Chemical differences in volatiles between Melittis melissophyllum L. subsp. melissophyllum and subsp. albida (Guss) P. W. Ball (Lamiaceae) determined by solid-phase microextraction (SPME) coupled with GC/FID and GC/MS.

Abstract

Melittis melissophyllum (Lamiaceae) is a perennial herb, typical of woody places, occurring in Italy with two subspecies, i.e., melissophyllum and albida. So far, the classification of these two taxa was only based on morphology, i.e., the presence of glandular trichomes, the dimension of the leaves, and the number of teeth on each side as the main discriminant characters. To find marker compounds to chemically discriminate the subsp. melissophyllum with respect to the subsp. albida, a solid-phase microextraction SPME analysis coupled with GC/FID (=flame ionization detector) and GC/MS was carried out. SPME proved to be a chemotaxonomically useful technique that permitted a clearly differentiation of the two subspecies at headspace level. The subsp. melissophyllum was characterized by high amount of the mushroom alcohol oct-1-en-3-ol and the phenolic coumarin, whilst the subsp. albida exhibited a high content in monoterpenes and sesquiterpenes, α-pinene, sabinene, and (E)-caryophyllene being the major compounds. Multivariate chemometric techniques, such as cluster analysis (CA) and principal-component analysis (PCA), were used to support chemical data and characterize the population according to the taxonomy. In addition, the micromorphology and distribution of glandular trichomes of both subspecies were studied by scanning electron microscopy (SEM).

Full text

Page 1

Chemical Differences in Volatiles between Melittis melissophyllum L. subsp.
melissophyllum and subsp. albida (Guss
Determined by Solid-Phase Microextraction (SPME) Coupled with GC/FID
and GC/MS
uss) P. W. Ballall (Lamiaceae)
by Filippo Maggi*a), Fabio Contib), Gloria Cristallic), Claudia Giulianid), Fabrizio Papac),
Gianni Sagratinic), and Sauro Vittoric)
a) School of Pharmacy, University of Camerino, Via Pontoni 5, I-62032 Camerino
(phone: þ39-0737-404506; fax: þ39-0737-404508; e-mail: filippo.maggi@unicam.it)
b) School of Environmental Sciences, University of Camerino, Centro Ricerche Floristiche
dell?Appennino, San Colombo, I-67021 Barisciano
c) School of Pharmacy, University of Camerino, Via Sant?Agostino 1, I-62032 Camerino
d) Department of Evolutionary Biology, University of Florence, Via La Pira 4, I-50121 Florence
Melittis melissophyllum (Lamiaceae) is a perennial herb, typical of woody places, occurring in Italy
with two subspecies, i.e., melissophyllum and albida. So far, the classification of these two taxa was only
based on morphology, i.e., the presence of glandular trichomes, the dimension of the leaves, and the
number of teeth on each side as the main discriminant characters. To find marker compounds to
chemically discriminate the subsp. melissophyllum with respect to the subsp. albida, a solid-phase
microextraction SPME analysis coupled with GC/FID (¼flame ionization detector) and GC/MS was
carried out. SPME proved to be a chemotaxonomically useful technique that permitted a clearly
differentiation of the two subspecies at headspace level. The subsp. melissophyllum was characterized by
high amount of the mushroom alcohol oct-1-en-3-ol and the phenolic coumarin, whilst the subsp. albida
exhibiteda highcontentinmonoterpenesandsesquiterpenes, a-pinene,sabinene,and(E)-caryophyllene
being the major compounds. Multivariate chemometric techniques, such as cluster analysis (CA) and
principal-component analysis (PCA), were used to support chemical data and characterize the
population according to the taxonomy. In addition, the micromorphology and distribution of glandular
trichomes of both subspecies were studied by scanning electron microscopy (SEM).
Introduction. – The family of Lamiaceae comprises many species producing aroma
and scented compounds. Among them, there is Bastard balm (Melittis melissophyllum
L., subfamily Lamioidaeae, subtribe Melittidinae), a perennial herb covered by
abundant protecting trichomes mainly on the quadrangular stems. The plant has
petiolate to subsessile, decussate, oblong to ovate, cordate to truncate at base, and
coarsely crenate or dentate leaves. Their similarity with those of Lemon balm (Melissa
officinalis L.) is at the basis of the species name, while the name of the genus is based on
the flowers which are visited by honey-producing pollinators. The flowers, of white,
pink, or purple bilabiate corolla much exceeding the bilabiate persistent calyx, are the
biggest in the Lamiaceae family. Bastard balm occurs in Middle, Southwestern,
Southeastern, and Eastern Europe [1].
In Italy, two subspecies occur: subsp. melissophyllum, 30- to 50-cm tall, with low-
density glandular trichomes represented by peltate (type A) and capitate (types B and
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)325
? 2011 Verlag Helvetica Chimica Acta AG, Z?rich

Page 2

C) ones [2], largest leaves 5–7(9) cm, and with 10–20 large teeth on each side [1],
mainly in the Northwestern and Central Italy [3]; and subsp. albida (Guss) P.W. Ball,
larger than the nominate one (40–70 cm tall), with abundant glandular short
trichomes, largest leaves of 6–15 cm, and with 20–30 large teeth on each side, mainly
in Southern Italy and Islands [1][3][4] (Fig. 1 and Table 1). Both subspecies grow in
coppiced or copse broadleaf woodlands belonging to the community of the class
Querco?Fagetea.
In the folk medicine of central Italy, M. melissophyllum, called ?Erba Lupa?, ?Erba
Limona?, or ?Cedrina?, was used as antispasmodic, and against insomnia and eye
inflammations [5]. More, it is still used in Abruzzo by shepherds during transhumance,
under a warm decoction, used as digestive, and to treat cough and sore throat [6]. In
spite of these properties, people used to make an aromatic tea with the fresh or dry
leaves to be drunk after meals as digestive and antispasmodic.
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)326
Fig. 1. Distribution of Melittis melissophyllum subspecies in Italy [3]

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)327
Table 1. Collection Localities of Melittis melissophyllum Populations Investigated in This Study
Popula-
tion
Subspecies
Collection site
Region
Date of
collection
GPS coordinates (WGS84)
Altitude
[m]
Voucher codesa)
1
melissophyllum
Morello, Sassoferrato (Ancona)
Marche
18/05/2009
N 43829’34’’; E 12848’37’’
440
CAME 16042
2
melissophyllum
Piedilapiaggia, Camerino (Macerata)
Marche
13/05/2009
N 43809’10’’; E 13807’18’’
590
CAME 13430
3
melissophyllum
Anversa degli Abruzzi (L?Aquila)
Abruzzo
27/05/2009
N 41858’59’’; E 13847’32’’
1277
APP 39504
4
melissophyllum
Civitella del Tronto (Teramo)
Abruzzo
08/06/2010
N 42844’36’’; E 13836’29’’
860
APP 41959
5
albida
Monte Vulture (Potenza)
Basilicata
06/07/2010
N 40856’08’’; E 15837’04’’
1300
APP 42031
6
albida
Celico (Cosenza)
Calabria
30/06/2010
N 39818’33’’; E 16823’02’’
1200
APP 42035
7
albida
Madonie, M. Balatelli (Palermo)
Sicily
04/06/2010
N 37854’29’’; E 13859’45’’
933
APP 42032
a) Accession number in: CAME, Herbarium Camerinensis, School of Environmental Sciences, University of Camerino, Camerino, Italy; APP, Herbarium of
Centro Ricerche Floristiche dell?Appennino, Barisciano, Italy.

Page 4

So far, the classification of M. melissophyllum subspecies was exclusively based on
morphology, i.e., the density of glandular trichomes, the dimension of the leaves, and
the number of teeth on each side as the main discriminant characters [1][4]; the white
color of corolla has not been anymore considered as a taxonomic feature of the susbsp.
albida.
The aim of this work was to use solid-phase microextraction (SPME) in order to
distinguish the two subspecies from each other by detecting marker compounds, and,
hence, to support the botanical classification.
SPME is an extraction technique widely applied for the characterization of the
volatile-fraction composition of many different samples as fruits, vegetables, plants, and
beverages [7]. It is a non-destructive and non-invasive method to evaluate aroma
compounds [8], permitting the use of considerably smaller amounts of plant material
than in other extraction techniques. Being a solvent-free technique, it avoids loss of
volatiles during the concentration of the extractive solutions. Moreover, the low
temperatures normally used during sampling avoid chemical changes in the natural
flavor pattern and the formation of artifacts, and give a better estimation of the aroma
profile as perceived by the human nose. Finally, the higher concentration capability of
this technique allows the identification of many compounds [9].
Accordingly, this technique is very eligible for chemotaxonomic studies, especially
in the case we have scant material to analyze, and we want to have an idea on what
really the plant material emits under conditions very close to environmental ones.
To our knowledge, no previous work has been reported on the comparative analysis
of volatile components emitted from M. melissophyllum subspecies, and it is the first
time that this technique is used for the analysis of subsp. albida headspace (HS).
Recently, we investigated the subsp. melissophyllum by performing a HS-SPME
optimization of the extraction parameters in order to enhance the release from the
plant material of oct-1-en-3-ol that is an C8alcohol responsible for the unique fungal
aroma and flavor of edible mushrooms [10]. Along with oct-1-en-3-ol and according to
literature data [11][12], another major headspace component was coumarin (¼2H-1-
benzopyran-2-one), the simplest compound of a large class of naturally occurring
phenolic constituents made of fused benzene and a-pyrone (¼2H-pyran-2-one) rings.
It is a pleasant smelling compound with a characteristic spicy odor of fresh hay,
woodruff, or vanilla, used as a flavoring and fragrance material in food, tobacco,
cosmetics, and toiletries [13], and nowadays subjected to some restrictions in food uses
[14][15], because it is considered to exert hepatotoxicity, and suspected to be
mutagenic and carcinogenic [16].
Finally, to support the data in literature [1][4], a micromorphological investigation
has been carried out with the aim to compare the indumentum of Melittis
melissophyllum subspecies from Italy.
Results and Discussion. – 1. Headspace Volatile Profiles. The HS volatiles of seven
Italian populations of M. melissophyllum belonging to two different subspecies are
reported in Table 2, while the GC/FID chromatograms are depicted in Fig. 2. A total of
122 volatiles were identified in the different populations, accounting for 94.3–98.9% of
the total volatiles.
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011) 328

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A great variability was observed in the qualitative composition of the HS of
different samples, since only 43 components out of 122 were in common among the two
subspecies; 19 were the components exclusively emitted by the subsp. albida, while 13
volatiles were detected only in the subsp. melissophyllum.
Major headspace fractions were represented by aliphatic alcohols (45.1–65.0%)
and aromatics (25.5–39.2%) in the subsp. melissophyllum, and by monoterpenes
(36.3–53.9%) and sesquiterpenes (27.5–47.5%) in the subsp. albida, with oct-1-en-3-ol
(1; 43.2–63.4%) and coumarin (2; 24.6–38.5%) as the most prevalent in the former,
and a-pinene (3; 4.0–41.8%) and (E)-caryophyllene (4; 8.7–32.3%) in the latter
(Fig. 3). Their chemical structures are given in Fig. 4. The method provided a good
reproducibility, since the relative standard deviations (RSDs) of three replicate
analyses for major-compound peak areas were: 0.3–10.0% for oct-1-en-3-ol (1), 5.0–
11.3% for coumarin (2), 8.0–15.0% for a-pinene (3), and 5.7–10.0% for (E)-
caryophyllene (4).
A similar variability was found within the subsp. albida; the population from
Basilicata gave quantitatively different results compared to the others (Calabria and
Sicily), being dominated by sabinene (5; 28.5%), while a-pinene (3; 4.0%) and (E)-
caryophyllene (4; 8.7%) were present in lower amounts. This population also showed a
high level of the monoterpene a-thujene (6.2%) that was missing in the others. Other
terpenoids that contributed to the HS of the subsp. albida were the sesquiterpenes a-
humulene (1.6–6.5%) and germacrene D (2.3–5.7%).
Among the volatiles detected only in the subsp. albida, there were a-thujene, thuja-
2,4(10)-diene, (E)-hex-2-enol, trans-muurola-4(14),5-diene, caryophylla-4(12),8(13)-
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011) 329
Fig. 2. SPME-GC/FID Chromatograms of the two M. melissophyllum subspecies: a) and b) subsp.
melissophyllum from Marche (1) and Abruzzo (4); c) and d) subsp. albida: from Basilicata (5) and Sicily
(7). Sample numbers refer to Table 1.

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)330
Table 2. Headspace Components Identified in M. melissophyllum subsp. melissophyllum and subsp. albida Using the PDMS Fibre
Entry
Componenta)
Calculated RIb)
RI Literaturec)
Melittis melissophyllum samplesd)
IDe)
subsp. melissophyllum
subsp. albida
RI HP-
Innowax
RI HP-5
NIST 08
Adams
1
2
3
4
5
6
7
1
a-Pinene (3)
1028
938
1028
939
0.4?0.06
0.5?0.04
4.7?0.44
2.4?0.28
4.0?0.14
41.8?3.83
22.1?2.32
Std
2
a-Thujene
1034
930
1035
930
6.2?0.68
MS, RI
3
Camphene
1066
951
1066
954
tr.f)
tr.
0.3?0.02
Std
4
Hexanal
1086
802
1087
801
0.1?0.03
0.1?0.03
0.2?0.04
0.1?0.03
0.2?0.01
0.2?0.00
0.4?0.00
MS, RI
5
b-Pinene
1103
978
1102
979
tr.
0.1?0.01
0.9?0.13
0.2?0.01
1.6?0.12
2.8?0.15
1.8?0.11
Std
6
Sabinene (5)
1119
976
1119
975
0.1?0.09
0.4?0.07
2.5?0.72
1.5?0.05
28.5?2.21
0.2?0.01
0.6?0.05
MS, RI
7
Thuja-2,4(10)-diene
1134
957
1137
960
1.4?0.62
0.2?0.06
MS, RI
8
Myrcene
1163
993
1163
990
0.5?0.17
tr.
0.2?0.01
0.2?0.00
0.3?0.00
Std
9
a-Phellandrene
1169
1005
1169
1002
tr.
1.6?0.20
0.7?0.05
Std
10
a-Terpinene
1178
1019
1178
1017
tr.
tr.
1.1?0.23
0.3?0.13
Std
11
Heptanal
1187
906
1187
902
0.1?0.03
0.1?0.03
0.1?0.01
tr.
0.2?0.14
MS, RI
12
Limonene
1195
1032
1194
1029
tr.
tr.
0.2?0.01
0.5?0.09
1.5?0.36
0.8?0.12
0.9?0.20
Std
13
2-Methylbutan-1-ol
1204
732
1205
732
0.3?0.09
0.2?0.20
MS, RI
14
b-Phellandrene
1205
1028
1205
1029
tr.
tr.
0.1?0.05
0.1?0.00
1.1?0.24
MS, RI
15
1,8-Cineole
1216
1034
1216
1031
tr.
tr.
tr.
0.2?0.05
Std
16
(E)-Hex-2-enal
1223
851
1223
855
0.5?0.02
0.9?0.10
0.5?0.01
0.6?0.05
4.5?0.87
1.4?0.21
2.1?0.13
MS, RI
17
2-Pentylfuran
1232
987
1232
988
tr.
tr.
tr.
tr.
tr.
tr.
MS, RI
18
(Z)-b-Ocimene
1238
1040
1238
1037
0.1?0.10
0.4?0.10
0.5?0.18
0.9?0.22
Std
19
g-Terpinene
1246
1063
1246
1059
tr.
0.1?0.01
0.1?0.06
0.2?0.14
2.5?0.20
0.8?0.14
Std
20
(E)-b-Ocimene
1255
1053
1254
1050
0.1?0.06
0.8?0.08
0.7?0.06
0.5?0.05
MS, RI
21
3-Octanone
1258
989
1258
983
0.7?0.08
0.6?0.03
0.4?0.05
0.2?0.01
MS, RI
22
p-Cymene
1273
1028
1273
1024
tr.
tr.
tr.
tr.
0.3?0.01
0.2?0.00
0.7?0.01
Std
23
Terpinolene
1284
1090
1284
1088
tr.
tr.
tr.
1.0?0.09
1.2?0.09
0.1?0.05
4.6?0.17
Std
24
Octanal
1287
1000
1287
998
0.1?0.01
tr.
tr.
0.1?0.01
0.1?0.01
Std
25
Tridecane
1298
1300
1300
1300
tr.
tr.
0.1?0.02
tr.
0.1?0.01
0.1?0.01
Std
26
Oct-1-en-3-one
1303
979
1303
977
0.1?0.01
0.1?0.01
0.1?0.01
0.2?0.01
0.1?0.01
MS, RI
27
6-Methylhept-5-en-2-one
1337
988
985
0.1?0.00
tr.
0.1?0.00
0.1?0.02
0.1?0.00
0.1?0.02
0.2?0.04
MS, RI

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)331
Table 2 (cont.)
Entry Componenta)
Calculated RIb)
RI Literaturec)
Melittis melissophyllum samplesd)
IDe)
subsp. melissophyllum
subsp. albida
RI HP-
Innowax
RI HP-5 NIST 08 Adams
1
2
3
4
5
6
7
28
2,6,10-Trimethyl-
dodecane
1341
1377
1344
1378
tr.
0.1?0.03
0.1?0.01
0.1?0.01 tr.
0.1?0.01 0.1?0.02 MS, RI
29
Hexan-1-ol
1352
867
1353
870
0.1?0.01
0.1?0.02
0.1?0.01
tr.
0.1?0.00 0.2?0.01 0.2?0.04 MS, RI
30
Oct-1-en-3-yl acetate
1379
1115
1379
1112
tr.
tr.
0.1?0.00
MS, RI
31
(Z)-Hex-3-enol
1383
860
1383
859
0.3?0.01
0.2?0.02
0.6?0.01
0.4?0.18 0.4?0.09 0.1?0.01 0.1?0.01 MS, RI
32
Nonanal
1392
1106
1392
1100
0.5?0.04 MS, RI
33
Octan-3-ol
1393
997
1393
991
0.3?0.00
0.8?0.04
0.5?0.05
1.0?0.21 0.5?0.04 0.3?0.02
MS, RI
34
(2E,4E)-Hexa-2,4-dienal
1400
910
909
0.1?0.01
0.1?0.01 MS, RI
35
(E)-Hex-2-enol
1404
856
1404
854
tr.
0.1?0.01 0.1?0.01 MS, RI
36
Oct-1-en-3-ol (1)
1449
982
1449
979
49.9?4.67 63.4?0.18 43.2?0.62 49.0?0.33 5.0?0.42 4.1?0.32 1.7?0.17 Std
37
a-Cubebene
1461
1349
1460
1348
0.1?0.01
0.3?0.05
0.1?0.03 0.2?0.01 0.1?0.01 0.1?0.02 Std
38
(2E,4E)-Hepta-2,4-dienal
1464
1010
1463
1007
0.1?0.05
0.2?0.01
0.1?0.01 0.2?0.02 tr.
tr
MS, RI
39
cis-Sabinene hydrate
1465
1071
1070
0.2?0.02
0.2?0.02
0.4?0.01
0.1?0.08 0.9?0.06 0.1?0.02 0.2?0.02 MS, RI
40
(Z)-Hex-3-enyl isovalerate 1476
1232
1480
1238
0.1?0.01
MS, RI
41
2-Ethylhexan-1-ol
1484
1030
1484
1028
0.1?0.03
0.3?0.09
tr.
tr.
0.1?0.00
0.4?0.14 MS, RI
42
a-Campholenal
1492
1129
1499
1126
0.1?0.04
1.6?0.24
MS, RI
43
a-Copaene
1494
1374
1494
1376
0.1?0.00
0.1?0.04
0.7?0.10
0.2?0.03 2.5?0.24 0.2?0.01 1.1?0.08 Std
44
Pentadecane
1499
1500
1500
1500
0.4?0.11
Std
45
Decanal
1500
1204
1500
1201
0.4?0.01
0.3?0.11
0.4?0.04
MS, RI
46
b-Bourbonene
1521
1383
1520
1388
0.1?0.00
0.1?0.00
0.2?0.01
0.2?0.06 0.6?0.02 0.8?0.04 0.8?0.06 MS, RI
47
Benzaldehyde
1523
959
1523
960
0.1?0.00
0.1?0.00
0.2?0.01
0.2?0.06 0.1?0.00 0.1?0.01 0.2?0.01 Std
48
Octa-3,5-dien-2-one
1524
1077
1522
1076
0.4?0.01
0.4?0.17
0.2?0.02 0.3?0.02 0.2?0.00 0.2?0.01 MS, RI
49
cis-Chrysanthenyl acetate
1530
1265
1265
0.1?0.01
MS, RI
50
b-Cubebene
1540
1388
1540
1388
0.1?0.00
0.2?0.04 0.5?0.02 0.3?0.01 0.3?0.00 MS, RI
51
(E)-Non-2-en-1-al
1541
1160
1541
1161
0.1?0.00
0.1?0.02
MS, RI
52
Linalool
1545
1101
1546
1096
0.4?0.02
0.6?0.17
0.6?0.00
0.5?0.10 0.2?0.03 0.3?0.01 0.3?0.01 Std
53
trans-Sabinene hydrate
1552
1098
1098
0.1?0.00
0.3?0.02 MS, RI
54
Octan-1-ol
1554
1070
1554
1068
tr.
tr.
0.1?0.01
0.1?0.01 0.1?0.00 0.1?0.01 MS, RI
55
Pinocarvone
1576
1163
1576
1162
0.2?0.00
0.2?0.08
0.2?0.03
0.1?0.01
0.7?0.02 0.4?0.03 MS, RI

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)332
Table 2 (cont.)
Entry Componenta)
Calculated RIb)
RI Literaturec)
Melittis melissophyllum samplesd)
IDe)
subsp. melissophyllum
subsp. albida
RI HP-
Innowax
RI HP-5 NIST 08 Adams
1
2
3
4
5
6
7
56
b-Ylangene
1580
1425
1576
1420
0.1?0.03 0.3?0.15 0.5?0.01
0.3?0.02
0.1?0.00 MS, RI
57
Isobornyl acetate
1582
1285
1285
tr.
tr.
0.1?0.01 Std
58
a-trans-Bergamotene
1583
1433
1583
1434
0.1?0.01 MS, RI
59
(E,Z)-Nona-2,6-dienal
1585
1154
1585
1154
tr.
0.1?0.03
0.1?0.01 tr.
MS, RI
60
b-Elemene
1588
1389
1588
1390
0.2?0.02 MS, RI
61
cis-Muurola-
4(14),5-diene
1593
1462
1463
tr.
0.1?0.00 0.2?0.05 0.3?0.01
0.2?0.01
0.1?0.01 MS, RI
62
(E)-Caryophyllene (4) 1599
1420
1599
1419
0.3?0.02 0.2?0.02 1.2?0.09
1.1?0.09
8.7?0.56 13.4?0.88 32.3?1.84
Std
63
Hexadecane
1599
1600
1600
1600
0.1?0.01 tr.
0.1?0.01 0.2?0.08 0.6?0.06
0.5?0.02
Std
64
Isophorone
1601
1116
1601
1118
0.1?0.03
MS, RI
65
Safranal
1617
1198
1617
1196
0.3?0.02 0.2?0.05 0.5?0.03
Std
66
b-Cyclocitral
1626
1220
1626
1219
0.2?0.00 0.3?0.08 0.2?0.01 0.3?0.06 0.2?0.02
0.2?0.01
0.4?0.03 MS, RI
67
Myrtenal
1641
1193
1641
1195
0.1?0.02 0.1?0.04 0.1?0.01
0.4?0.01
0.1?0.04 Std
68
Benzeneacetaldehyde
1655
1044
1655
1042
0.1?0.08 0.4?0.12 0.2?0.04 0.1?0.00 0.3?0.02
0.3?0.01
0.3?0.03 Std
69
Alloaromadendrene
1648
1459
1648
1460
0.2?0.02
MS, RI
70
trans-Pinocarveol
1657
1141
1657
1139
0.6?0.02
MS, RI
71
g-Gurjunene
1661
1471
1659
1477
0.4?0.01 0.3?0.07
MS, RI
72
(E)-b-Farnesene
1661
1456
1661
1456
0.1?0.03
2.2?0.09
0.7?0.05 MS, RI
73
trans-Muurola-
4(14),5-diene
1667
1493
1493
0.2?0.01
0.2?0.01
0.1?0.00 MS, RI
74
a-Humulene
1678
1453
1678
1454
0.1?0.00 0.1?0.05 0.4?0.04 0.2?0.15 1.6?0.42
5.3?0.62
6.5?0.38 Std
75
Salicylaldehyde
1682
1040
1682
1039
0.1?0.02 tr.
0.2?0.02 0.1?0.02
MS, RI
76
Geranial
1686
1272
1686
1267
0.2?0.01 MS, RI
77
b-Selinene
1690
1488
1694
1490
0.2?0.01
0.2?0.01 MS, RI
78
a-Amorphene
1694
1484
1692
1484
0.5?0.02 0.1?0.01 0.3?0.01
0.1?0.00
0.2?0.04 MS, RI
79
Heptadecane
1700
1700
1700
1700
0.2?0.02 0.1?0.07
0.1?0.02 0.3?0.01
0.4?0.02
Std
80
Viridiflorene
1701
1499
1701
1496
0.1?0.00
0.1?0.00
MS, RI
81
a-Terpineol
1702
1189
1702
1188
0.1?0.00 tr.
0.1?0.00 0.3?0.00 1.0?0.12
0.2?0.05
0.1?0.00 Std
82
Dodecanal
1712
1405
1712
1408
0.1?0.01 tr.
0.1?0.03 0.1?0.04 0.1?0.00
0.1?0.01
0.1?0.02 Std

Page 9

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)333
Table 2 (cont.)
Entry
Componenta)
Calculated RIb)
RI Literaturec)
Melittis melissophyllum samplesd)
IDe)
subsp. melissophyllum
subsp. albida
RI HP-
Innowax
RI HP-5
NIST 08
Adams
1
2
3
4
5
6
7
83
Germacrene D
1719
1480
1718
1485
0.2?0.00
0.1?0.03
1.6?0.10
2.0?0.32
5.7?0.85
4.7?0.42
2.3?0.05
MS, RI
84
Verbenone
1721
1208
1721
1205
0.2?0.07
0.2?0.01
0.1?0.02
Std
85
a-Zingiberene
1725
1493
1725
1493
0.4?0.02
tr.
0.3?0.02
0.2?0.07
0.3?0.01
MS, RI
86
a-Muurolene
1732
1495
1733
1500
0.7?0.02
0.1?0.02
0.7?0.06
0.2?0.05
0.5?0.02
0.3?0.01
0.2?0.03
MS, RI
87
Bicyclogermacrene
1740
1502
1738
1500
0.1?0.01
0.2?0.12
0.2?0.02
MS, RI
88
(E,E)-a-Farnesene
1747
1505
1747
1505
0.2?0.15
0.6?0.01
0.7?0.02
Std
89
d-Cadinene
1767
1522
1767
1523
0.1?0.02
0.1?0.02
0.2?0.03
0.1?0.07
1.1?0.01
0.4?0.02
0.5?0.04
MS, RI
90
g-Cadinene
1771
1512
1771
1513
0.1?0.01
0.1?0.07
0.5?0.02
0.3?0.02
0.2?0.04
MS, RI
91
7-epi-a-Selinene
1774
1516
1522
0.1?0.03
tr
MS, RI
92
ar-Curcumene
1782
1481
1782
1480
0.5?0.01
0.1?0.02
0.4?0.11
0.1?0.03
0.2?0.01
0.1?0.01
MS, RI
93
b-Sesquiphellandrene
1779
1520
1778
1522
0.1?0.00
tr.
0.1?0.00
tr.
MS, RI
94
Methyl salicylate
1796
1193
1798
1191
0.2?0.03
0.2?0.03
0.1?0.04
0.1?0.01
0.1?0.01
0.2?0.08
MS, RI
95
Myrtenol
1797
1195
1796
1195
0.5?0.02
MS, RI
96
Octadecane
1799
1800
1800
1800
tr.
0.1?0.01
0.6?0.17
0.1?0.01
0.2?0.01
0.3?0.01
0.3?0.06
Std
97
Tridecanal
1823
1509
1824
1510
0.1?0.01
0.1?0.02
0.1?0.07
0.1?0.03
tr.
0.1?0.01
0.1?0.01
MS, RI
98
1-Methylethyl
dodecanoate
1831
1831
0.1?0.05
MS, RI
99
cis-Calamenene
1845
1530
1845
1529
0.1?0.04
tr.
MS, RI
100
trans-Carveol
1837
1217
1835
1216
0.2?0.02
MS, RI
101
Hexanoic acid
1855
975
1854
973
0.1?0.01
0.1?0.06
0.2?0.06
0.1?0.07
0.1?0.00
0.2?0.02
0.1?0.05
MS, RI
102
p-Cymen-8-ol
1856
1186
1856
1182
tr.
0.5?0.00
MS, RI
103
Geranylacetone
1863
1456
1862
1455
0.2?0.00
0.3?0.06
0.2?0.03
0.2?0.02
0.2?0.01
0.2?0.01
0.2?0.08
MS, RI
104
Nonadecane
1900
1900
1900
1900
0.1?0.01
0.1?0.04
0.1?0.08
0.1?0.03
0.1?0.01
0.1?0.01
Std
105
Phenylethyl alcohol
1925
1117
1925
1107
0.1?0.00
0.2?0.02
0.2?0.03
tr.
0.1?0.00
0.2?0.00
0.1?0.02
MS, RI
106
(E)-b-Ionone
1956
1484
1956
1488
0.4?0.04
0.4?0.11
0.5?0.04
0.4?0.01
0.1?0.02
0.1?0.02
0.4?0.04
Std
107
Dodecan-1-ol
1969
1471
1969
1470
tr.
0.1?0.04
0.2?0.13
tr.
0.2?0.01
0.1?0.00
0.2?0.05
Std
108
Docosane
1998
1999
2000
2000
tr. 3
tr.
0.1?0.00
tr.
0.1?0.00
tr.
tr
Std
109
Caryophyllene oxide
2010
1581
2008
1583
0.2?0.02
0.2?0.04
0.3?0.00
0.2?0.05
0.4?0.02
0.5?0.01
1.1?0.53
Std
110
Salvial-4(14)-en-1-one
2029
1596
1594
0.1?0.03
tr.
tr.
tr.
tr
MS, RI
111
Isopropyl myristate
2039
1829
2040
1827
0.1?0.01
0.1?0.03
0.2?0.06
0.1?0.03
0.1?0.02
0.1?0.02
0.4?0.05
MS, RI

Page 10

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)334
Table 2 (cont.)
Entry Componenta)
Calculated RIb)
RI Literaturec)
Melittis melissophyllum samplesd)
IDe)
subsp. melissophyllum
subsp. albida
RI HP-
Innowax
RI HP-5 NIST 08 Adams
1
2
3
4
5
6
7
112
Humulene epoxide II
2057
1606
1608
0.1?0.07
0.1?0.01
0.1?0.00
0.2?0.01 MS, RI
113
Tridecan-1-ol
2075
1569
2076
1571
0.1?0.01
0.1?0.04
0.2?0.13 tr.
0.1?0.00 tr.
tr
MS, RI
114
Heneicosane
2099
2100
2100
2100
tr.
tr.
tr.
tr.
tr.
0.1?0.00 Std
115
6,10,14-Trimethylpenta-
decan-2-one
2132
1846
2131
1845
0.2?0.03
0.2?0.05
0.4?0.00
0.1?0.01
0.2?0.01 tr.
0.2?0.00 MS, RI
116
Spathulenol
2135
1575
2135
1578
0.1?0.03
MS, RI
117
Methyl dihydrojasmonate 2275
1653
2276
1650
0.1?0.01 tr.
0.1?0.03 tr.
0.1?0.01 tr.
tr
MS, RI
118
2,4-Bis(1,1-dimethyl-ethyl)phenol
2314
1511
2316
1514
0.1?0.02 tr.
0.1?0.07
0.1?0.01
0.1?0.01
0.2?0.02
0.1?0.01 MS, RI
119
Caryophylla-4(12),8(13)-
dien-5-olg)
2312
1640
1639
0.1?0.00
0.3?0.01
0.3?0.00 MS, RI
120
Dihydroactinidiolide
2376
1529
2375
1532
tr.
0.4?0.11 tr.
0.1?0.01
0.1?0.01
0.1?0.02
0.1?0.00 MS, RI
121
Kaurene
2391
2039
2393
2043
0.7?0.17 MS, RI
122
Coumarin (2)
2456
1437
2456
1434
38.5?4.30 24.6?2.37 28.3?1.88 26.0?2.95
0.4?0.03
0.2?0.01
0.3?0.02 Std
Total identified [%]
98.6
98.9
98.0
96.4
98.7
94.3
96.2
Identified compounds
75
73
81
93
89
85
87
Grouped compounds
Monoterpenes
2.0
2.8
11.5
7.6
53.6
53.9
36.3
Sesquiterpenes
3.2
1.2
7.7
7.0
27.5
28.5
47.5
Aliphatic alcohols
50.8
65.0
45.1
50.6
6.6
5.3
3.1
Aromatics
39.2
25.5
29.1
26.7
1.1
1.1
1.2
Others
3.4
4.5
4.3
4.3
8.2
4.6
7.1
a) Compounds are listed in order of their elution from a HP-Innowax column (detection at GC/FID); percentage values are means of three determinations?
standard deviation.b) Retentionindex(RI) on HP-Innowaxand HP-5 column, respectively,experimentally determinedusinghomologousseriesofC8–C26alkanes.
c) Relative RI taken from NIST 08 [21] and Adams [20] for polar and nonpolar column, respectively.d) Sample numbers corresponding to populations as reported in
Table 1.e) Identification (ID) methods: Std, by comparison of the tRand MS of available authentic standard; MS, by comparison of the mass spectrum with those of
the computer mass libraries WILEY 275, ADAMS, and NIST 08; RI, by comparison of the RI value with those reported in literature [20][21].f) tr., Traces (mean
value below 0.1%).g) Correct isomer not identified.

Page 11

dien-5-ol, kaurene, whereas, in the subsp. melissophyllum, oct-1-en-3-yl acetate,
decanal, safranal, salicylaldehyde, and b-sesquiphellandrene were detected.
Noteworthy is the detection, in the HS of both subspecies, of 100 volatiles in scant
amounts (below 1%).
According to previous works [2][10], these findings confirmed that the subsp.
melissophyllum can be considered an important source of the mushroom-like alcohol
oct-1-en-3-ol, offering an interesting opportunity for its use as flavoring ingredient in
food products. At the same time, high-level occurrence of the phenolic coumarin in this
subsp. was also confirmed [11][12].
The subsp. albida showed a different phytochemical volatile profile, with terpenoids
(monoterpene and sesquiterpene hydrocarbons) being much more abundant than in
the subsp. melissophyllum. This confirms the literature data [1][4] about the higher
density of glandular trichomes that make the subsp. albida much richer in terpenoids,
usually segregating in these structures. On the other hand, it is interesting to observe
that the subsp. albida shows low levels of oct-1-en-3-ol (1) and lacks almost completely
the phenolic coumarin that has been shown to cause hepatoxicity in animals.
To determine and verify the variations of HS components in different populations
of M. melissophyllum, the composition data were analyzed by cluster analysis (CA)and
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011) 335
Fig. 3. Changes of main chemical volatile groups in the headspace of the two M. melissophyllum
subspecies. Values, in percentages, are means of four and three locality measurements (samples 1–4 and
5–7), respectively, that are, in turn, means of three determinations each. *: p<0.004; **: p<0.001; ***:
p<0.0001.
Fig. 4. Structures of major compounds identified in the headspace of M. melissophyllum subsp. albida, 3–
5, and melissophyllum, 1 and 2

Page 12

principal-component analysis (PCA). CA led to a dendrogram shown in Fig. 5. The
results show thatthe populations belonging to subsp.melissophyllum arewellseparated
from those of subsp. albida, while similarity among populations of the latter is minor.
A grouping similar to the hierarchical CA was that obtained by PCA. In fact, a
significant association was found between the HS composition and taxonomy of the
seven populations. The 2D graphical representation of principal-component analysis is
shown in Fig. 6, and represents 93.60% of the total variance in the data set.
Populations on the right hand side of the PCA score plot were those belonging to
the subsp. melissophyllum (Fig. 6,a); they were positively correlated with components
on the right hand side of the loading plot that mostly contributed to 80.14% of total
variance, i.e., coumarin (2) and, to a greater extent, oct-1-en-3-ol (1; Fig. 6,b). These
populations were close to each other, being qualitatively and quantitatively similar in
major volatile components. Hence, the presence of oct-1-en-3-ol (1) in amounts higher
than 40%, and coumarin (2) in amounts higher than 20% appears to be characteristic of
the central-Italy populations of the subsp. melissophyllum. In contrast, populations on
the left hand side of the PCA score plot (Fig. 6,a) were those belonging to the subsp.
albida, which were influenced most by components in the same position of the loading
plot such as a-pinene (3), sabinene (5), and (E)-caryophyllene (4) that mostly
contributed to 13.46% of total variance (Fig. 6,b). In this case, their correlation was
Fig. 5. A dendrogram of seven populations of M. melissophyllum in central and southern Italy based on
Euclidean distances and UPGMA clustering method. Sample numbers correspond to populations of
origin indicated in Table 1.
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)336

Page 13

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011) 337
Fig. 6. a) Score plot (PCA) for main variation among M. melissophyllum subspecies. Samples are
represented by numbers corresponding to populations of origin of Table 1. b) The PCA loading plot for
headspace volatile compounds which explains 74.99% of the variation on horizontal axis (PC1) and
17.17% on the vertical axis (PC2). Subsp. melissophyllum (oct-1-en-3-ol and coumarin chemotype) and
subsp. albida (terpene-rich chemotype) are distinct chemical forms according to the four most important
variables identified.

Page 14

lower than that of the other subspecies, with population from Basilicata (Sample 5)
being slightly different from the others due to the high content in sabinene (5).
Therefore, the presence of monoterpenes and sesquiterpenes in amounts higher than 30
and 20%, respectively, and the almost absence of coumarin (below 0.5%) appear to be
characteristic of the southern-Italy populations belonging to the subsp albida.
The variability of data was generated mostly by the content of oct-1-en-3-ol (values
of eigenvectors: 0.98; ?0.09) and coumarin (values of eigenvectors:0.94; ?0.10) in the
first PC, and by a-pinene (values of eigenvectors: ?0.78; ?0.57), sabinene (values of
eigenvectors: ?0.33; 0.92), and (E)-caryophyllene (values of eigenvectors: ?0.83;
?0.20) in the second PC (Table 3).
2. Morphological Studies. The vegetative and reproductive organs of both
subspecies are covered by a dense indumentum consisting of non-glandular multi-
cellular uniseriate trichomes and glandular hairs of three different types (A, B, and C)
(Fig. 7,a–e, and Table 4).
Table 3. CorrelationCoefficients between SevenMajorCompoundsofM.melissophyllumHeadspaceand
Scores of the First Two Principal Components
Major compoundPrincipal components
PC1PC2
a-Pinene (3)
Sabinene (5)
Oct-1-en-3-ol (1)
(E)-Caryophyllene (4)
a-Humulene
Germacrene D
Coumarin (2)
Eigenvalue
% of variance
Cumulative %
?0.78
?0.33
0.98
?0.83
?0.89
?0.79
0.94
1176.52
80.14
80.14
?0.57
0.92
?0.09
?0.20
?0.38
0.37
?0.10
197.67
13.46
93.60
Table 4. Distribution of the Different Types of Glandular Trichomes on the Vegetative and Reproductive
Organs of Melittis melissophyllum Subspeciesa)
StemLeaf CalyxCorolla
adaxabaxadaxabaxadaxabax
subsp. mellissophyllum
A
þ
B
þ
C
þ þ
subsp. albida
A
þ þ
B
?
C
þ þ þ
a) Symbols: ?, absent; ?, sporadic; þ, present; þ þ, abundant; þ þ þ, very abundant.
?
þ þ
?
þ
þ
?
?
?
þ þ
þ
þ
þ þ
þ
?
?
þ
þ
þ þ
?
þ þ þ
?
þ þ þ
þ
?
?
?
þ þ
þ
þ
þ þ þ
þ
?
?
þ
þ þ
þ þ þ
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)338

Page 15

These trichomes have been described in detail in [2]; however their morphology is
briefly mentioned below for convenience.
Type A are typical peltate hairs with one elongated basal epidermal cell forming a
well-developed stalk. Types B and C are capitate hairs showing a short unicellular or a
long 2–3-celled stalk, respectively. Type A trichomes produce exclusively terpenes,
whereas capitate-type hairs present a complex secretion of both hydrophilic and
lipophilic substances, in which the essential oil represents a minor fraction.
In general, glandular and non-glandular trichomes are more abundant in subsp.
albida with respect to subsp. melissophyllum (Fig. 7,a–e, and Table 4).
The main differences are with respect to the vegetative organs. Indeed, type-A
peltate hairs are densely distributed on the leaf abaxial side of subsp. albida (Fig. 7,c),
whereas they are scattered in subsp. melissophyllum (Table 4).
Moreover, the subspecies albida lacks, unlike subsp. melissophyllum, type-B hairs
on stems. These taxa show instead similar trichome distribution patterns on the
reproductive organs (Table 4), where capitate hairs are much more abundant than the
peltate ones (Fig. 7,d and e). Therefore, the calyx indumentum, generally significant in
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011) 339
Fig. 7. a) Leaf adaxial surface of Melittis melissophyllum subsp. melissophyllum showing type-B capitate
hairs along the veins. b) Leaf adaxial surface of M. melissophyllum subsp. albida showing type-B capitate
hairs along the veins. c) Particular of the leaf abaxial surface of M. melissophyllum subsp. albida showing
type-A peltate trichomes. d) Calyx abaxial surface of M. melissophyllum subsp. melissophyllum showing
types A, B, and C trichomes. e) Calyx and corolla surfaces of M. melissophyllum subsp. albida showing a
dense indumentum. Scale bars: 1 mm (a, b, and e); 50 mm (c); and 200 mm (d).