Foliar and Cortex Oleoresin Variability of Silver Fir (Abies alba Mill.) in Albania§ Gazmend Z eneliab, Christina Tsitsimpikouc, Panos V. Petrakisd, George Naxakis3, Dalip Habilib and Vassilios Roussis * 0 a Mediterranean Agronom ic Institute o f Chania, P. O. Box 85, 73100, Chania, Greece b Forest and Pasture Research Institute. P. O. Box 8367 Tirana, Albania c School of Pharmacy, Departm ent of Pharmacognosy, University o f Athens, Panepistimioupolis Zografou, Athens 15771, Greece. Fax: ++30 1 7274592. E-mail:
[email protected] d Ministry of Agriculture, Department of Informatics and Biodiversity, Group of Natural R esource M onitoring, Aharnon 381, 111 43 Athens, Greece • Author for correspondence and reprint requests
Z. Naturforsch. 56c, 5 3 1 -5 3 9 (2001); received January 9/March 5, 2001 A bies alba, O leoresin, Terpenes Terpene com position o f needle and cortical oleoresin from lateral shoots were analyzed by GC/MS for four Silver fir (A bies alba Mill.) populations scattered in natural species range in Albania. More than sixty compounds were detected in the needle oleoresin, which was characterized by a high content of a-pinene, camphene, ß-pinene, limonene and bornyl ace tate. Three m onoterpenes, a-pinene, ß-pinene and limonene, and two sesquiterpenes, ßcaryophyllene and germacrene D, comprised the majority of cortical oleoresin. The terpene composition differences among the populations that led to the recognition o f two chemotypes. The needle oleoresin from the provinces of Puka, Bulqiza and Llogara were character ised by high amounts of ß-pinene, camphene and a-pinene and low amounts of limonene, while that from D renova had high amounts of ß-pinene and limonene. A similar pattern was found in the cortical oleoresin with the exception of camphene that was a minor contributor. Geographical and seasonal variation between the populations was, also, investigated. Multi variate analysis o f both needle and cortical oleoresin separated D renova (southeastern pop ulation) from the other sites. W hen both major m onoterpenes and sesquiterpenes were con sidered four chemical profiles could be attributed. Based on their chemical profiles, the populations can be divided into two groups: • Populations with high content of ß-pinene and a-pinene but a low content of limonene (Puka, Bulqiza and Llogara), typical of most of A. alba populations in all its distribution range. • Population with a high content of limonene and a moderate content of ß-pinene and apinene (D renova).
naceae , are widely distributed across both Eastern
Introduction The genus A bies , which is regarded to be com plex in comparison with other genera of the family Pinaceae, was established by Miller in 1754 [type species: E uropean Silver fir (A bies alba)]. There are 39 species, 23 varieties and 8 hybrids of the genus A bies (Liu, 1971). All these taxa are native to cool tem perate and boreal regions of the N orth ern Hemisphere and, like other genera of the Pi-
§ This paper is based on a M.Sc thesis defended by the senior author in the M editerranean Agronomic Insti tute o f Chania, Greece. 0939-5075/2001/0700-0531 $ 06.00
and Western parts of the world. Silver fir, an ecologically valuable and indige nous tree species in many European m ountain for ests, is currently one of the most im portant coni fers in Albania, occupying an area of about 16060 ha or 9.3% of conifer forests. Natural occurring fir forest in Albania, comprise pure fir forest and mixed forest with other conifers or broadleaf. Sil ver Fir populations of the southeastern area of dis tribution are very variable species and regularly had a higher variation than other populations (Wolf, 1990; Wolf, 1994). Extensive studies have been conducted on the morphological, anatomical characters and iso-
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532
G. Zaneli et al. ■N eedle and Cortical Oleoresin C om position o f A bies alba
zymes of fir species in an attem pt to examine the genetic structure of fir populations in Albania and the kind of existing variation (Mitrushi, 1955; Mitrushi, 1966; Habili, 1985; Dano, 1998; Misiri 1999). Recently, several classes of chemical compounds have increasingly been used at all levels of the tax onomic hierarchy, the exact level depending upon the particular compounds employed. Terpenoids, in general, are most useful at the lower level (spe cific and infraspecific) (Stuessy, 1990). Conifer oleoresin composition affords a readily available source of genotypic information, which is much less susceptible to environm ental variation. It rep resents a useful device in chemotaxonomy of coni fers and in characterizing trees of different seed or even clonal origin within a single species (Kossuth et al. 1988; Lockhart, 1990; Lang, 1994; Gallis and Panetsos, 1997; Canard et al. 1997; Gallis et al, 1998). Needle oleoresin, on the other hand, which is a prim ary resin, synthesized in primary tissues, represents another resin system and constitutes a very powerful tool for the study of variation among populations and chemotaxonomic differ ences between species from the same family or ge nus (Fady et al., 1992). The value of the resin from different tissues as taxonomic tools lies in the fact that many of them are under the strong genetic control of relatively few num ber of genes and they are not greatly influenced by environmental factors (Hanover, 1990). Terpenes have been used to solve problems re lated to population and ecological genetics of fir trees as well as m arkers for genetic research into inheritance (Mitsopoulos and Panetsos, 1987; Wolf, 1992; Fady et al., 1992; Lang 1994). M onoterpenes have also been used as a tool in the studies of general adaptation and growth of certain geno
types of fir (Gaudlitz et al., 1988; Wolf, 1992). The m onoterpene composition of the volatiles from the fir needle and cortical oleoresin show a high degree of variation from tree to tree (Paule et al., 1988; Wolf, 1992). To our knowledge, however, no studies have been conducted on terpene variation of Silver fir grown in Albania. The aim of the p re sent study was to determ ine qualitative and quan titative differences in the needle and cortical oleo resin of the naturally grown Silver fir located in different geographical regions in Albania during two seasons. Chemotaxonomic investigation on this species was also attem pted in order to inter pret the origin of variation. Experimental Sampling
Collection Sites Plant material from natural distributions of sil ver fir forest was sampled at four locations: Lum ebardhe (Puka district), Liqeni i Zi (Bulqiza dis trict), Drenova - Bozdovec (Korea district) and Llogara (Vlora district). These areas cover a re presentative sample of the natural population of this species in Albania (Table I). Trees, 15-30 years of age, at least 50 m apart, in good health and without visible symptoms of disease or insect infestation, were selected for this study. Samples were taken considering only last year branches starting from the lower longitudes and progressing to the higher ones. The first sampling was perform ed during the dormancy period, while the second sampling was conducted when the trees were in full vegetation. Winter sampling took place between 15th Novem
Table I. Geographical and topographic characteristics of collection sites. Geographic Position Location* Puka B
u l q iz a
L lo g ar a D renova
Longitude 42° 41° 40° 40°
07' 27' 12' 35'
Topographic Characteristics Latitude
Altitudinal range (m)
Orientation of compartment**
A spect of Slope (°)
20° 10' 2 0 ° 18'
8 0 0 -8 5 0 1540-1600 9 8 0-1050 9 0 0-1000
SW w w SW
23 2 0 -2 5 25 2 1 -2 7
19° 35' 20° 48'
* Plant material from natural distributions of silver fir forest was sampled at four locations: Lumebardhe (Puka district), Liqeni i Zi (Bulqiza district), Drenova - Bozdovec (Korea district) and Llogara (Vlora district). ** W west, SW south-west.
G. Zaneli et al. • N eedle and Cortical Oleoresin Composition of A bies alba
ber and 5th D ecem ber 1998. Summer sampling started on 24th May 1999 and was completed on 8 th June. Twenty trees per area were sampled and each of them was labelled with num bers that were used for the second collection. The samples (branches one or two years old) were kept in dry ice during transportation and then in a deep freezer (-2 0 ° C). Laboratory procedures
Cortical oleoresin sample collection Cortical rather than xylem oleoresin was sam pled as many previous investigations showed that the former contained more major constituents and was less affected by the location in the tree. After the branch was removed from the freezer, drops of oleoresin were collected within 2 - 3 minutes from cortical tissue by excising branch buds at about 10 mm or less from the tips. Exuded oleoresin was placed in screw-cap vials, diluted with 0.25 ml cy clohexane and stored in a refrigerator (4° C) un til analysed. Extraction of needle oleoresin via hydrodistillation The needles were manually separated from the branches and 50 grams of fresh needles were dis tilled in 65 ml of water. A steam distillation m ethod with a Clevenger apparatus was used for distillation of the needles after removal of woody parts. The distillation process lasted three hours. The needle oleoresin collected in cyclohexane (Holubova et al., 2000) was dried over Na 2 S 0 4. Subsequently, the samples were stored in a refrig erator (4 °C) until analysed.
533
260° C, respectively. 0.5 |xl of each sample were injected using an HP 7673 auto-sampler (split ratio 1/ 20 ). The qualitative analysis was perform ed on a HP5890 GC coupled with a VG TR IO 2000 Mass Spectrometer. Chromatographic conditions were similar to those described above. Mass spectra re corded at 70 eV. Identification of the chemical constituents was based on comparison of the re tention times (RT) values and mass spectra with those obtained from authentic standards and/or the NIST/NBS, Wiley 275 library spectra and A d ams’s Book (1995). Statistical procedures
The Statistical Package for Social Sciences (SPSS) for Windows version 8.0 software (SPSS Inc. Chicago, 1997) was used for the statistical analysis. The following differential statistical methods were employed: • One way analysis of variance (one way ANOVA) was used to test the hypothesis of equality of all population means, while multivariate analy sis of variance (MANOVA) was used to test differ ences among the groups • Hierarchical cluster analysis with Ward methods and Squared Euclidean distance was used to clas sify groups of objects judged to be similar accord ing to a distance or similarity measure. • Canonical variate analysis by Wilks-Lambda method was used to analyse the correlation be tween two groups of variables (attributes) in the same set of objects simultaneously. Results and Discussion
Quantitative and qualitative analyses
Needle oleoresin
Q uantitative oleoresin analysis was carried out on a Hewlett Packard Gas C hrom atoghraph HP 5890 series II equipped with a flame ionization de tector (FID). The GC was equipped with a polar fused silica capillary column HP-FFAP (30 m x 0.25 mm i.d., 0.33 ^m film thickness). Helium was used as the carrier gas (flow rate = 1.7 ml/min at 60° C). Initial column tem perature was 60° C for 3 min, then increased at a rate of 3° C/min up to 240° C, and finally held at 240° C for 20 min. Injec tor and detector tem perature were 230° C and
Hydrodistillation of the needles of A. alba re sulted to a pleasant smelling, colorless to pale yel low oil. The geographical variation in the needle oleoresin yield for the investigated populations, is presented in Table II. The yields reported in the present study are within the range previously re ported for the fir species (G uenther, 1952; Habili 1985; Glowacki, 1994). The different yield ob tained from trees of different geographical sites can be explained by the influence of ecological factors (Fady et al ., 1992; Simic et al., 1996). In the
534
G. Zaneli et al. • Needle and Cortical O leoresin Composition of A bies alba
Table II. Variation in essential oil yield from different provenances. Provenance*
Bulqiza Drenova Llogara Puka
Summer C ollection 5
Winter Collection** Range (ml/g)
Mean (%) (ml/g fresh tissue)
Range (ml/g)
Mean (% ) (ml/g fresh tissue)
0 .5 0 -0 .8 8 0 .4 0 -0 .7 8 0 .3 6 -0 .5 8 0 .4 2 -0 .7 6
0.656 0.566 0.484 0.560
0 .1 3 -0 .4 0 0 .1 7 -0 .4 0 0.1 7-0.3 3 0 .1 7 -0 .4 0
0.280 0.278 0.207 0.260
*: LSD test detected that significant variations exist among the provenances under investigation. #: One way analysis of variance (A nova) showed that the yield of essential oil of the Bulqiza provenance differed significantly from that of the Llogara and the Drenova provenances, but no significant differences existed among Puka and other provenances. $: The yields of essential oil from the summer collection showed that only Bulqiza and Llogara differed significantly from each other.
present study, the variation may be attributed to abrupt changes in climatic conditions prevailing in the mainland of Albania. The decrease in needle resin yield from autumn to spring is also supported by other previous investigations (Guenther, 1952; Schonwitz et al., 1990). More than sixty compounds were detected and identified in the needle oleoresins of A. alba, rep resenting approximately 92-96% of the oleoresin (Table III). The two major classes, mono- and sesquiterpenoids, were equally represented by 34 and 36 com pounds, respectively, accounting together for more than 75% of the total needle oleoresin, as is de picted in Table III. Most of the previous studies dealt only with m onoterpenes (Wolf, 1990; Wolf, 1992; Paule et a l, 1988). Sesquiterpenes in the nee dle oleoresin of A. alba have only been studied by Isidorov et al. (1996) and Roussis et al. (2000). Within the class of monoterpenoids, 13 monoterpene hydrocarbons, 9 m onoterpenes alcohol and 1 2 other oxygenated m onoterpenes (including ke tones, esters, ethers and miscellaneous com pounds) were detected. The second class, sesquiterpenoids, was represented by 2 1 sesquiterpene hydrocarbons and 15 oxygenated sesquiterpenes. No qualitative variation was recorded between populations. Generally, only a few compounds were present in all individuals and these major contributors are the compounds present in the highest proportions. M onoterpenes are abundant in all needle oleo resins. Positive correlation is found to exist be tween the relative amounts of a- and ß-pinene and
ß-pinene and limonene and this correlation is sig nificant at alpha 0.01%. The correlation of the rel ative amounts of a-pinene and limonene is nega tive but not significant. ß-Pinene was the major constituent in needle oleoresin in both seasons and all populations. A decrease in the am ount of this compound was observed from winter to summer. In general, the content of m onoterpenes decreases from winter to summer while the concentration of sesquiterpenes increases. The a-pinene content among the investigated populations in both sea sons was low. It seems that, from the major com pounds, limonene is the one that can distinguish the populations. The Tukey HSD test showed that individuals of the D renova population, which yielded the highest am ount of this compound, dif fered significantly from all the populations in the winter collection, and significantly from Puka and Bulqiza but not significantly from Llogara in the the summer collection. Oxygenated m onoterpenoids, although repre sented by many compounds (2 1 ), they account for only 10.3% to 22% of the total percentage. Among them, borneol and a-terpineol and bornyl acetate were the only ones present in all popula tions and individuals analyzed. Bornyl acetate, which was found in high quantities in the winter samples and shows significant variation between the northern populations (Bulqiza and Puka) and southern populations (Drenova and Llogara), does not show any significant variation among the populations in the summer collection. The mean concentration of this compound greatly decreased in the summer collection. There are only a few
535
G. Zaneli et al. • N eedle and Cortical Oleoresin Composition o f A bies alba Table III. Percentage contribution of the identified m etabolites in the needle oleoresin from A. alba. C om pound*
Santene Tricyclene a-P inene C am phene ß-Pinene M yrcene a-P hellandrene Lim onene Z -ß-O cim ene a-T erpinolene
Kovats indices
Winter
888
3.2 (1 .3 3 )A 1.8 (0.24) 9.5 (0.45) 17.2 (1.65) 24.7 (1.74) 1 . 0 (0.06) 0 . 2 ( 1 .1 1 ) 11.4 (2.73) 0.2 (0.14) 0 . 8 (0.06)
926 939 953 980 991 1005 1031 1040 1088
1098 1112
1125 1139 1144 1148 1165 1173 1177 1189 1257 1285 1350
( 0 .1 1 ) tr 0.1 (0.24) tr 0.1 (0.23) 0 . 2 (0 . 1 1 ) 1.0 (0.09) 0.1 (0.25) tr 1.6 (0.45) 0 . 2 (0.08) 17.6 (1.42) 0 . 6 (0.08)
1365 1418 1420 1447 1454 1467 1473 1476 1480 1485 1490 1494 1499 1503 1513 1524 1532 1538 1542
£ -N erolid ol 10-epi-y-Eudesm ol E pi-a-C adinol C ubenol H im achalol a-C adinol £ ,£-F arn eso l
1564 1619 1640 1642 1647 1653 1722
tr (0 .1 2 ) (0.09) (0.23) (0.16) (0.19) (0.45) (0.13) tr (0.46) tr (0 . 1 1 ) (0.16)
0 .2
0.5 1.0 0 .2
0.2 5.5 0.2 3.5 1 .8 0 .2
0.3 (0.11) (1.18) 0 . 2 (0.18) 0.1 (0.33) 5.7 (0.46) tr tr 0.5 (0.11) 0 . 2 (0 . 1 1 ) 0.6 (0.13) 0 . 6 (0 . 1 2 ) 0 . 6 (0 .1 2 ) 0.7 (0.10) 0 . 2 (0.18) 0 . 8 (0 .1 0 ) 2 . 2 ( 0 .2 1 ) 0.2 (0.46) 0.2 (0.55) 0 . 2 (0.18)
1 0 .8
2.3 0.1 0.3 0.4 0.3
tr (0.31) (0.25) (0.14) (0.22) (0.10) tr
Summer
Winter
Summer
2.9 (0.45) 1.7 (0.25) 11.7 (0.85) 16.0 (1.37) 30.7 (1.17) 1.0 (0.67) 0.2 (0.34) 1 1 .5 (0 .0 9 ) 0 . 1 (0.06) 0.8 (0.24)
0.8 (0.09) 0.7 (0.1) 8.4 (0.9) 4.8 (0.12) 18.4 (1.4) 0 . 8 (0 .1 ) 0 . 2 (0.08) 9.8 (0.5) tr 1.7 (0.25)
2.6 (0.45) 1.6 (0.64) 10.9 (0.95) 15.5 (1.14) 29.5 (1.64) 1.1 (0.42) 0 . 1 (0.06) 9.6 (1.12) 0 . 2 (0.06) 0 . 8 (0 .1 2 )
0.9 (0.1) 0.7 (0.09) 9.9 (1.14) 5.2 (0.94) 16.0 (1.45) 0.7 (0.04) 0 . 2 ( 0 .1 1 ) 6.9 (0.74) tr 1.6 (0.46)
42.7
77.2
46.1
72.2
0.1 (0.33) tr tr tr tr tr 0.4 (0.68) 0.1 (0.45) tr 1.4 (0.45) 0 . 1 (0 . 1 2 ) 7.1 (0.73) 0.4 (0.46)
tr 0.2 (0.13) 0.3 (0.23) 0 . 6 ( 0 .1 1 ) 0 . 1 (0.18) 0 . 2 (0 .1 0 ) 3.4 (0.25) 0 . 1 (0.08) tr 4.0 (0.43) tr 1 . 6 (0.16) 0.2 (0.13)
(0.18) tr tr tr tr 0 . 2 (0.16) 0.6 (0.13) tr tr
5.68 0.35
tr 0.4 0.5 0.4 0.1 (0.13) 0.2 (0.17) 2.3 (0.35) 0.1 (0.43) tr 5.8 (0.63) tr 2.1 (0.42) 0.2 (0.13)
0.92 (0.11) tr tr tr tr 0.16 (0.13) 1.7 (0.19) tr 0.1 (0.56) 2.0 (0.41) 1.0 (0.19) 13.0 (1.13) 1.4 (0.13)
10.5
11.3
10.1
12.7
20.9
0.6 (0.14) 12.8 (1.65) 0.3 (0.13) tr 7.2 (0.74) 0.4 (0.10) 0.4 (0.11) 0.8 (0.09) 0.1 (0.24) 1.0 (0.19) 0.3 (0.14) 1 . 0 (0.18) 1.2 (0.14) 0.4 (0.08) 1 . 2 (0.18) 3.5 (0.31) 0.3 (0.11) 0.3 (0.12) 0.3 (0.11)
tr 1.2 (0.14) 0 . 2 (0.16) 0 . 1 (0.28) 0 . 6 (0 .1 2 ) tr tr tr 0.3 (0.13) 0 . 2 (0.18) 0.3 (0.11) 0 . 2 ( 0 .2 0 ) 0.4 (0.21) 0 . 1 (0.28) 0 . 1 (0.26) 0.5 (0.11) 0.1 (0.27) tr tr
(0.18) 7.2 (0.65) 0.3 (0.13) 0 . 1 (0.28) 4.7 (0.44) 0.3 (0.14) 0.3 (0.13) 0.9 (0.11) 0.3 (0.12) 0.7 (0.10) 1 . 2 (0 .1 0 ) 0.7 (0.09) 1.2 (0.13) tr 0 . 1 (0.26) 2 . 8 ( 0 .2 2 ) 0.2 (0.14) 0.3 (0.11) 0 . 1 (0.28)
32.6
5.1
23.0
3.3
25.2
(0.18) 0.7 (0.08) 0.3 (0.12) 0.1 (0.25) tr 4.2 (0.52) 1.0 (0.17)
0.4 (0.08) 4.8 (0.41) 0 . 6 ( 0 .1 0 ) 0.2 (0.19) 0 .6 (0 .1 1 ) 0.3 (0.14) tr
0.4 (0.15) 3.9 (0.34) 0 . 2 (0.18) tr 0.2 (0.19) 4.2 (0.44) 0.3 (0.11)
tr 1.4 (0.14) tr 0.5 (0.09) 0.3 (0.12) 0.42 (0.07) tr
0.2 (0.17) 1.1 (0.14) 0 . 2 (0.18) tr tr 2.7 (0.21) 0.8 (0.09)
_ 0 .6
0.3
0 .2
0 .2 0 .1 0 .2
0.1 0.5 0.1 0.1
(0 . 1 2 ) tr tr (0.18) tr tr tr (0 . 1 1 ) tr (0.16) ( 0 .6 6 ) (0.18) (0.48) tr (0.16) (0.15) tr (0.33)
25.6
3.1
(0.28) 1.8 (0.19) 0 . 2 (0.16) tr tr 2.8 (0.24) 0.7 (0.09)
tr 5.4 (0.55) tr 0.3 (0.11) 0 . 8 (0 . 1 2 ) 0.3 (0.14) 0.3 (0.13)
0 .1
W inter
40.3
14.4
2.8
Sesquiterpene hydrocarbons
Summer
77.0
47.9
0 .2
tr 0.7 (0.11) tr tr 0.4 (0.18) tr tr tr 0.1 (0.38) 0 . 1 (0.26) tr 0.2 (0.14) 0 . 2 ( 0 .1 1 ) tr tr 0.3 (0.12) 0 . 1 (0.28) tr tr
W inter
Puka
Llogara
1.0 (0.07) 2.1 (0.13) 0.3 (0.06) 0.7 (0.23) 1.3 (0.53) 0.3 (0.08) 15.2 (2.61) 1 0 . 1 ( 2 . 1 ) 8.7 (1.15) 5.4 (0.72) 10.8 (1.43) 3.0 (0.75) 16.4 (2.32) 31.6 (2.72) 14.5 (1.67) 0.6 (0.09) 1.2 (0.07) 0.6 (0.09) 0.2 (0.14) 0.2 (0.72) 0.1 (0.31) 6.5 (0.73) 1 7 .5 (2 .1 1 ) 1 0 .2 (1 .9 1 ) tr tr# tr 0.6 (0.07) 1.0 (0.5) 0 . 8 (0.06)
22.05
Oxygenated monoterpenes N eryl acetate E -C aryophyllene p -M e n th - l-e n -9 -o l-a c e ta te a-H im achalene a-H u m ulene 9-epi-E-C aryophyllene y-G urjunene y-M uurolene G erm acrene D ß-Selinene c/s-ß-G uaiene a-S elin en e ß-H im achalene G erm acrene A y-C adinene 6 -Cadinene C a d in a -1 ,4 -diene a-C adinene a-C alacorene
Summer
70.3
Monoterpene hydrocarbons L inalool endo-Fenchol a-C am pholenal frans-Pinocarveol Cam phor C am phene hydrate B orneol m -P in ocam p h o n e T erpin-4 - o l a-T erpineol Linalool acetate B ornyl acetate a-Terpinyl acetate
D renova
Bulqiza
0 .2
0 .2
1 .2 1 0 .1 1
0 .2
0 .8 0 .2 0 .2
0.3
0 .1
0.4 0 .2
0.3 0 .1
0.3
tr ( 0 .2 1 ) (0.16) (0.18) (0.13) tr tr tr (0.28) tr (0.10) (0.16) tr tr (0.13) (0.26) tr (0.11)
(0 .1 1 ) 0.4 (0.16) 0.5 (0.13) 0.9 (0.11) 0 . 2 (0.18) 0.4 (0.38) 5.4 (0.62) 0.3 (0.18) 0.4 (0.14) 6.5 (0.78) tr 2.4 (0.18) 0.4 (0.18)
0 .2
21.4 0.3 9.4 0.7 0.2 5.2 0 .2
0.4 0.7 0.2 0.7 0.4 0.5 1.0 0.3 0.7 2 .0 0 .2 0 .2
0.1
(0.24) (1.12) (0.11) (0.14) (0.44) (0.18) (0.14) (0.16) (0.09) (0.13) (0.10) (0.20) (0.19) (0.13) (0.09) (0.18) (0.16) (0.18) (0.24)
Oxygenated sesquiterpenes
4.8
12.1
9.4
15.7
7.7
18.2
3.5
10.7
Total (%)
100
100
100
99.9
100
100
99.9
100
* : Every compound was present at least in one individual tree in amounts s:0.1% . A : Numbers in parenthesis represent standard deviation. # : Compound present in less than 0.1%. $ : The LSD (least significant difference test) showed that significant differences existed among the provenances.
536
G. Zaneli et al.
reports on the presence of bornyl acetate in genus A bies (Guenther, 1952; Bagci et al., 1999; Ross et al., 1996). Strong positive correlation existed be
tween camphene and bornyl acetate, which was significant at 0.1 level. On the other hand, a decrease in the mean concentrations of camphene and bornyl acetate was associated with an increase in the content of borneol and a-terpineol and a negative correlation existed between these com pounds. The most im portant sesquiterpene hydrocar bons are, namely, E-caryophyllene, a-humulene, a- and ycadinene and ß-himachalene. Drenova differed significantly from Llogara in both seasons when the content of ^-caryophyllene was consid ered. ANOVA revealed that there was no signifi cant variation among the investigated populations for the remaining sesquiterpenes. All populations had a low content of £-caryophyllene and a-humulene with a strong positive correlation, but their mean concentrations increased during the sum mer. E-Caryophyllene had been reported only by Roussis et al. (2000) for A. abies individuals. To our knowledge there has been no other report on its presence in European Silver Fir. From oxygenated sesquiterpenes, 10-epi-y-eudesmol was characterized by significant variation among populations, as shown in Tukey HDS m ultiple’s range and its tendency to increase from north to the south could be observed. The investigated populations in Albania pro duced higher amount of ß-pinene than those from Southeastern Europe and Calabria (Wolf and Bungart, 1992), whereas the production of a-pinene and camphene was lower com pared to that of Eastern and South-Eastern Europe (Wolf, 1990; Wolf, 1994; Wolf and Bungart, 1992). It is interest ing to note the total absence of ß-phellandrene. This compound was found to be a m ajor one in the study reported by Wolf (1994) with a mean concentration of 5.4% but with very high ampli tude (0% to 54% of total needle oleoresin content). As it was generally observed, very high variation existed within each population both in summer and winter collection. Nevertheless, based on the major contributors, a chemical profile could be de duced for each of the populations. While con sidering the chemical profiles in this investigation, trace components were largely ignored as they
N eedle and Cortical Oleoresin Composition of A bies alba
could be probably the result of incomplete block ing of recessive alleles, low level activities of non specific enzymes or chemical ärtefacts“ caused by chemical transformations during extraction and isolation (Franz. 1993). These chemical profiles, particularly those of the winter collection, ap peared qualitatively the same for the populations of Puka, Bulqiza and Llogara, and agreed with re sults previously reported (Roussis et al ., 2000): ß-pinene > camphene > a-pinene > limonene
On the contrary, the chemical profile of D ren ova population was different, but in accordance with a chemical profile for the Southeastern E uro pean populations reported by Wolf (1990): ß-pinene > a-pinene > limonene > camphene Multivariate analysis was carried out using Ca nonical Discriminant Analysis and Cluster Analy sis for the m ajor compounds (> 1 %) and for two classes of terpenes both for winter and summer collection. The results for the winter collection showed significant differences up to the third func tion. The total variance proportion for the first two functions was 8 8 %, with 65.9% for the first (ca nonical correlation 0.889) and 22% for the second (canonical correlation 0.889). Appropriate plot ting indicated several groupings of the popula tions. The distribution centers of Llogara and Drenova appeared very close, whereas Puka and Bulqiza were very well separated both from each other and from Drenova and Llogara. On the other hand, the results from the summer collection didn’t produce a good separation for the investi gated populations, as several cases were misclassified. Cortical oleoresin
Cortical oleoresin composition was different from the needle oleoresin profile. This study con firmed the existence of two resins that have dif ferent terpene proportions, which has been noted previously for other conifer species (Sjordin et al., 1996; D orm ont et al ., 1998; Kleinhentz et al ., 1999; Wainhouse et al., 2000). The results of the analyses revealed the presence of thirty-seven compounds, including m ono-and sesquiterpenoids. Thirty-three of these compounds were identified, representing approximately 95-97% of the cortex oleoresin. Generally, the number of compounds in the summer collection samples was smaller than the
537
G. Zaneli et al. ■N eedle and Cortical Oleoresin Composition o f A bies alba
num ber of compounds found in the winter collec tion. This decrease in the number of compounds was higher in Puka and smaller in Drenova. The highest variation in terms of the number of com pounds was observed for the sesquiterpenes, while oxygenated sesquiterpenes exhibited the highest seasonal variation. The cortical oleoresin of silver fir in both sea sons was found to contain mainly m onoterpene hydrocarbons with a mean yield of 62-82% . The compositions of the studied populations of silver fir were similar to those reported by Lang (1994) and Vendramin et al. (1997), all stressing the im portance of a-pinene, ß-pinene and limonene. Ox
ygenated m onoterpenes had a mean concentration smaller than 2%. As shown in Table IV, Llogara had the highest mean concentration of m onoter penes and regardless the change in the mean con centration of single compounds, there were no sig nificant differences between the seasons. Drenova shows the highest variation in monoterpene content as the mean increased from 64.23% in the winter collection to 85.83% in the summer collec tion. a-Pinene is one of the major compounds found in all populations. A general increase in the content of this compound from winter to summer could be observed. Drenova was the lowest pro-
Table IV. Percentage contribution of the identified m etabolites in the cortical oleoresin from A. alba. Com pound*
Kovats indices
a-P inene Cam phene ß-Pinene M yrcene Lim onene a-T erpinolene
939 953 980 991 1031 1088
Bulqiza Winter
71.0 1285
0 .2
Oxygenated monoterpenes a-L ongipinene Neryl acetate £-C aryophyllene a-H im achalene a-H um ulene y-M uurolene Germ acrene D ß-m -G u aijene a-S elinene ß-Him achalene y-Cadinene 6 -C adinene a-C adinene
Summer
1351 1365 1418 1447 1454 1477 1480 1490 1494 1499 1513 1524 1538
Winter
Summer
W inter
Puka
Summer
Winter
Summer
26.3 (1 .4 5 )A 42.9 (4.11) 12.2 (1.42) 23.1 (2.15) 36.0 (3.11) 38.6 (3.65) 37.3 (3.65) 47.1 (4.23) 0.7 (0.14) 0.9 (0.10) 0.3 (0.15) 0.4 (0.11) 0.6 (0.09) 0.4 (0.08) 0.9 (0.10) 0.7 (0.11) 35.7 (2.11) 26.6 (3.10) 14.9 (1.22) 18.3 (1.15) 32.6 (3.24) 29.9 (2.33) 36.6 (3.06) 30.0 (3.21) 1.4 (0.18) 1.7 (0.18) 1 . 6 ( 0 .2 2 ) 2 . 6 ( 0 .2 2 ) 1.5 (0.18) 1.6 (0.17) 1.4 (0.14) 1.4 (0.13) 6.9 (0.64) 10.2 (1.43) 35.3 (3.14) 41.4 (4.44) 10.2 (1.04) 10.3 (1.18) 6.7 (0.63) 5.5 (0.53) tr # tr tr tr 0 . 1 (0.26) tr tr 0 . 1 (0.28)
Monoterpene hydrocarbons Bornyl acetate
Llogara
D renova
82.5
64.2
85.8
81.1
80.9
(0.16)
tr
1.1 (0.24)
tr
tr
-
0.2
tr
1.1
tr
tr
0.3 (0.13) tr 8.9 (0.86) tr 3.9 (0.35) 0.3 (0.11) 15.2 (1.54) 0 . 2 (0.16) tr tr 0.2 (0.17) 0.9 (0.15) 0.6 (0.09)
tr 0.1 (0.27) 3.9 (0.33) tr 1.9 (0.11) tr 6.9 (0.66) tr tr tr 0.1 (0.29) 0 . 1 ( 0 .2 2 ) 0.3 (0.13)
13.5
0.3 (0.12) 0.2 (0.31) 5.0 (0.51) 0.3 (0.12) 2.4 (0.21) 0.6 (0.09) 12.5 (1.23) 0.6 (0.09) 0.3 (0.13) 0.2 (0.19) 0 . 6 (0.08) 1 . 1 (0.16) 0.6 (0.07)
Sesquiterpene hydrocarbons
0 .1 0 .1
3.6 1.8 0.1 9.2 0.2 0 .1
0.3 0.3 0 .2
(0.26) (0.28) (0.33) tr (0.19) (0.27) (0.95) tr (0.19) ( 0 .2 1 ) (0.10) (0.11) (0.16)
83.1
84.7
(0.18)
tr
-
0.2
tr
tr tr 3.2 (0.38) tr 1 . 6 (0.18) tr 1 2 . 1 (1.26) tr tr tr 0 . 1 (0.26) tr 0.4 (0.13)
0.3 (0.11) (0.26) 3.4 (0.33) 0.3 (0.14) 1 . 6 (0.18) 0.3 (0.13) 5.9 (0.56) 0.7 (0.11) tr 0 . 2 (0.16) 0.2 (0.17) 0.6 (0.09) 0.3 (0.14)
15.2
18.0
14.8
(0.16) 1.4 (0.32) 0.4 (0.16) 0.2 (0.24)
tr 0.4 (0.24) tr 0.3 (0.15)
3.9 1 .8
0.4 6 .6
0 .1
0.1 0.3 0.6 0.5
tr tr (0.31) tr (0.18) (0.10) (0.61) tr ( 0 .2 2 ) (0.23) (0.16) (0.09) (0.10)
0 .2
0 .1
3.6 1 .8
8.4 0.1
0 .2
0.1 0 .2
tr tr (0.33) tr ( 0 .1 1 ) tr (0.81) (0.23) tr tr (0.18) (0.25) (0.16)
25.7
16.0
30.8
(0.16) 1.4 (0.16) 0.2 (0.15) tr
tr 1.1 (0.14) tr 0 . 1 ( 0 .2 2 )
(0.26) 2.0 (0.24) tr 0.1 (0.25)
Oxygenated sesquiterpenes
2.11
1.2
3.4
0.3
3.3
1.0
1.4
0.2
Total (%)
99.0
99.7
99.5
99.6
99.6
99.9
99.5
99.7
£ -N erolid ol 10-epi-y-Eudesm ol epi-a-C adinol £ , £-Farnesol
1564 1619 1640 1722
0 .2
0 .1
0 .2
tr (0.18) tr tr
0 .2
* : Every compound was present at least in one individual tree in amounts >0.1% . A : Numbers in parenthesis represent standard deviation # : Compound present in less than 0.1%.
0 .8 0 .2 0 .2
tr (0.16) (0 . 1 2 ) (0.18)
14.8
0 .2
tr tr tr (0 .1 1 )
538
G. Zaneli et al. ■N eedle and Cortical Oleoresin Composition o f A bies alba
ducer of ß-pinene and the highest of limonene. Llogara showed the highest within-population vari ation for a-pinene and ß-pinene for both the winter and summer collection. A tendency for an increase in limonene content from north to south was ob served whereas a north to south decreasing trend could be observed for camphene. A strong correla tion existed between the relative amounts of a-pinene, ß-pinene and limonene. This correlation was positive and significant at alpha = 0.05% for a-pi nene and ß-pinene. Negative correlation existed be tween the content of ß-pinene and limonene and between a-pinene and limonene. This correlation was significant at the 0.01 level. Strong positive cor relation between m onoterpene proportions may in dicate common biosynthesis (Shaw et al., 1982). In grand fir (A bies grandis Mill) the common biosyn thetic route was found to be catalyzed by a single enzyme and led to the formation of the major monoterpenes (Bohlmann et al., 1997). A large number of sesquiterpenes were present in low quantities in the cortical oleoresins. High variation existed in the quantities of these com pounds in all populations and between seasons. ECaryophyllene was among the most im portant ses quiterpenes. Significant variation existed among populations. Individuals from Drenova contained the highest amounts of f-caryophyllene in both the
summer and winter collection. O ther important sesquiterpenes detected were germacrene D and the three isomers of cadinene: a-cadinene, y-cadinene and 6 -cadinene. It is interesting to note the strong positive correlation found between E-caryophyllene and a-humulene. These compounds come from the same biosynthetic route and have similar structures (Croteau, 1986). A strong positive corre lation is also found between these two compounds in the needles of Pinus caribaea (Barnola et al., 1997), the needles of Cedrus libani (A.) Rich, and Cedrus atlantica Manet (Canard et al., 1997). Among the group of oxygenated sesquiterpenes, only 1 0 -epi-y-eudesmol was found in amounts higher than 1 % of the total content.
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Acknowledgem ents
This work was financially supported by the M editerranean Agronomic Institute of Chania. G. Zeneli wishes to thank M. Aliaj and A. Kurti for their help during collections. G.Z acknowledge the help of Prof. K. Panetsos, (University of Thessaloniki-Greece); Dr. B.Fady (INRA-France), Dr. H. Wolf, (Sachs. Landesanstalt für Forsten GraupaGerm any), and Dr. K. J. Lang (Lehrstuhl für Forstbotanik München Germany), for providing articles of their work.
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