is not degraded during the reaction, and as it was not coeluted with other products of wood acid degradation. Furthermore, it has a molar absorption coefficient and solubility properties close to those of ellagic acid. The same reaction was run with wood extracts and extracted wood meals. The added amount of 1-naphthol was determined in order to match the quantity of ellagic acid produced. Wood meals were kept well mixed throughout the reaction with magnetic stirring. The tubes were placed in an oil-bath at 120 ° during 160 min. The solutions were analysed by HPLC on a Merck LiChrospher RP1Be (5 #m) column (25 cm x 4 mm i.d.) with the following elution conditions: linear gradient from 0 to 90% solvent B; solvent A: H 2 0 - H 3 P O 4 (990:1); solvent B: MeOH-H3PO 4 (990:1); gradient duration: 30 rain; flow speed: 1 ml min- 1. Detection: UV 280 nm. Rts were 25.0 and 28.8 min for eUagic acid and 1naphthol respectively. Identity of ellagic acid was checked by cochromatography with commercial standard and by its UV spectra obtained with a diode array detector (Hewlett Packard 1040 A) on line with the HPLC system. Results are expressed in castalagin equivalents (CE) provided that 1 mol of ellagic acid corresponds to 1 tool of castalagin. Acknowledgements--We sincerely thank Mr Lacheze (Office National des For6ts at Rambouillet) who provided the wood samples and Dominique Jouin for excellent technical assistance.

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