High Diversity of Indigenous Populations of Dalmatian Sage ( Salvia officinalis L.) in Essential-Oil Composition by Marija Jug-Dujakovic ´* a ), Mihailo Ristic ´ b ), Dejan Pljevljakus ˇic ´ b ), Zora Dajic ´-Stevanovic ´ c ), Zlatko Liber d ), Katarina Hanc ˇevic ´ a ), Tomislav Radic ´ a ), and Zlatko S ˇ atovic ´ e ) a ) Institute for Adriatic Crops and Karst Reclamation, Split, Put Duilova 11, HR-21000 Split (phone: þ 385-21-434402; fax: þ 385-21-316584; e-mail: [email protected]) b ) Institute for Medicinal Plant Research (Dr. Josif Panc ˇic ´), Tadeus ˇa Kos ˇc ´us ˇka 1, RS-11000 Belgrade c ) Faculty of Agriculture, University of Belgrade, Nemanjina 6, RS-11000 Belgrade d ) University of Zagreb, Faculty of Science, Marulic ´ev trg 9, HR-10000 Zagreb e ) University of Zagreb, Faculty of Agriculture, Svetos ˇimunska 25, HR-10000 Zagreb Essential oils of 25 indigenous populations of Dalmatian sage ( Salvia officinalis L.) that represent nearly half of native distribution area of the species were analyzed. Plantlets collected from wild populations were grown in the same field under the same environmental conditions and then sampled for essential-oil analysis. The yield of essential oil ranged from 1.93 to 3.70% with average of 2.83%. Among the 62 compounds detected, eight (cis-thujone, camphor, trans-thujone, 1,8-cineole, b-pinene, camphene, borneol, and bornyl acetate) formed 78.13–87.33% of essential oils of individual populations. Strong positive correlations were observed between camphor and b-pinene, b-pinene and borneol, as well as between borneol and bornyl acetate. The strongest negative correlation was detected between camphor and trans-thujone. Principal component analysis (PCA) on the basis of eight main compounds showed that first main component separated populations with high thujone content, from those rich in camphor, while the second component separated populations rich in cis-thujone from those rich in trans-thujone. Cluster analysis (CA) led to the identification of three chemotypes of S. officinalis populations: cis- thujone; trans-tujone, and camphor/b-pinene/borneol/bornyl acetate. We propose that differences in essential oils of 25 populations are mostly genetically controlled, since potential environmental factors were controlled in this study. Introduction. – Dalmatian sage or common sage ( Salvia officinalis L.) from Lamiaceae family is one of ca. 1,000 Salvia species [1]. It is a perennial subshrub cultivated in temperate regions all around the world [2], often escaped from the cultivation and naturalized [ 3] . S. officinalis is native to the east side of Adriatic [4] and Ionian seas with a habitat reaching south into northwest Greece [5]. Well-known enclaves with indigenous populations exist in south Serbia [6] and in west Macedonia [7]. S. officinalis is a herb and medicinal plant, widely used in food, pharmaceutical, and cosmetic industries. Sage enjoys the reputation of being a panacea because of its wide range of medical effects [8]. It was scientifically established that Dalmatian sage has gastroprotective [9], antidiabetic [10], anti-obesity [11], anti-inflammatory [12], antispasmatic [13], virucidal [14], fungicidal, and bactericidal [8] [15] [16] properties. It was also shown that this plant improves mild to moderate Alzheimer)s disease and decreases irritability [17], improves mood and cognitive performance, reduces anxiety CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 2309 # 2012 Verlag Helvetica Chimica Acta AG, Zürich
Transcript
High Diversity of Indigenous Populations of Dalmatian Sage (Salviaofficinalis L.) in Essential-Oil Composition
by Marija Jug-Dujakovic*a), Mihailo Risticb), Dejan Pljevljakusicb), Zora Dajic-Stevanovicc),Zlatko Liberd), Katarina Hancevica), Tomislav Radica), and Zlatko Satovice)
a) Institute for Adriatic Crops and Karst Reclamation, Split, Put Duilova 11, HR-21000 Split(phone: þ385-21-434402; fax: þ385-21-316584; e-mail: [email protected])
b) Institute for Medicinal Plant Research �Dr. Josif Pancic�, Tadeusa Koscuska 1, RS-11000 Belgradec) Faculty of Agriculture, University of Belgrade, Nemanjina 6, RS-11000 Belgrade
d) University of Zagreb, Faculty of Science, Marulicev trg 9, HR-10000 Zagrebe) University of Zagreb, Faculty of Agriculture, Svetosimunska 25, HR-10000 Zagreb
Essential oils of 25 indigenous populations of Dalmatian sage (Salvia officinalis L.) that representnearly half of native distribution area of the species were analyzed. Plantlets collected from wildpopulations were grown in the same field under the same environmental conditions and then sampled foressential-oil analysis. The yield of essential oil ranged from 1.93 to 3.70% with average of 2.83%. Amongthe 62 compounds detected, eight (cis-thujone, camphor, trans-thujone, 1,8-cineole, b-pinene, camphene,borneol, and bornyl acetate) formed 78.13–87.33% of essential oils of individual populations. Strongpositive correlations were observed between camphor and b-pinene, b-pinene and borneol, as well asbetween borneol and bornyl acetate. The strongest negative correlation was detected between camphorand trans-thujone. Principal component analysis (PCA) on the basis of eight main compounds showedthat first main component separated populations with high thujone content, from those rich in camphor,while the second component separated populations rich in cis-thujone from those rich in trans-thujone.Cluster analysis (CA) led to the identification of three chemotypes of S. officinalis populations: cis-thujone; trans-tujone, and camphor/b-pinene/borneol/bornyl acetate. We propose that differences inessential oils of 25 populations are mostly genetically controlled, since potential environmental factorswere controlled in this study.
Introduction. – Dalmatian sage or common sage (Salvia officinalis L.) fromLamiaceae family is one of ca. 1,000 Salvia species [1]. It is a perennial subshrubcultivated in temperate regions all around the world [2], often escaped from thecultivation and naturalized [3]. S. officinalis is native to the east side of Adriatic [4] andIonian seas with a habitat reaching south into northwest Greece [5]. Well-knownenclaves with indigenous populations exist in south Serbia [6] and in west Macedonia[7].
S. officinalis is a herb and medicinal plant, widely used in food, pharmaceutical, andcosmetic industries. Sage enjoys the reputation of being a panacea because of its widerange of medical effects [8]. It was scientifically established that Dalmatian sage hasgastroprotective [9], antidiabetic [10], anti-obesity [11], anti-inflammatory [12],antispasmatic [13], virucidal [14], fungicidal, and bactericidal [8] [15] [16] properties.It was also shown that this plant improves mild to moderate Alzheimer�s disease anddecreases irritability [17], improves mood and cognitive performance, reduces anxiety
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 2309
� 2012 Verlag Helvetica Chimica Acta AG, Z�rich
of young people [18], improves memory and attention of older people [19], and inhibitsHIV-1 reverse transcriptase [20].
The quantity and composition of S. officinalis essential oil is mostly affected bygenotype [21], yet also by environmental factors [22 –24], physiological stage, i.e.,season [25– 30], ratio of leaves/flowers/stems used for distillation [23], and drying [31].
Indigenous populations of S. officinalis have been studied regarding influence ofphysiological stage, season, locality [24] [29] [30] [32– 35], and also distillation [36] andextraction [37] on the yield and composition of essential oil. Those studies wereconducted on relatively small number of populations.
The aim of this study was to examine the content, composition, and diversity ofessential oil, and to determine chemotypes and relation to geography of S. officinalisbased on the analysis of oils from 25 populations. By taking cuttings from 25populations of S. officinalis, we investigated the entire northern half of the species-indigenous habitat. This is the first comprehensive research of this type of nativeDalmatian sage populations.
Results and Discussion. – Essential-Oil Analysis. Twenty five indigenous popula-tions of Salvia officinalis L. (Fig. 1 and Table 1) were chosen for essential-oil analysis.Plantlets from all the collected populations were grown in the same field under thesame environmental conditions and then sampled for essential-oil analysis. The samplesfor oil analysis were taken in semi-dormant vegetative stage to avoid influence ofphenophase [26].
The essential-oil yield of the Salvia officinalis populations sampled ranged from1.9% in population from Vratnik (P07) to 3.7% (P10, Dugi otok) with average of 2.8%.Previous studies could be divided into two groups, those that analyzed indigenouspopulations ranging from 1.2 to 3.5% oil content [24] [30] [32], and those dealing withcultivated S. officinalis where oil yield ranged from 0.4 to 2.2% [22] [27]. Nine of thepopulations gave an essential-oil yield higher then 3% (Table 1). In previouslyexamined wild sage plants from mountain Velebit, and islands Pag and Hvar at similartime of the year, essential-oil yield ranged from 1.4 to 3.5% with average of 2.6% [24].The high percentage of essential oil in the populations analyzed here could beexplained by the timing of harvest in August, while the plants were in vegetative phaseafter dry and hot period. A long dry period in vegetative phase may be a basis for higherproduction of essential oil in S. officinalis [38]. It is known that aromatic plants produceessential oils also as a defense against drought and heat. That is traditionally the periodwhen Dalmatian sage is collected commercially from the wild in Dalmatia.
Sixty-two compounds were detected in essential oils across all sage populationsanalyzed, 58 of which were identified. Thirty-six compounds were common to oils ofevery population (Table 2). It has been suggested that the components of essential oilsare predominantly determined by the plants genotype [39].
The most abundant compounds were cis-thujone, camphor, and trans-thujone(found in concentration higher than 35% at least in one population). Analysis ofessential oils of 24 populations confirmed [26] [40] that thujones and camphor are mainconstituents of S. officinalis essential oil, while population P21, Hvar, had somewhathigher content of 1,8-cineole than camphor.
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)2310
Total thujone content varied from 12.5% (P06, Losinj) to 62.8% (P17, Biokovo)with average of 38.2%. In 93 samples of essential oil of wild Dalmatian sage analyzedby Vernazza from four regions of sage gathering within Dalmatia, the total thujonecontent ranged from 32.8 to 60.3% [34]. The quality of essential oil of Dalmatian sagewas determined mainly by its thujone content with a high percentage of thujoneindicating good quality oil [24].
The trade distinguished between two types of oils: �high-test oils� and �low-test oils�(41.6 –61.2% and 22.0– 39.7%, resp.) [41]. The sage oil samples have been divided onthe basis of this system [22]. Using this classification, the essential oil of our testedpopulations could be divided into three groups: eleven �high-test� populations; ten�low-test�, and four populations with total thujones from 12.5 to 16.3%.
Compounds found at concentration of higher than 5% in any population werechosen for further analysis and were hereafter referred as main compounds. Those werecis-thujone, camphor, trans-thujone, 1,8-cineole, b-pinene, camphene, borneol, andbornyl acetate (Table 2). They constituted 78.1 to 87.3% of essential oils of individualpopulations.
In this research, a-humulene (0.6 – 2.2%), viridiflorol (1.5 –3.4%), manool (0.31–1.2%), and b-caryophylene (0.1 – 1.5%) were detected in small amounts within the oilanalyzed (Table 2), even though 25 populations were examined. In recent studies thesecompounds were often detected in higher amounts in essential oils of S. officinalis: b-caryophylene (10.2%) [42]; a-humulene (13.6%) [35], and some of them were maincompounds, i.e., viridiflorol (24.0 and 19.5%) [35] [43] and manool (20.9%) [42].
Besides classification on the basis of the total thujones, other methods to categorizeare based on the most abundant compounds in the oil. We applied a proposed system[44] to classify Dalmatian sage oils into chemotypes based on their relative contents ofcis- and trans-thujone, 1,8-cineole, and camphor, which was supported by results of
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)2312
Table 1. Dalmatian Sage (Salvia officinalis L.) Indigenous Populations Included in the Analysis
Population Accession No.a) Locality Latitude (N) Longitude (E) Elevation [m] EO Yield [%]b)
a) Accession No. from the Collection of Medicinal and Aromatic Plants, Zagreb, Croatia, as available atthe Croatian Plant Genetic Resources Database (http://cpgr.zsr.hr); voucher specimens are depositedwith the Herbarium Croaticum (ZA), Zagreb, Croatia (IDs 26292–26316). b) EO Yield: the essential-oil yield, calculated as percentage of the dry weight of the leaves
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 2313
Tabl
e2.
Che
mic
alC
ompo
sitio
nof
the
Ess
entia
lO
ilsE
xtra
cted
from
25D
alm
atia
nSa
ge(S
alvi
aof
fici
nalis
L.)
Indi
geno
usP
opul
atio
ns,P
01–
P25
No.
Com
poun
dna
me
Com
posi
tion
[%]
P01
P02
P03
P04
P05
P06
P07
P08
P09
P10
P11
P12
P13
P14
P15
P16
P17
P18
P19
P20
P21
P22
P23
P24
P25
1ci
s-Sa
lven
e0.
70.
30.
50.
20.
40.
20.
50.
30.
20.
30.
50.
50.
40.
50.
30.
50.
40.
60.
20.
50.
40.
40.
30.
30.
32
tran
s-Sa
lven
e0.
1tr
.a )tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.3
Tric
ycle
netr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.4
a-T
huje
ne0.
10.
20.
10.
30.
20.
30.
20.
30.
20.
30.
2tr
.tr
.tr
.0.
2tr
.–
tr.
0.4
0.2
tr.
0.2
0.1
tr.
0.2
5a
-Pin
ene
tr.
tr.
tr.
tr.
tr.
tr.
0.1
tr.
0.1
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
0.1
tr.
6C
amph
ene
9.9
8.0
2.0
5.7
2.1
4.4
3.7
4.8
3.0
4.5
4.8
1.7
1.0
2.7
3.7
7.2
4.8
4.2
9.7
4.9
6.7
5.7
5.2
8.5
8.3
7b
-Pin
ene
5.3
6.3
5.1
10.0
5.6
11.9
6.9
9.3
5.6
9.9
7.7
5.1
3.7
2.7
6.7
3.1
1.2
2.9
10.4
5.5
3.2
7.0
4.8
3.5
6.0
8M
yrce
ne1.
10.
91.
12.
00.
91.
81.
81.
71.
31.
61.
20.
80.
41.
01.
20.
80.
60.
92.
21.
10.
71.
21.
21.
21.
49
a-P
hella
ndre
ne1.
21.
11.
11.
20.
91.
31.
21.
21.
31.
01.
21.
31.
11.
31.
31.
21.
01.
01.
11.
01.
21.
21.
21.
31.
110
a-T
erpi
nene
0.2
0.2
0.2
0.1
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.3
0.2
0.3
0.2
0.3
0.2
0.2
0.2
0.2
0.2
0.2
0.2
11p-
Cym
ene
0.8
0.8
0.8
0.5
0.8
0.5
0.6
0.7
0.8
0.6
1.0
0.9
1.2
1.3
0.8
1.1
1.3
1.1
0.6
1.0
1.1
0.8
1.0
1.1
0.8
12L
imon
ene
2.6
2.8
2.3
3.4
2.2
4.2
2.6
3.0
2.5
3.1
2.9
2.7
2.2
1.9
2.7
1.8
1.1
1.3
3.1
1.7
2.0
2.5
2.4
2.1
2.5
131,
8-C
ineo
le11
.86.
110
.010
.07.
28.
012
.19.
48.
113
.18.
97.
77.
110
.110
.75.
09.
115
.712
.212
.49.
311
.09.
48.
211
.914
g-T
erpi
nene
0.2
0.2
0.3
0.2
0.2
0.2
0.2
0.2
0.4
0.2
0.3
0.3
0.3
0.3
0.3
0.4
0.3
0.3
0.2
0.3
0.3
0.2
0.3
0.4
0.3
15ci
s-Sa
bine
nehy
drat
etr
.–
tr.
tr.
–tr
.0.
1tr
.tr
.–
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
0.1
tr.
16Te
rpin
olen
e0.
30.
40.
30.
40.
30.
50.
30.
30.
30.
40.
30.
30.
30.
20.
30.
30.
20.
20.
40.
20.
20.
30.
30.
30.
317
tran
s-Sa
bine
nehy
drat
e0.
60.
70.
50.
80.
61.
10.
70.
50.
60.
60.
50.
50.
50.
40.
60.
40.
60.
40.
50.
70.
50.
50.
70.
50.
418
cis-
Thu
jone
(b-t
hujo
ne)
28.4
27.9
32.5
12.3
28.6
11.0
27.6
20.3
29.5
12.3
29.3
30.1
34.9
49.7
23.4
49.3
17.9
46.3
13.4
38.7
13.6
20.0
18.9
10.3
31.6
19tr
ans-
Thu
jone
(a-t
hujo
ne)
3.9
3.9
4.6
1.6
8.2
1.5
3.4
5.8
11.8
4.0
3.2
18.4
15.4
10.2
10.1
8.9
44.9
6.5
1.7
5.6
40.0
13.3
27.7
38.5
5.4
20ci
s-p-
Men
th-2
-en-
1-ol
––
––
––
––
––
tr.
––
0.1
–0.
1–
tr.
–tr
.–
––
––
21n.
i.b)
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
0.1
tr.
tr.
tr.
tr.
tr.
tr.
tr.
0.11
22a
-Cam
phol
enal
tr.
tr.
tr.
tr.
0.2
tr.
tr.
0.2
0.1
0.1
tr.
0.4
0.4
tr.
0.2
tr.
0.3
tr.
tr.
tr.
0.6
0.3
0.4
0.5
tr.
23Is
othu
jan-
3-ol
0.2
0.2
0.2
tr.
0.2
0.1
0.2
0.1
0.2
0.1
0.1
0.2
0.3
0.2
0.2
0.2
0.3
0.2
tr.
0.2
0.3
0.2
0.2
0.5
0.2
24C
amph
or18
.929
.526
.936
.531
.029
.523
.023
.820
.331
.321
.413
.618
.25.
217
8.4
5.5
10.0
31.2
15.1
7.1
22.5
13.7
9.9
18.9
25tr
ans-
Pin
ocam
phon
e0.
20.
30.
30.
20.
40.
40.
50.
30.
2–
0.4
0.4
0.4
0.2
0.2
0.3
0.1
0.2
0.2
0.2
0.1
0.2
0.11
–0.
226
Bor
neol
1.9
2.5
2.2
4.0
3.0
6.2
2.1
4.6
2.4
6.3
4.3
3.1
2.1
1.5
5.7
1.4
0.9
1.1
2.6
2.3
2.2
2.4
2.3
1.4
1.7
27Te
rpin
en-4
-ol
0.4
0.5
0.5
0.4
0.5
0.6
0.5
0.5
0.5
0.6
0.6
0.5
0.7
0.5
0.5
0.5
0.6
0.6
0.5
0.6
0.5
0.5
0.6
0.6
0.5
28p-
Cym
en-8
-ol
tr.
0.1
tr.
0.1
tr.
0.2
0.1
0.1
tr.
0.2
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
29a
-Ter
pine
ol0.
20.
20.
20.
30.
20.
40.
30.
30.
20.
50.
20.
20.
20.
10.
20.
10.
20.
10.
20.
20.
30.
20.
30.
20.
130
Myr
teno
l0.
20.
3tr
.0.
10.
20.
50.
50.
20.
2tr
.0.
20.
10.
10.
1tr
.0.
20.
30.
10.
1tr
.0.
40.
10.
20.
4tr
.31
cis-
Car
veol
tr.
tr.
tr.
tr.
tr.
0.1
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
tr.
–tr
.tr
.tr
.tr
.tr
.tr
.tr
.32
Isob
orny
lfo
rmat
etr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.tr
.–
tr.
tr.
tr.
tr.
tr.
tr.
–
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)2314
Tab
le2
(con
t.)
No.
Com
poun
dna
me
Com
posi
tion
[%]
P01
P02
P03
P04
P05
P06
P07
P08
P09
P10
P11
P12
P13
P14
P15
P16
P17
P18
P19
P20
P21
P22
P23
P24
P25
33N
eral
––
––
––
––
––
tr.
tr.
––
tr.
–tr
.–
–tr
.–
––
––
34(E
)-H
ex-2
-eny
lis
oval
erat
etr
.–
tr.
–tr
.–
tr.
tr.
–tr
.–
tr.
–tr
.–
tr.
0.1
––
–tr
.tr
.tr
.tr
.–
35n.
i.–
tr.
––
tr.
––
tr.
–tr
.–
––
tr.
––
tr.
––
tr.
0.1
tr.
tr.
0.1
–36
Isot
huja
n-3-
olac
etat
e–
––
––
––
–tr
.–
–tr
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some studies [22]. Since there is a continuous variation of almost all of the maincompounds across the analyzed population, this classification was inadequate foressential oils of 25 populations analyzed in this research, because only two out ofresulted 16 chemotypes corresponded to five proposed chemotypes. Therefore, wechose multivariate statistical methods, by which discrete chemotype categories could beestablished, and applied them to the eight main compounds.
A few of the main essential-oil compounds appeared to be highly correlated amongthemselves (Table 3). Strong positive correlations (r¼0.70; P<0.001) have beenobserved between camphor (C02) and b-pinene (C05), b-pinene (C05) and borneol(C07), as well as between borneol (C07) and bornyl acetate (C08). The strongestnegative correlation (r¼ �0.66; P<0.001) was detected between camphor (C02) andtrans-thujone (C03).
The principal component analysis (PCA) of eight main essential-oil compounds in25 Dalmatian sage populations revealed that three principal components hadeigenvalues greater than 1 and explained jointly 81.20% of the total variation(Table 4). Strong positive correlations (r>0.70; P<0.001) were observed between thefirst principal component and camphor (C02), b-pinene (C05), borneol (C07), andbornyl acetate (C08). The second principal component was strongly negativelycorrelated with cis-thujone (r¼ �0.79; P<0.001) and positively correlated with trans-thujone (r¼0.74; P<0.001). Camphene (C06) was the only essential-oil constituentstrongly correlated with the third principal component (r¼0.80; P<0.01). The biplotconstructed by two principal components showing populations and essential-oilcompounds (as vectors) is presented in Fig. 2. The first principal component, explaining46.78% of the total variation, separated populations with high camphor (C02) contentfrom those rich in thujones. Along the second principal component, explaining 18.09%of the total variation, populations characterized by a high cis-thujone (C01) contentwere separated from those with high trans-thujone (C03) content.
UPGMA (Unweighted pair-group method using arithmetic averages) was used forclassifying populations in chemotypes. Average Euclidean distance between pairs of
Table 3. Pearson�s Correlation Coefficients among the Eight Main Essential-Oil Compounds ofDalmatian Sage (Salvia officinalis L.)a)
a) The significance of the correlations is indicated as follows: ***, significance at the P<0.001 nominallevel; **, significance at the 0.001<P<0.01 nominal level; *, significance at the 0.01<P<0.05 nominallevel; ns, not significant at the P>0.5.
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 2315
populations calculated by using eigenvectors of the first three components was 3.31,ranging from 0.29 (P02, Kamenjak/P22, Vis) to 7.63 (P06, Losinj/P17, Biokovo).UPGMA Dendrogram based on Euclidean distance matrix shown in Fig. 3 revealedthree main clusters, A, B, and C. The highest value of Pseudo F (PSF) statistics wasobtained for three clusters, indicating that the populations could be grouped in threedistinct chemotypes. Fourteen populations characterized by a high cis-thujone contentgrouped in cluster A (P07, P20, P03, P02, P22, P01, P25, P05, P09, P12, P13, P14, P16,and P18); four trans-thujone populations in cluster B (P21, P24, P17, and P23), andseven populations high in camphor/b-pinene/borneol/bornyl acetate combination incluster C (P04, P10, P06, P08, P11, P15, and P19). Attribution to a particularchemotype was not determined only by a single compound. Although an individualpopulation can be assigned to certain chemotype named by one component, it does notnecessarily mean that this particular component has the highest percentage. Popula-tions P02 (Kamenjak), P22 (Vis), and P05 (Cres) that were classified in cluster A hadhigher percentages of camphor than cis-thujone, but they were separated by first maincomponent, because total thujone content was higher than camphor. Populations P11(Otisina) and P15 (Vinisce) from cluster C had higher cis-thujone content thancamphor, but they had also higher percentages of b-pinene, borneol, and bornylacetate.
Suitability of classification of S. officinalis populations in three clusters by clusteranalysis (CA) was checked by discriminant analysis (DA). Based on eight maincompounds of essential oil 23 of 25 populations (92%) were successfully classified inthree clusters. Several studies have previously made extensive use of statistical methodsto interpret different aspects of the metabolism of aromatic plants, demonstrating theusefulness of multivariate statistical methods such as PCA, CA, and DA [45] [46].
Analysis of variance (ANOVA) revealed that the chemotypes differed significantly(P<0.01) in six out of eight main essential-oil compounds (Table 5) as well as in total
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)2316
Table 4. Component Loadings of the Eight Essential-Oil Compounds on the First Three PrincipalComponentsa)
a) The significance is indicated as follows: ***, significance at the P<0.001 nominal level; **, significanceat the 0.001<P<0.01 nominal level; *, significance at the 0.01<P<0.05 nominal level; ns, not significantat the P>0.5.
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 2317
Fig
.2.
Bip
loto
fth
eP
CA
Bas
edon
the
Eig
htM
ain
Ess
entia
l-O
ilC
ompo
unds
of25
Indi
geno
usP
opul
atio
nsof
Dal
mat
ian
Sage
(Sal
via
offi
cina
lisL
.)
thujone content and cis-thujone/trans-thujone ratio, but not in the total essential-oilcontent. Chemotype A had the highest cis-thujone (C01) content and chemotype B
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)2318
Fig. 3. UPGMA Dendrogram of Cluster Analysis on 25 Indigenous Populations of Dalmatian Sage(Salvia officinalis L.) Using the First Three Principal Components. Three major clusters (A, B, and C) are
indicated.
trans-thujone (C03), while the chemotype C showed the highest values for camphor, b-pinene, borneol, and bornyl acetate. Chemotype C had significantly lower values oftotal thujons than chemotypes A and B. The cis-thujone/trans-thujone ratio wassignificantly lower in chemotype B than both in A and C.
Such differences in essential oils of 25 populations show that this trait is mostlygenetically controlled, since they were planted in the same environment. Results ofresearch with Salvia fruticosa [47] showed that the basis of variations in the essential-oilcomposition depends more on the genetic background than on the other factors.Comparison of seed-grown wild populations of S. officinalis in the culture in Israel withthose in the wild in Dalmatia revealed similarities and resulted in conclusion thatgenetic variability could be used for selection and breeding purposes [21].
Correlation between population location, i.e., latitude, longitude, and altitude, andtotal percentage of essential oil, percentage of each of the main compounds, percentageof total thujone, cis-thujone/trans thujone ratio (Table 6) displayed strong positivecorrelation of camphor (C02 ; r¼0.591; 0.001<P<0.01) with latitude and negative (r¼�0.591; 0.001<P<0.01) with longitude, while it was opposite with trans-thujone(C03). Having in mind that population locations are in direction northwest�southeast,because that is how the Adriatic coast extends, correlation of latitude and longitude isalmost absolute. Other correlations were not significant or weak. In previous research[24] [35], where a much smaller sample area of sage populations was studied, theinfluence of altitude on yield of essential oil of Dalmatian sage native populations wasreported, while there was no influence of latitude, longitude, or altitude on biochemicaldiversity of studied populations. In addition, samples in that study were taken directlyfrom the wild, where ecological influence has to be taken into account [23].
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 2319
Table 5. Content of the Eight Main Essential-Oil Compounds of Three Chemotypes (A, B, and C) ofIndigenous Dalmatian Sage (Salvia officinalis L.)
a) The significance of the ANOVA�s F-test is indicated as follows: ***, significance at the P<0.001nominal level; **, significance at the 0.001<P<0.01 nominal level; *, significance at the 0.01<P<0.05nominal level; ns, not significant at the P>0.5. b) The contents [%] of the main essential-oil componentsare represented as means� standard deviations. Means within the same raw followed by different lettersin parentheses are significantly different at P<0.05 on the basis of Tukey�s test.
Work on a related species, Salvia lavandulifolia, in Spain, could distinguishpopulations into two groups with a well-defined geographical distribution according tochemical composition of their essential oils [48]. Interestingly, there is no cleargeographical border between populations of three different chemotypes in our study.Fourteen cis-thujone populations are scattered through whole research area, while fourtrans-thujone populations are clustered on the south-east of the area studied.Investigations of micro habitats of each population could possibly resolve populationsgeographically by oil content, because the influence of environmental conditions on thenature of plant chemical composition has an important role in plant adaptation andspeciation [49].
Conclusions. – Essential oils of 25 indigenous populations of Dalmatian sage (Salviaofficinalis L.), sampled across half of the species� native habitat, were analyzed. Theessential-oil content ranged from 1.9 to 3.7% with average of 2.8%. Sixty-twocompounds were detected, 58 of which were identified, while 36 compounds werepresent in oil of each population. The most abundant compounds were cis-thujone,camphor, and trans-thujone. The eight main compounds, which were found atconcentration higher than 5% in any single population, were used for chemotypeidentification: cis-thujone, camphor, trans-thujone, 1,8-cineole, b-pinene, camphene,borneol, and bornyl acetate. Strong positive correlations were observed betweencamphor and b-pinene, b-pinene and borneol, as well as between borneol and bornylacetate. The strongest negative correlation was detected between camphor and trans-thujone. PCA and UPMGA were suitable tools for determining chemotypes among 25populations of S. officinalis analyzed: A, cis-thujone; B, trans-thujone, and C, camphor/b-pinene/borneol/bornyl acetate. Chemotypes differed significantly in six out of eightmain essential-oil compounds as well as in total thujone content and cis-thujone/trans-thujone ratio, but not in the total essential-oil content. Such differences in essential oil
Table 6. Pearson�s Correlation Coefficients between the Essential-Oil Componds and the GeographicVariables for Populations of Indigenous Dalmatian Sage (Salvia officinialis L.)
a) The significance is indicated as follows: ***, significance at the P<0.001 nominal level; **, significanceat the 0.001<P<0.01 nominal level; *, significance at the 0.01<P<0.05 nominal level; ns, not significantat the P>0.5.
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)2320
across the populations sampled show that this trait is mostly genetically controlled,since they were planted in the same environment. No clear geographical borderbetween populations of three different chemotypes was observed.
This study was supported by the Ministry of Science, Education and Sports of the Republic of Croatia(Project Nos. 178-1191193-0212 and 119-1191193-1232) and by Ministry of Science and TechnologicalDevelopment of the Republic of Serbia (Grant No. 173021).
Experimental Part
Plant Material. Salvia officinalis L. branches were collected from 25 wild indigenous populations(Fig. 1 and Table 1) from Croatia (23 populations), and Bosnia and Herzegovina (two populations) inearly spring of 2006. Plantlets were prepared by rooting of apical stem parts in the greenhouse. Plantationof 750 genotypes representing 25 populations was established in October 2006 following row-columndesign, in experimental field of Institute for Adriatic Crops, Split Duilovo (N 43821’02’’, E 16830’17’’), inrows 1-m-apart and with 0.5 m between plants. Plants were grown with no irrigation, fertilizing, ortrimming. Accession Nos. are MAP02343–MAP02367 from the Collection of Medicinal and AromaticPlants, Zagreb, Croatia, as available at the Croatian Plant Genetic Resources Database (http://cpgr.zsr.hr); voucher specimens are deposited with the Herbarium Croaticum (ZA), Zagreb, Croatia(IDs 26292–26316).
The distal parts of leafy shoots from ten plants per population were collected in the beginning ofAugust 2008 and dried in a shaded room at ca. 308. After drying, healthy leaves were separated fromstems and, together with apical stalks, preserved in paper bags until analysis.
Essential-Oil Extraction. The essential oils were isolated from dried plant material by hydro-distillation, according to the standard procedure reported in the Fifth European Pharmacopoeia usingClevenger-type apparatus. Duration of distillation was 2 h. Oil samples were dried with anh. Na2SO4,dissolved in EtOH, and analyzed by GC/FID and GC/MS.
Gas Chromatography. GC/FID Analysis of the oils was carried out on a HP-5890 Series II GCapparatus (Hewlett-Packard, D-Waldbronn), equipped with split-splitless injector and automatic liquidsampler (ALS), attached to HP-5 column (25 m�0.32 mm, 0.52 mm film thickness) and fitted to flame-ionization detector (FID). Carrier gas (H2) flow rate was 1 ml/min; split ratio, 1 : 30; injector temp., 2508 ;detector temp., 3008, while column temp. was linearly programmed from 40–2608 (at rate of 48/min).Solns. of essential-oil samples in EtOH (ca. 1%) were consecutively injected by ALS (1 ml, split mode).Area-percent reports, obtained as result of standard processing of chromatograms, were used for thequantification purposes.
Gas Chromatography/Mass Spectrometry (GC/MS). The same anal. conditions as those mentionedfor GC/FID were employed for GC/MS analysis, along with column HP-5MS (30 m�0.25 mm, 0.25 mmfilm thickness), using HP G 1800C Series II GCD system (Hewlett-Packard, Palo Alto, CA, USA).Instead of H2, He was used as carrier gas. Transfer line was at 2608. Mass spectra were acquired in EImode (70 eV), in the m/z range of 40–450. Sample solns. in EtOH (ca. 1%) were injected by ALS (200 nl,split mode).
The components of the oil were identified by comparison of their mass spectra with those fromWiley275 and NIST/NBS libraries, using different search engines. The exper. values for retention indiceswere determined by using calibrated Automated Mass Spectral Deconvolution and Identification Systemsoftware [50], compared to those from available literature [51], and they were used as additional tool tosupport MS findings.
Correlations. The relationships among the eight main essential-oil compounds were assessed byPearson�s correlation coefficient as implemented in PROC CORR in SAS [52].
Principal Components Analysis (PCA). The PCA based on eight main essential-oil compounds wasperformed using PROC PRINCOMP procedure in SAS. The biplot was constructed by two principalcomponents showing populations and essential-oil compounds (as vectors).
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 2321
Cluster Analysis (CA). The standardized scores of the first three principal components weremultiplied by the root of their eigenvalues, and the Euclidean distance matrix between all pairs ofpopulations was calculated to be used in CA. The average linkage method (i.e., UPGMA) of PROCCLUSTER in SAS was applied in order to determine the optimal number of clusters by calculating andplotting Pseudo F (PSF) statistics. Populations were classified in groups representing distinct chemo-types.
Discriminant Analysis (DA). Eight main essential-oil constituents were evaluated for the perform-ance as discriminant criterions for the correct classification of populations into their respective groups(i.e., chemotypes), by estimating the probabilities of misclassification with cross-validation using PROCDISCRIM procedure in SAS.
ANOVA. Univariate analysis of variance using PROC GLM in SAS was conducted to test the meandifferences between chemotypes for the eight main essential-oil compounds as well as for the totalessential-oil content, total thujones, and cis-thujone/trans-thujone ratio. The percentages werenormalized by logarithmic transformation. Post-hoc comparisons of the population means were carriedout using Tukey�s student range test at P<0.05.
Correlations between Essential-Oil Compounds and Geographic Variables. The values of each mainessential-oil compound were correlated with the longitudes, latitudes, and altitudes of the collecting sitesusing Pearson�s correlation coefficient.
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