Industrial Crops and Products 21 (2005) 7179

Composition and chemical variability of the oleoresin of Pinus nigra ssp. laricio from Corsica
Serge Rezzi, Ange Bighelli , Vincent Castola, Joseph Casanova
UMR CNRS 6134, Equipe Chimie et Biomasse, Universit de Corse, Route des Sanguinaires, 20000 Ajaccio, France Received 15 April 2003; accepted 23 December 2003

Abstract Eighteen samples of oleoresin of Pinus nigra ssp. laricio were harvested in six locations in Corsica. Eight diterpene acids, six neutral diterpenes and -pinene were identied and their contents calculated by 13 C NMR spectroscopy, following a procedure recently developed in our laboratories. The results were submitted to chemometric analysis (k-means and principal component analysis) and two clusters were distinguished with respect to the contents of levopimaric acid and dehydroabietic acid. 2004 Elsevier B.V. All rights reserved.
Keywords: Pinus nigra ssp. laricio; Oleoresin; Chemical composition; Chemical variability; Diterpenes; 13 C NMR

1. Introduction Pine oleoresin is an important forestry product, which is traditionally obtained by tapping the bark (bark chipping) of pine tree and collection of the resulting exudate. Oleoresins are complex mixtures of acidic and neutral diterpenes together with a more or less important fraction of volatile compounds (monoand sesquiterpenes). In the industry, the crude oleoresin is converted by steam distillation into gum turpentine (volatile compounds) and gum rosin (diterpenes), both gums in turn are processed into chemical industrial products such as food gums, adhesives, coatings, printing inks, disinfectants, cleaners, pharmaceuticals, fragrances and avoring.
Corresponding author. Tel.: +33-4955-24123; fax: +33-4955-24142. E-mail address: ange.bighelli@univ-corse.fr (A. Bighelli).

Several studies have been carried out on the characterization of pine oleoresin. The main components are neutral monoterpenes (mostly -pinene) and diterpene acids. Neutral diterpenes and sesquiterpenes are also present at appreciable to moderate amounts. Several compositions, exhibiting bicyclic diterpenes (labdanes), tricyclic diterpenes (abietanes and pimaranes) and macrocyclic diterpenes (cembranes) as main components have been reported in the literature depending on the pine species and the geographical origins (Iconomou and Valkanas, 1966; Tobolski and Zinkel, 1982; Hazo glu, 1983; Walter et al., 1985; Weimann and Lange, 1987; Lange and Weimann, 1987, 1988, 1989; Fiebach and Kraemer, 1993; Lange and Stevanovic Janeic, 1993; Lange et al., 1994a,b; Cheung et al., 1994; Lange and Spanoudaki, 1995; Song et al., 1995; Sunzel et al., 1997; Coppen et al., 1998; Arrabal et al., 2002).

0926-6690/$ see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2003.12.008

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Pinus nigra Arnold (European black pine) is native to Europe and Asia. The species is divided into ve subspecies, among them P. nigra ssp. laricio (Poir.) Maire (=P. laricio Poir.), is widespread in Calabria and Sicily (Italy) and Corsica (France). In Corsica, P. nigra ssp. laricio constitutes forests covering 22000 ha. It is a tree up to 50 m high with a straight trunk and grey to dark brown bark growing on supramediterranean and mountainous terrain at altitudes of 9001800 m (Gaussen et al., 1993; Gamisans and Marzocchi, 1996). This tree is exploited for timber production under the control of the Ofce National des For ets (ONF). As part of our ongoing work on the chemical characterization of the extractives of species growing wild in Corsica (Castola et al., 2000, 2002; Rezzi et al., 2001a,b), the aim of this study was to characterize the oleoresin of P. nigra ssp. laricio, to observe the homogeneity or conversely the chemical variability of its composition, and to compare the composition with those of other species of pine.

digital resolution of 0.382 Hz/pt; line broadening (LB) of 1 Hz was applied before Fourier transform. The spectra of the resins were recorded with 200250 mg of raw material diluted in 2 ml of CDCl3 added with 1012 mg (weighted at the precision of 0.1 mg) of diglyme (internal standard for quantitative determinations), 5000 acquisitions were accumulated for each sample. 2.3. Identication and quantitative determination of diterpenes NMR was carried out on the whole sample without previous separation of the components. Identication of the components was led according to an experimental procedure and a computerized method developed in our laboratories and adapted to diterpenes (Rezzi et al., 2002). The components were identied by comparison of the values of the carbon chemical shifts in the mixture spectrum with those of reference spectra compiled in a computerized data bank. Each compound is identied by taking into account three parameters, directly available from the computer program: (i) the number of observed signals with respect to that expected, (ii) the difference between the chemical shift of each signal in the mixture and in the reference ( ), (iii) the number of overlapped signals of carbons belonging to two components which possess fortuitously the same chemical shift. The mass percentage (% mC ) of each diterpene was determined following the procedure recently developed by our group which provided satisfactory accuracy and precision (Rezzi et al., 2002). From the 13 C NMR spectrum, the mean value of the area of the protonated carbons of each diterpene (AC ) was calculated and compared to that of the two methylenes of diglyme (AD ) which was used as internal standard. The calculated amount mC (mg) of each diterpene, was determined using the formula (1) and its subsequent mass percentage was then calculated. mC = 2 AC M C m D AD M D (1)
13 C

2. Material and methods 2.1. Plant material Eighteen samples of oleoresin of P. nigra ssp. laricio, obtained by tapping the bark of individual adult trees growing wild in the six most important forests of Corsica (Aitone, Ghisoni, Melo, Valdoniellu, Verde and Vizzavona, three samples per location) were collected in July and August 1999 (Fig. 1). 2.2.
13 C

NMR spectra

All the 13 C NMR spectra were recorded on a Bruker AC 200 Fourier Transform spectrometer operating at 50.323 MHz for 13 C, equipped with an Aspect 3000 computer and a 10 mm probe. The spectra were recorded in deuteriochloroform, with all shifts () referred to internal tetramethylsilane (TMS), with the following parameters: pulse width (PW): 3 s (ip angle 30 ); acquisition time: 1.3 s and relaxation delay D1 : 0.1 s (total recycling time 1.4 s). The data were zero lled from 32 to 64 K data table with a spectral width (SW) of 12,500 Hz (250 ppm); composite phase decoupling (CPD) of the proton band;

where mD is the amount (mg) of diglyme, MD the molecular weight of diglyme, and MC the molecular weight of the considered diterpene. The factor 2 is due to the symmetry of diglyme.

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Fig. 1. Sampling of the oleoresins of Pinus nigra ssp. laricio from Corsica.

2.4. Chemometric analysis Principal component analysis (PCA) was performed using Statgraphics Plus 2.1 (Uniwin Plus, France), K-means clustering was performed using K-means partitioning program (Pierre Legendre, Canada).

3. Results and discussion Identication and quantitative determination of the major components of 18 samples of oleoresin were

carried out by using a method recently developed in our laboratories and based on the computer aided analysis of the 13 C NMR spectrum of the crude oleoresin (Rezzi et al., 2002). The results were submitted to chemometric analysis. In the 18 samples, we identied and quantied, using 13 C NMR spectroscopy, 15 compounds (Fig. 2), i.e. eight resinic acids (abietic acid 1, dehydroabietic acid 2, neoabietic acid 3, palustric acid 4, levopimaric acid 5, isopimaric acid 6, pimaric acid 7a, and sandaracopimaric acid 7b), six neutral diterpenes (pimaral 7c, pimarol 7d, isopimaral 8, isocembrol 9a, 4-epi-isocembrol 9b and cembrene

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S. Rezzi et al. / Industrial Crops and Products 21 (2005) 7179

H H COOH 1 H COOH 2

H H COOH 3

H H COOH 4 R2 R3 H H R1 7a-7d H H CHO 8 H COOH 5

H H COOH 6 R1 R2

9a-9b

10

11

Fig. 2. Compounds identied in the oleoresins of P. nigra ssp. laricio from Corsica. 1: abietic acid, 2: dehydroabietic acid, 3: neoabietic acid, 4:palustric acid, 5: levopimaric acid, 6: isopimaric acid, 7a: pimaric acid (R1 = COOH, R2 = CH=CH2 , R3 = CH3 ), 7b: sandaracopimaric acid (R1 = COOH, R2 = CH3 , R3 = CH=CH2 ), 7c: pimaral (R1 = CHO, R2 = CH=CH2 , R3 = CH3 ), 7d: pimarol (R1 = CH2 OH, R2 = CH=CH2 , R3 = CH3 ), 8: isopimaral, 9a: isocembrol (R1 = OH, R2 = CH3 ), 9b: 4-epi-isocembrol (R1 = CH3 , R2 = OH), 10: cembrene, and 11: -pinene.

10) and one monoterpene (-pinene 11), which represented from 75.5 to 88.7% of the whole composition of each resin (Table 1). According to the intensities of the unassigned signals in the 13 C NMR spectrum, all the unidentied compounds were minor components. The acidic fraction, which represented from 46 to 66% of the identied compounds, was the major one while neutral components accounted for 1736% of the composition.

Seven diterpene acids, abietic acid (4.213.5%), dehydroabietic acid (2.317.8%), neoabietic acid (2.811.8%), levopimaric acid (2.726.0%), palustric acid (6.815.0%), pimaric acid (2.15.7%) and isopimaric acid (1.87.6%) were present in the 18 samples. Similarly, the neutral oxygenated diterpenes isocembrol (1.810.5%) and 4-epi-isocembrol (1.711.3%) and the monoterpene -pinene (3.616.8%) were identied in all samples. Conversely, sandaracopimaric

Table 1 Chemical composition of the 18 resins of P. nigra ssp. laricio harvested in six locations of Corsica Components Vizzavona 1 Abietic acid 1 Dehydroabietic acid 2 Neoabietic acid 3 Palustric acid 4 Levopimaric acid 5 Isopimaric acid 6 Pimaric acid 7a Sandaracopimaric acid 7b Pimaral 7c Pimarol 7d Isopimaral 8 Isocembrol 9a 4-epi-isocembrol 9b Cembrene 10 -Pinene 11 Total
a

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Ghisoni 3 (I) 4.2 3.2 8.2 12.9 26.0 3.6 3.6 0.7 1.8 1.7 16.8 82.7 4 (I) 6.7 6.2 11.8 10.0 19.5 5.7 4.2 1.0 1.2 0.7 1.0 6.6 7.4 6.7 88.7 5 (II) 8.8 8.8 11.0 15.0 11.7 4.0 4.6 4.5 7.5 1.6 4.2 81.7 6 (II) 8.7 13.1 7.0 11.5 13.2 4.2 2.8 1.6 0.9 0.9 1.0 4.0 4.2 8.7 81.8

Verde 7 (I) 7.7 6.0 8.2 7.9 15.9 4.3 4.5 1.2 5.0 5.7 12.2 78.6 8 (I) 5.6 2.3 10.9 12.2 21.0 4.3 5.1 1.6 1.3 0.6 5.0 5.3 13.0 88.2 9 (II) 7.5 12.7 7.3 9.5 12.7 5.0 5.4 1.5 1.0 0.9 4.4 5.0 6.7 79.6

Melo 10 (II) 4.3 12.7 6.7 10.0 9.8 4.4 4.9 10.5 11.3 10.9 85.5 11 (I) 6.5 5.8 9.2 9.5 22.7 1.8 2.3 4.8 5.0 13.0 80.6 12 (I) 4.6 4.3 9.9 12.9 24.9 4.1 5.0 1.3 1.7 5.8 6.4 3.6 84.5

Aitone 13 (II) 9.5 7.8 5.3 7.1 8.7 7.6 4.6 0.9 0.8 6.4 6.3 15.7 80.7 14 (I) 4.7 3.7 8.9 8.4 17.0 3.7 2.6 0.8 0.7 8.9 9.9 15.0 84.3 15 (II) 7.0 8.3 7.1 7.4 10.0 2.8 2.6 0.9 9.9 10.0 16.4 82.4

Valdoniellu 16 (II) 13.5 14.5 3.6 6.8 2.7 6.4 2.1 8.6 9.2 8.1 75.5 17 (II) 8.3 17.8 2.8 8.6 4.9 5.9 4.9 1.0 4.8 5.4 11.1 75.5 18 (II) 9.7 10.6 9.0 11.3 10.8 6.6 5.6 1.2 1.5 0.8 0.8 4.2 5.5 4.2 81.8

(II)a

2 (II) 9.2 8.9 7.0 8.4 7.3 4.5 5.7 0.7 1.3 0.9 6.2 7.2 14.9 82.2

5.5 8.7 9.0 9.3 12.8 5.2 5.5 0.6 1.2 10.5 11.0 5.5 84.8

Sample (cluster).

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20

Cluster I Cluster II

axis 2 (20,4%)

2 16 17

13

15 14 7 6 9 18 1 5 4 12
30

3 11 8

10

-10 -10 axis 1 (58,1%)

Fig. 3. Principal component analysis scatterplot of 18 samples of resin of Pinus nigra ssp. laricio from Corsica.

acid (0.61.6%), pimaral (0.81.5%), and isopimaral (0.61.7%) were identied respectively in 11, 11 and 8 samples, while pimarol (0.70.9%) was detected in three samples and cembrene (1.6%) was present only once at a content observable by NMR. The results of the 18 analyses were submitted to chemometric analysis in order to distinguish clusters. K-means analysis, taking into account the mass percentages of the 14 diterpenes and -pinene in the 18 samples, suggested the existence of two principal clusters within the resins of P. nigra. This partitioning was thus corroborated by principal component analysis in which the rst two axes accounted for 58.1 and 20.4%, respectively (Fig. 3). Samples belonging to cluster I (seven samples) were characterized by a high content of levopimaric acid (mean: M = 21.0%, standard deviation: S.D. = 3.8) (Fig. 4). The other important components were -pinene (M = 11.5%, S.D. = 4.7), palustric acid (M = 10.5%, S.D. = 2.1), neoabietic acid (M = 9.6%, S.D. = 1.4), 4-epi-isocembrol (M = 5.9%, S.D. = 2.5), abietic acid (M = 5.7%, S.D. = 1.3) and isocembrol (M = 5.4%, S.D. = 2.1). The content of the remaining components did not exceed 5%. In samples belonging to cluster II (11 samples), the mean content of levopimaric acid (M = 9.5%, S.D. = 3.4) is much less important than in cluster I (Fig. 4). It is of the same magnitude than those of other acids: dehydroabietic acid (M = 11.3%, S.D. = 3.2), palustric acid (M = 9.5%, S.D. = 2.4) and abietic acid (M = 8.4%, S.D. = 2.4). The contents of neutral

diterpenes 4-epi-isocembrol (M = 7.5%, S.D. = 2.5) and isocembrol (M = 6.7%, S.D. = 2.7) were a little bit more important than in cluster I. -Pinene was also present at appreciable amount (M = 9.7%, S.D. = 4.5). The proportion of the other products was less than 5% (Fig. 4). Only a few studies reported the chemical composition of resins of P. nigra. They concerned: (i) the acidic fraction of the resin of pines from Greece (Iconomou and Valkanas, 1966), Spain and Yugoslavia (Tobolski and Zinkel, 1982) and, Russia and Bulgaria (Hazo glu, 1983); and (ii) the neutral diterpene fraction of resins of P. nigra of Austrian origin (Lange and Weimann, 1987, 1989). It appears that, the chemical composition of samples belonging to cluster I differs from most of that reported in the literature. Indeed, in the samples from Yugoslavia and Spain (Tobolski and Zinkel, 1982), levopimaric acid represented 56% of the acidic fraction while, in our samples, this acid reached 1626% of the whole composition i.e. 2743% of the identied acid diterpenes. In the samples from Greece (Iconomou and Valkanas, 1966) and Russia (Hazo glu, 1983), abietic acid and isopimaric acid represented, respectively, 16.723.0 and 9.620.0% of the composition of the acidic fraction while in our samples, these two compounds reached only 4.27.7 and 1.85.7% of the global composition i.e. 7.012.8 and 3.09.5% of the acidic compounds. Conversely, the chemical composition of the acidic fraction of samples from Bulgaria is quite similar to that of our

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Fig. 4. Mean chemical composition of samples of clusters I and II. Grey: mean, hachured: standard deviation.

samples belonging to cluster I (Hazo glu, 1983). Concerning the samples of cluster II, they could be distinguished from Greek, Spanish and Yugoslavian resins in which dehydroabietic acid was identied only at very low concentrations, while this compound was present at higher contents (7.817.8% of the whole composition) in our samples. However, the chemical composition of the acidic fraction of the resin of P. nigra from Russia (Hazo glu, 1983) is close to that of samples of cluster II. The comparison of Corsican pine resin with the resin of P. nigra of Austrian origin is more difcult since in the last one, only the chemical com-

position of the minor neutral diterpene fractions is reported (Lange and Weimann, 1987, 1989). Nevertheless, samples from Corsica (clusters I and II) in which isocembrol and 4-epi-isocembrol represented 1.810.5 and 1.711.3%, respectively, of the whole composition, differed from those of Austrian origin in which these two diterpene alcohols were not reported. Otherwise, a few studies concerning the chemical composition of resins of several species of Pinus have been published. The resins of P. nigra ssp. laricio of Corsica could be distinguished from those of P. pinaster (Arrabal et al., 2002) P. montezumae

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S. Rezzi et al. / Industrial Crops and Products 21 (2005) 7179 populations of Thai and Filipino Pinus merkusii de Vriese. Flav. Fragr. J. 13, 3339. Fiebach, K., Kraemer, L.R., 1993. Resins, Natural in Ulmanns Encyclopedia of Industrial Chemistry, vol. A23. VCH Publishers, Bremen, pp. 7388.. Gamisans, J., Marzocchi, J.F., 1996. La Flore Endmique de la Corse. Edisud, Aix-en-Provence. Gaussen, H., Heywood, D.H., Chater, A.O., 1993. In: Pinus, L., Tutin, T.G., Burges, N.A., Chater, A.O. (Eds.), Flora Europaea, vol.1. Cambridge University Press, Cambridge. Hazo glu, H., 1983. Wood Extractives of Pinus silvestris L., Pinus nigra Arn. and Pinus brutia Ten. with special reference to nonpolar components. Holzforshung 37, 321326. Iconomou, N., Valkanas, G., 1966. ber die Zusammensetzung des harzbalsams einiger Pinus-Arten Griechenlands. Pharmaceutica Acta Helvetiae 41, 5963. Lange, W., Weimann, G., 1987. Zusammensetzung der Neutralteile des Balsamkolophoniums von Pinus silvestris L., Pinus nigra austriaca Endl. und Pinus pinaster Ait. Holz als Roh-und Werkstoff. 45, 345349. Lange, W., Weimann, G., 1988. Composition of Pinus sylvestris L. gum oleoresin from different sources. Holz als Roh- und Werkstoff. 46, 157161. Lange, W., Weimann, G., 1989. Die Zusammensetzung der Diterpenkohlenwasserstoffe des Harzbalsams von Pinus nigra austriaca Endl., Pinus silvestris L. und Pinus pinaster Ait. Holzforshung 43, 359362. Lange, W., Stevanovic Janeic, T., 1993. Chemical composition of some Pinus sylvestris L. oleoresins from southern Serbia, Bosnia and Makedonia. Holzforschung 47, 207212. Lange, W., Spanoudaki, M., Stevanovic Janeic, T., 1994a. The composition of Balkan white pine (Pinus peuce Griseb.) oleoresin. Holzforschung 48, 368370. Lange, W., Stevanovic Janeic, T., Spanoudaki, M., 1994b. Cembratrienols and other components of white bark pine (Pinus heldreichii) oleoresin. Phytochemistry 36, 12771279. Lange, M., Spanoudaki, M., 1995. The composition of an oleoresin of Pinus montezumae Lamb. from Guatemala. Holz als Rohund Werkstoff 53, 6871. Rezzi, S., Bighelli, A., Mouillot, D., Casanova, J., 2001a. Composition and chemical variability of the needle essential oil of Pinus nigra subsp. laricio from Corsica. Flav. Fragr. J. 16, 379383. Rezzi, S., Cavaleiro, C., Bighelli, A., Salgueiro, L., Proena da Cunha, A., Casanova, J., 2001b. Intraspecic chemical variability of the leaf essential oil of Juniperus phoenicea subsp. turbinata from Corsica. Biochem. Syst. Ecol. 29, 179 188. Rezzi, S., Bighelli, A., Castola, V., Casanova, J., 2002. Direct identication and quantitative determination of acidic and neutral diterpenes using 13 C NMR spectroscopy. Application to the analysis of oleoresin of Pinus nigra. Appl. Spectrosc. 56, 312317. Song, Z., Liang, Z., Liu, X., 1995. Chemical characteristics of oleoresins from Chinese pine species. Biochem. Syst. Ecol. 23, 517522.

(Lange and Spanoudaki, 1995), P. sylvestris (Lange and Weimann, 1988; Lange and Stevanovic Janeic, 1993), P. massoniana (Weimann and Lange, 1987), P. merkusii (Weimann and Lange, 1987), P. luchensis (Weimann and Lange, 1987) by the occurrence of isocembrol and 4-epi-isocembrol. They differed also from the 22 species belonging to the Strobus, Parrya and Pinus sections from China (P. sibirica, P. bungeana, P. taiwanensis, etc.), in which these two macrocyclic alcohols were not reported (Song et al., 1995). Conversely, isocembrol has been identied in the resins of P. peuce (Lange et al., 1994a) and P. heldreichii (Lange et al., 1994b). However, these two pine species can be differentiated from P. nigra ssp. laricio since their resins exhibited higher contents of cembrol and isopimaric acid (P. peuce) or limonene (P. heldreichii). From these results, we conclude that the oleoresins of P. nigra ssp. laricio from Corsica can be classied into two clusters in function of the contents of levopimaric acid and dehydroabietic acid and both compositions could be distinguished from most of the other P. nigra as well as those of other Pinus species reported in the litterature.

Acknowledgements We are indebted to Collectivit Territoriale de Corse (CTC) and Agence De lEnvironnement et Ma trise de lEnergie (ADEME) for a research grant (SR).

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