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NPC
Natural Product Communications
Composition of Essential Oils from Seeds of Abies koreana
2013
Vol. 8
No. 2
227 - 230
Anna Wajs-Bonikowskaa,*, Karol Olejnika, Radosław Bonikowskia and Piotr Banaszczakb
a
Technical University of Lodz, Faculty of Biotechnology and Food Sciences, Institute of General Food Chemistry,
Stefanowskiego 4/10, 90-924 Lodz, Poland
b
Arboretum - Rogów Forestry Experimental Station, Warsaw University of Life Sciences, 95-063 Rogów, Poland
anna.wajs@p.lodz.pl
Received: October 26th, 2012; Accepted: December 3rd, 2012
The essential oils from seeds of nine Abies koreana specimens have been studied using GC-MS-FID and NMR methods, leading to the determination of 96
volatiles, which constituted over 99% of the oils. The hydrodistilled oils of fresh, resinous scent were isolated with yields in the range of 3.8-8.5%. The results
showed that the essential oil of Korean fir seeds contained 70-95% monoterpenes and 1-20% oxygenated monoterpenes as the dominant groups. The numerous
sesquiterpenes, diterpenes and their oxygenated derivatives constituted only 2-8% of the oil. The major component of the seed essential oil was limonene (4172 g/100g); the laevorotary form of this terpene predominated. A. koreana seeds seem to be a rich source of both essential oil and (-)-limonene, whose average
enantiomeric excess was above 95%.
Keywords: Abies koreana, Fir, Seed, Essential oil, Terpenes, Volatiles, Limonene.
The genus Abies (Pinaceae) comprises about 50 species of
evergreen conifers, ranged in Europe, North and Central America,
Asia, and North Africa, occurring in mountains over most of the
range. A. koreana is a fir native to the higher mountains of South
Korea. This species was discovered in 1907 on the volcanic island
of Cheju (South Korea) and one year later was introduced into
France. From a Korean natural forest this fir was introduced into
Polish Arboreta in 1929 [1]. Nowadays, A. koreana is cultivated as
an ornamental plant in many places, in temperate climates. Korean
fir is grown both for its foliage and for its abundant blue cones. The
winged ripe seeds are released when the cones disintegrate at
maturity about 5-6 months after pollination, in Poland usually in
September [1,2]. A. koreana, like other species of Abies, has been
used in folk medicine to treat colds, stomachache, indigestion, and
vascular and pulmonary diseases [3]. Abies needle oils are used
industrially by soap and perfume manufacturers to add pleasant
odors to their products [4]. A. koreana needle essential oil has been
previously investigated [5]. The volatile composition of intact cones
and twigs has also been published [5c-d]. However, to our
knowledge, no studies on the volatiles from the seeds of Korean fir
have been reported. Therefore, the present investigations were
carried out to isolate and identify the compounds of seed essential
oils. This paper is the second in a series in which we analysed the
fir seed volatiles [6].
The hydrodistilled oil of Korean fir seeds has a characteristic
pleasant, balsamic, resinous scent with a fresh lemon note. The
yield of essential oils was in the range of 3.2-8.5%.
Hydrodistillation of the seeds of individual woods was performed in
triplicate; the results (calculated as an average yield with standard
deviation value) are presented in Table 1. It is noticeable that the
yield of essential oils from the seeds of trees no. 2-9 are at a similar
level, while the yield from tree no. 1 was 37% higher than the
lowest yield (tree no. 4). The yield differences may be caused by
different degrees of seed ripeness or different sun exposure of
individual trees. However, the eight Korean firs tested were of the
same age (woods no. 1-8) and grown in the same area. The average
essential oil yield was 4.9%, which was much higher than those of
the needles (0.9-1.0%) and cones (0.42%) reported in the literature
[5-8]. It is noticeable that the yields from seeds of A. koreana,
growing in Poland are not much lower than those of hydrodistilled
oils from the seeds of other fir species: Abies maroccana (5.3%,
north Morocco), [7] A. nordmanniana (6.0%, Georgia) [8], and
A. alba (7.4%, south Poland) [6]. In total, 96 compounds were
identified, of which, to the best of our knowledge, 51 are new
records for Korean fir. The identified compounds presented in
Table 1 constituted 99.1-99.9% of the 9 examined oils; the volatiles
new for A. koreana seeds are marked with asterisks.
The hydrodistilled oils contained 15 monoterpene hydrocarbons,
which constituted 70.4-94.9% of the seed oils. The main compound
was limonene, which formed 39.8% to 69.6% of the total. This was
also one of the main components of A. koreana cone oils, but
occurred at a lower concentration (25.6%) than in the examined
seeds [5c]. The other major monoterpenes in the tested essential oils
were: α-pinene (7.3-38.6%), camphene (0.7-12.6%), -pinene (1.87.6%) and -myrcene (1.3-1.9%). They were also predominant in
the oils of Korean fir cones, needles and twigs [5]. In order to
provide the real concentration of the main oil constituents, we
carried out a methodology based on the true quantitation. This
experiment (listed in Table 1) showed that the amount of limonene
(41.0-71.7 g/100 g essential oil) corresponded to almost 40%-70%.
The concentrations (g/100 g) of other monoterpenes were for α- and
-pinene 7.4-39.4 g/100 g and 1.8-7.8 g/100 g, respectively. The
monoterpene hydrocarbons identified in the nine individual trees of
A. koreana were qualitatively the same, but differed in the
percentages of their major components.
The second identified group was oxygenated monoterpenes. These
17 compounds constituted 1-20% of the oils. Predominant was
bornyl acetate, the structure of which, as well as that of geranyl
acetate, were confirmed by NMR spectroscopy; the amount of
bornyl acetate varied strongly between the specimens from 0.4% to
18.5%. The quantitative determination of this ester was carried out
through the calibration curve, and the concentration (Table 1) was
0.6-28.5 g/100 g. This compound was also a predominant
component of needle oil from the Korean fir grown in Poland
(30.2%) [7] and in Korea on Jeju Island (30.4%) [5b]. Borneol
228 Natural Product Communications Vol. 8 (2) 2013
Wajs-Bonikowska et al.
Table 1: The composition of essential oils from seeds of 9 specimens of Abies koreana
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Tree no.
Compound
Santene
Tricyclene
α-Thujene
α-Pinene [%]
α-Pinene [g/100g]
Camphene
Sabinene
β-Pinene [%]
-Pinene [g/100g]
β-Myrcene
δ-2-Carene
α-Phellandrene
δ-3-Carene
p-Cymene
Limonene [%]
Limonene [g/100g]
-Terpinene
Terpinolene
α-Fenchol
Limonene oxide
trans-Pinocarveol
cis-Verbenol
-Terpineol
Borneol
Myrtenal
α-Terpineol
Myrtenol
Verbenone
trans-Carveol
Fenchyl acetate
cis-Carveol
Bornyl acetate [%]
Bornyl acetate [g/100g]
α-Terpinyl acetate
Neryl acetate
α-Cubebene
Geranyl acetate
-Ylangene
α-Copaene
-Elemene
-Longipinene
Longifolene
α-Cedrene
(E)- -Caryophyllene
-Elemene
-Copaene
cis-Muurola-3,5-diene
Cadina-3,5-diene
α-Humulene
cis-Muurola-4(15),5-diene
α-Acoradiene
(Z,Z)-α-Farnesene
ar-Curcumene
-Muurolene
Germacrene D
-Selinene
-Humulene
4-epi-Cubebol
α-Selinene
α-Muurolene
-Bisabolene
-Cadinene
cis/trans-Calamenene
δ-Cadinene
-Bisabolene
ω-Cadinene
α-Cadinene
-Elemol
-Calacorene
(E)-Nerolidol
Germacrene-1(10),5-dien-4-ol
Caryophyllene oxide
Globulol
Longiborneol
Cedrol
10-epi- -Eudesmol
Selin-6-en-4-ol
-Eudesmol
1-epi-Cubenol
τ-Cadinol
τ -Muurolol
trans-10-Hydroxycalamenene
1(10)-Spirovetiven-7 -ol
-Eudesmol
cis-10-Hydroxycalamenene
α-Cadinol
α-Eudesmol
Intermedeol
Tetradecan-1-ol
α-Bisabolol
RIexp.
881
922
925
935
946
968
974
984
995
990
1008
1014
1033
1053
1082
1102
1120
1128
1132
1137
1154
1175
1177
1183
1186
1202
1210
1215
1273
1336
1343
1353
1361
1379
1382
1392
1407
1415
1421
1426
1433
1434
1448
1449
1459
1464
1466
1468
1473
1477
1483
1487
1491
1494
1498
1500
1506
1515
1521
1522
1526
1533
1539
1542
1548
1551
1572
1575
1581
1598
1602
1607
1615
1619
1629
1636
1637
1638
1642
1649
1647
1648
1653
1657
1664
1676
1
2
3
4
tr
0.9
tr
17.3
17.6
9.1
tr
3.5
3.6
1.5
tr
0.2
0.8
tr
51.8
53.4
tr
0.2
tr
tr
0.1
0.1
0.6
0.7
tr
tr
tr
tr
tr
tr
tr
9.4
14.4
0.1
tr
tr
0.1
tr
tr
0.1
tr
tr
tr
0.1
0.1
tr
tr
tr
0.1
tr
tr
tr
tr
0.1
tr
0.3
0.1
tr
0.2
0.1
0.1
0.2
tr
0.4
tr
tr
tr
tr
tr
0.1
tr
tr
tr
tr
tr
tr
0.1
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
1.3
tr
tr
tr
tr
tr
38.6
39.4
0.4
tr
7.6
7.8
1.8
tr
tr
0.8
tr
43.5
44.8
tr
0.1
0.2
tr
0.1
0.1
0.1
tr
tr
tr
tr
0.1
tr
tr
tr
0.5
0.8
tr
tr
tr
0.4
tr
tr
tr
tr
0.3
tr
0.2
0.2
tr
tr
tr
0.1
tr
tr
tr
tr
tr
tr
0.4
0.2
tr
0.3
0.1
0.1
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
0.1
tr
tr
tr
tr
tr
tr
0.1
tr
tr
tr
2.6
tr
tr
tr
0.4
tr
9.5
9.7
5.3
tr
1.8
1.8
1.7
tr
0.3
0.5
tr
67.3
69.3
tr
0.1
0.1
tr
0.1
0.2
0.3
0.3
tr
tr
tr
tr
tr
0.1
tr
6.5
9.9
tr
tr
tr
tr
tr
tr
0.1
tr
0.1
tr
0.2
tr
tr
tr
tr
0.1
tr
tr
tr
tr
0.1
tr
0.3
0.2
tr
0.3
0.2
0.1
0.3
tr
0.5
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
0.1
tr
tr
0.1
tr
tr
tr
0.1
tr
tr
tr
2.4
tr
tr
tr
tr
tr
20.0
20.4
0.7
tr
4.4
4.5
1.6
tr
0.3
0.1
tr
64.7
66.6
tr
tr
0.1
tr
0.1
0.2
0.1
0.1
tr
tr
tr
tr
tr
tr
tr
1.1
1.7
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
0.1
0.1
tr
tr
tr
0.1
tr
tr
tr
tr
tr
tr
0.4
0.2
tr
0.3
0.1
tr
0.1
tr
0.2
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
0.2
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
3.9
tr
tr
5
Content [%]
tr
0.3
tr
11.3
11.5
2.9
tr
2.5
2.6
1.8
tr
0.2
0.1
tr
69.6
71.7
tr
0.1
tr
tr
0.1
0.1
0.1
0.2
tr
tr
tr
tr
tr
tr
tr
3.3
5.0
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
0.9
0.2
tr
tr
tr
0.5
tr
tr
tr
tr
0.1
tr
0.3
0.2
tr
0.3
0.2
tr
0.3
tr
0.5
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
0.1
tr
tr
0.1
tr
tr
tr
0.1
tr
tr
tr
2.8
tr
tr
6
7
8
9
Identif. method
tr
0.6
tr
17.3
17.6
7.9
tr
3.2
3.3
1.7
tr
tr
0.4
tr
49.3
50.8
tr
0.1
0.1
tr
0.1
0.1
0.4
0.5
tr
tr
tr
0.1
tr
tr
tr
10.1
15.5
tr
tr
tr
0.1
tr
tr
0.2
tr
0.1
tr
0.2
0.1
tr
tr
tr
0.1
tr
tr
tr
tr
0.1
0.1
0.4
0.2
tr
0.4
0.2
0.1
0.3
tr
0.5
tr
tr
tr
tr
tr
0.1
tr
tr
tr
tr
tr
tr
0.2
tr
tr
0.1
tr
tr
tr
0.1
tr
tr
tr
3.7
tr
tr
tr
1
tr
15.1
15.4
12.6
tr
2.6
2.7
1.4
tr
tr
0.9
tr
39.8
41.0
tr
0.2
tr
tr
0.2
0.3
0.6
0.9
tr
tr
0.1
0.1
tr
0.1
tr
15.7
24.0
0.1
tr
tr
tr
tr
tr
0.1
tr
tr
tr
0.2
0.1
tr
tr
tr
0.1
tr
tr
tr
tr
0.1
0.1
0.4
0.2
tr
0.4
0.2
0.1
0.2
tr
0.4
tr
tr
tr
tr
tr
0.2
tr
tr
tr
tr
tr
tr
0.2
tr
tr
0.1
tr
tr
tr
0.1
tr
tr
tr
4.5
tr
tr
tr
0.2
tr
7.3
7.4
4.7
tr
2
2.0
1.3
tr
0.2
0.5
tr
54.0
55.6
tr
0.2
0.1
tr
0.1
0.1
0.3
0.7
tr
tr
tr
tr
tr
tr
tr
18.6
28.5
tr
tr
tr
0.5
tr
tr
0.1
tr
tr
tr
0.2
0.1
tr
tr
tr
0.1
tr
tr
tr
tr
0.1
0.1
0.4
0.1
tr
0.3
0.1
0.1
0.1
tr
0.2
tr
tr
tr
tr
tr
0.1
tr
tr
tr
tr
tr
tr
0.2
tr
tr
tr
tr
tr
tr
0.1
tr
tr
tr
5.2
tr
0.3
tr
tr
tr
17.5
17.9
0.2
tr
5.3
5.4
1.9
tr
0.1
1
tr
68.6
70.7
tr
0.1
0.1
0.1
0.1
0.2
tr
tr
tr
tr
tr
0.1
tr
tr
tr
0.4
0.6
tr
tr
tr
0.4
tr
0.1
tr
tr
tr
tr
0.1
0.1
tr
tr
tr
tr
tr
tr
tr
tr
0.1
tr
tr
0.1
tr
tr
0.1
tr
0.3
tr
0.4
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
0.1
tr
tr
tr
0.1
tr
tr
tr
0.8
0.1
0.2
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
¹H,RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
¹H,RI,MS
RI,MS
RI,MS
RI,MS
¹³C,¹H,RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
¹H,RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
¹³C,¹H,RI,MS
RI,MS
¹³C,¹H,RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
¹³C,¹H,RI,MS
RI,MS
RI,MS
¹H,RI,MS
RI,MS
RI,MS
¹H,RI,MS
RI,MS
RI,MS
¹H,RI, MS
RI,MS
RI,MS
Abies koreana seed essential oil
Natural Product Communications Vol. 8 (2) 2013 229
87
* Eudesm-7(11)-en-4α-ol
88
Manoyl oxide
89
* Isopimara-8,15-diene
90
* Abietatriene
91
* Abieta-7,13-diene
92
* (8α,12Z)-Abienol
93
* Abieta-8(14),13(15)-diene
94
* trans-Totarol
95
* Dehydroabietal
96
* Abietal
Monoterpene hydrocarbons
Oxygenated monoterpenes
Sesquiterpene hydrocarbons
Oxygenated sesquiterpenes
Diterpene hydrocarbons
Oxygenated diterpenes
Nonterpenes
1677
2007
2027
2059
2100
2134
2166
2239
2252
2275
0.1
tr
tr
tr
tr
tr
tr
tr
tr
tr
85.3
11.1
1.9
1.6
0
0
tr
99.9
0.1
tr
tr
tr
tr
tr
tr
tr
tr
tr
92.8
1.5
1.9
2.9
0
0
tr
99.1
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
86.9
7.6
2.5
2.7
0
0
tr
99.7
tr
tr
tr
tr
tr
tr
tr
0.1
tr
tr
91.8
1.7
1.6
4.1
0
0.1
tr
99.3
tr
tr
0.1
tr
0.1
tr
tr
0.1
tr
tr
88.8
3.8
3.5
3.1
0.2
0.1
tr
99.5
tr
tr
0.1
tr
0.1
tr
tr
tr
tr
tr
80.5
11.5
3.0
4.2
0.2
0
tr
99.4
tr
tr
tr
tr
0.1
tr
tr
tr
tr
tr
73.6
18.1
2.6
5.1
0.1
0
tr
99.5
tr
tr
tr
tr
0.1
0.1
tr
0.1
tr
0.1
70.4
20.4
2.0
5.9
0.1
0.3
tr
99.1
tr
tr
tr
tr
0.1
tr
tr
0.1
tr
0.2
94.7
1.4
1.3
1.2
0.1
0.3
tr
99.1
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
RI,MS
Total identified
Yield of essential oil [%]
8.52
4.34
3.88
3.19
5.22
3.80
5.53
4.93
(standard deviation)
(0.41)
(0.52)
(0.53)
(1.14)
(0.03)
(0.26)
(0.51)
(0.34)
*- new for A. koreana seed volatiles; tr - <0.05%; RIexp. - experimental retention indices calculated on non-polar column;
(tr-0.9%), -terpineol (tr-0.6%), cis-verbenol (0.1-0.3%) and transpinocarveol (0.1-0.2%) are also dominant volatiles among the seed
monoterpenoids, but they occur at levels lower than 1% in the oils
of individual woods. Surprisingly, borneol was identified previously
as the main volatile in the needle oil (27.9%) of fir grown in Korea,
but at Mt Dukyu, Muju [5a].
The enantiometric distribution of four major monoterpene
hydrocarbons of the seed essential oils: limonene, α- and -pinene
and camphene (Table 2) was determined on a Chirasil-Dex CB
fused silica chiral column. It was found that the laevorotary isomer
was dominant for all examined monoterpenes. What is most
interesting is that the almost enantiomerically pure (-)-limonene was
found in A. koreana seed oil. The enantiomeric excess of (-)limonene was above 97%. Such a high concentration of the
laevorotary form of this terpene is also characteristic of A. koreana
needles [7] and other fir seeds, e.g: A. alba seeds [6] and A.
maroccana seeds [7]. The relatively high enantiomeric excess of the
(–) form was also noticed for α-pinene (an average 84%). The ratio
of the laevorotary to dextrorotary form of -pinene and camphene
was 76%: 24% and 90%: 10%, respectively. The excess of
laevorotary enantiomers of the above monoterpenes was also
observed previously in Korean fir needles [5d].
Table 2: Enantiomer ratio and enantiomeric excess (ee) of the main A. koreana seed
monoterpenes.
Tree no.
1
Compound
(-)-Limonene
96.6
(+)-Limonene
3.4
ee
93.2
(-)-α-Pinene
94.1
(+)-α-Pinene
5.9
ee
88.2
(-)- -Pinene
66.7
(+)- -Pinene
33.3
ee
33.4
(-)-Camphene 90.1
(+)-Camphene
9.9
ee
80.2
SD – Standard deviation
2
3
4
96.6
3.4
93.2
84.8
15.2
69.6
90.9
9.1
81.8
75.0
25.0
50.0
97.3
2.7
94.6
95.7
4.3
91.4
75.0
25.0
50.0
92.6
7.4
85.2
97.5
2.5
95.0
97.6
2.4
95.2
66.7
33.3
33.4
83.3
16.7
66.6
5
6
7
Content [%]
99.5 96.4 97.1
0.5
3.6
2.9
99.0 92.8 94.2
95.0 87.8 89.2
5.0 12.2 10.8
90.0 75.6 78.4
66.7 62.5 68.8
33.3 37.5 31.2
33.4 25.0 37.6
90.3 92.3 91.4
9.7
7.7
8.6
80.6 84.6 82.8
8
9
Mean ± SD
97.2
2.8
94.4
87.9
12.1
75.8
94.5
5.5
89.0
93.3
6.7
86.6
97.9
2.1
95.8
94.5
5.5
89.0
91.7
8.3
83.4
100.0
0.0
100.0
97.3 ± 0.9
2.7 ± 0.9
94.7 ± 1.9
91.8 ± 4.5
8.2 ± 4.5
83.7 ± 8.9
75.9 ± 12.8
24.1 ± 12.8
51.9 ± 25.5
89.8 ± 7.0
10.2 ± 7.0
79.6 ± 14.0
Although sesquiterpene hydrocarbons and their oxygenated
derivatives are the most numerous group in the seed oils, consisting
of 31 and 24 compounds, respectively, they constituted only 1.32.6% and 1.2-5.9% of the seed oils, respectively. Intermedeol (0.85.2%) and
-caryophyllene (0.1-0.9%) were the major
sesquiterpenes; the great majority of the others were found only in
traces. However, structures of seven terpenes, isolated by flashchromatography, were additionally confirmed by NMR
spectroscopy (Table 1). What is interesting is that intermedeol,
identified as the main sesquiterpenoid in the seeds, prevailed also in
needles and twigs of Korean fir [5c]. The identified compounds,
with many different sesquiterpene skeletons, generally had
muurolane, cadinane, eudesmane, elemane, cubebane, bisabolane,
caryophyllane, and germacrane skeletons; the most numerous had
muurolane (9 compounds) and cadinane (7 volatiles) skeletons.
However, farnesenes, curcumenes, acoradienes and spirovetivenes
were identified as isomer pairs in many of the examined essential
oils, but at concentrations lower than 0.05%.
Diterpene hydrocarbons and their oxygenated derivatives (9
compounds) were identified in quantities varying from tr-0.5%,
with abietal, trans-totarol, abieta-7,13-diene and isopimara-8,15diene being the main diterpenes in some of the examined tree oil
specimens. Eight of them were identified for the first time in A.
koreana.
In summary, the qualitative composition of all oils was exactly the
same, but quantitative differences between them were observed; for
many volatiles these differences were very significant, irrespective
of the population examined. For example, tree no. 1-8 grown in the
neighborhood and introduced to the Arboretum from a Korean
natural forest from the same seed packet. It is worth noting that
when the amount of monoterpene hydrocarbons in the seed oils
decreased the concentration of their oxygenated derivatives at the
same time increased (Figure 2). This relationship was observed
between the main compounds, like limonene and bornyl acetate, as
well as α-pinene, camphene and borneol, while the amount of
sesquiterpene hydrocarbons and their oxygenated derivatives did
not change as strongly (2-8%) as the monoterpene group did.
Concerning the previous publication in which the composition of
essential oils from needles of 20 A. koreana specimens (the same
population as ours) were examined, the qualitative composition was
very similar, while especially the content of the main volatiles like
camphene (2.4-24.5%), limonene (1.9-37.3%), bornyl acetate (10.742.8%) and intermedeol (0.1-25.6%) differed significantly [5d]. As
a result of chemical polymorphism of an aroma plant, huge
differences can be observed in the composition of essential oils,
even among one population. These differences may arise from the
fact that biosynthesis of terpenoids is controlled by genetic factors.
This process could also be a result of ontogenetic variability and
could be influenced by many environmental factors. These
correlations have been previously very well documented [9].
Moreover, like other conifer tissues, seed tissues appear to rely on
the reserves of essential oils to protect against the attack by insects
and fungal pathogens. The differences in the quantitative
composition of volatiles of the examined individual woods may be
caused by insect attack or different environmental conditions [10].
In summary, the seeds of A. koreana constitute a rich source of
essential oil (up to 8.5%) that may be potentially interesting for the
perfume industry. The essential oil contains the laevorotary
enantiomer of limonene in almost pure form and can be used to
isolate this monoterpene hydrocarbon.
230 Natural Product Communications Vol. 8 (2) 2013
Experimental
Plant material: Seeds were collected from 9 specimens of Abies
koreana E.H. Wilson growing in central Poland, identified by the
director of Rogów Arboretum. The material (seeds with cones) was
hand-picked in September 2009. The samples were stored in tight
plastic bags in a freezer (-24°C) until needed. Before
hydrodistillation, the seeds were separated from cones and ground
in a mill. Eight trees, about 30-years old (assayed as 1-8), were
grown in Rogów Arboretum, in the same (about 200m2) area, while
one tree, about 15-years old, was grown in a Łódź suburb. The
voucher specimen of plant material was deposited in the Institute of
General Food Chemistry, Lodz, Poland.
Isolation of essential oil: The fir seeds were crushed using a simple
mill. The essential oils were isolated from the crushed seeds by
hydrodistillation for 4 h using a Clevenger-type apparatus.
Hydrodistillation of the seeds was performed 3 times in parallel.
However, the mass of seeds of tree no. 8 was too small to calculate
the yield of essential oil.
Isolation of essential oil components: To isolate the volatiles, the
seed oil (20.8 g) – mixture of hydrodistilled oils from all tested
trees, was fractionally distilled under reduced pressure using a 20
cm Vigreux column. Eight fractions (1.1 – 1.8) were obtained
containing only monoterpenes: mainly limonene, and α- and pinene. From fraction 1.6 (4.2 g) limonene (89%) was identified by
1
H NMR spectroscopy. The residue from fractional distillation (2.3
g) was next flash-chromatographed (FC) on silica gel 60 (0.0400.063 mm, Merck) with n-hexane and increasing amounts of diethyl
ether. The separation was monitored by TLC and GC-MS. Twenty
fractions (2.1 – 2.20) were obtained and analysed by GC-MS.
Structures of 7 volatiles from the following fractions were
confirmed using NMR spectroscopy. Frs. 2.4 (31 mg): -bisabolene
(47%); 2.9 (300 mg): bornyl acetate (85%); 2.11 (42 mg): geranyl
acetate (68%); 2.13 (18 mg): (E)- -caryophyllene oxide (42%); 2.15
(19 mg): 1-epi-cubenol (20%); 2.16 (66 mg): (E)-nerolidol (48%);
2.17 (431 mg): intermedeol (87%). The repeated FC of sample 2.2
(326 mg) gave a fraction containing cis- and trans-10hydroxycalamenene; their structures were confirmed additionally by
1
H NMR spectroscopy.
Wajs-Bonikowska et al.
Main component quantification: Quantification of the main
components of the essential oil was carried out using a calibration
curve. Calibration curves were prepared for limonene α-pinene, pinene and bornyl acetate by diluting each standard in n-heptane, at
4 different concentrations, each concentration being injected in
triplicate. The application of this analytical procedure allowed the
accurate determination of the concentration (g/100 g) of all tested
components to be made. The linear regression equation of the
examined compounds had the coefficient of determination (R2) of
0.999.
Chromatographic analysis: Essential oils were analysed by GCMS-FID. The analyses were performed on a Trace GC Ultra column
coupled with a DSQII mass spectrometer (Thermo Electron).
A simultaneous GC-FID and MS analysis was performed using a
MS-FID splitter (SGE Analytical Science). Mass range was 33-550
amu, ion source-heating: 200°C, ionization energy: 70eV. Operating
conditions: capillary column Rtx-1 MS (60 m × 0.25 mm i.d., film
thickness 0.25 m), temperature program: 50 (3 min) - 300°C
(30 min) at 4°C/min. Injector and detector temperatures were 280°C
and 300°C, respectively. Carrier gas was helium (constant pressure:
300 kPa). Resolution of (-) and (+) enantiomers of limonene, α- and
-pinene, and camphene was performed on a Chirasil-Dex CB
column having the following dimensions: 30 m × 0.25 mm i.d.,
0.25 m df. Temperature program: 50 (3 min)-220°C (30 min) at
4°C/min. Injector and detector temperatures were 240°C and 250°C,
respectively. Carrier gas was nitrogen at the flow rate of
1.0 mL/min.
Identification of compounds: The identification of compounds was
based on a comparison of their MS with computer mass library
NIST 08, Wiley Registry of Mass Spectral Data 8th Edn, and
MassFinder 4.1, along with the relative retention indices (RI, nonpolar column). The identification was also based on a comparison
of 1H- and 13C-NMR spectra with literature data and a home-made
NMR data base. The identification of limonene, camphene, and αand -pinene enantiomers was based on (-)- and (+)-standards of
these terpenes.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
Bugala W. (1983) Trees and bushes, 3rd edn. PWRiL, Warsaw, 80-82.
Chojnowska E. (2000) Conifers in the garden. Multico Oficyna Wydawnicza, Warsaw, 171-172, 201-202.
Yesilda E, Honda G, Sezik E, Tabata M, Fujita T, Tanaka T, Takeda Y, Takaishi Y. (1995) Traditional medicine in Turkey; V: Folk medicine in the
inner Taurus Mountains. Journal of Ethnopharmacology, 46, 133-152.
Kim YG. (1997) Studies on the extractive of Abies koreana. Mokchae Konghak, 25, 1-9.
(a) Jeong SI, Lim JP, Jeon H. (2007) Chemical composition and antibacterial activities of the essential oil from Abies koreana. Phytotherapy
Research, 21, 1246-1250; (b) Yoon K, Weon-Jong Y, Sang-Suk K, Tae-Heon O, Nam Ho L, Chang-Gu H. (2009) Abies koreana essential oil
inhibits drug-resistant skin pathogen growth and LPS-induced inflammatory effects of murine macrophage. Lipids: ProQuest Agricultural Journal,
44, 471-476; (c) Baran Sz, Stephan von Reuss H, König WA, Kalemba D. (2007) Composition of the essential oil of Abies koreana Wils. Flavour
and Fragrance Journal, 22, 78-83; (d) Baran Sz, (2006) Biodiversity of essential oils in Abies. Ph.D. Thesis, Technical University of Lodz, Poland.
Wajs A, Urbańska J, Zaleśkiewicz E, Bonikowski R. (2010) Composition of essential oil from seeds and cones of Abies alba. Natural Product
Communication, 5, 1291-1294
Bazdi B, Oller Lopez JL, Cuerva JM, Oltra J. (2006) Composition of the essential oil from the seeds of Abies maroccana. Journal of Essential Oil
Research, 18, 160-161.
Sagareishvili TG. (1999) Composition of the essential oil of Abies nordmanniana, Chemistry of Natural Compounds, 35, 586
(a) Biodiversity Convention signed in Rio de Janeiro 5.06.1992 (Journal of Laws of the Republic of Poland 2002 no. 184, position 1532); (b) Hunt
RS, von Rudloff E. (1974) Chemosystematic studies in the genus Abies, part I. Canadian Journal of Botany, 52, 477-487; (c) von Rudloff E. (1976)
Chemosystematic studies in the genus Abies, part II. Canadian Journal of Botany, 54, 1926-1931; (d) von Rudloff E, Hunt RS. (1977)
Chemosystematic studies in the genus Abies, part III. Canadian Journal of Botany, 55, 3087-3092.
Person M. (2003) Chemodiversity and functions of monoterpene hydrocarbons in conifers. Ph.D. Thesis, Royal Institute of Technology (KTH),
Stockholm.
Natural Product Communications Vol. 8 (2) 2013
Published online (www.naturalproduct.us)
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225
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Book Review
Medicinal Plants of China, Korea, and Japan: Bioresources for Tomorrow’s Drugs and Cosmetics by Chritophe Wiart, PharmD, PhD
Stephen Pyne
281
Natural Product Communications
2013
Volume 8, Number 2
Contents
Original Paper
Page
Drimendiol, A Drimane Sesquiterpene with Quorum Sensing Inhibition Activity
Cristian Paz, Gerardo Cárcamo, Mario Silva, José Becerra, Homero Urrutia and Katherine Sossa
Microbial Transformation of Curcumol by Aspergillus niger
Chen Li-Xia, Zhang Hui, Zhao Qian, Yin Shi-Yu, Zhang Zhong, Li Tian-Xian and Qiu Feng
Chemopreventive Effect of Sarcophine-diol on NOR-1-Induced TPA-Promoted Skin Carcinogenesis in Female HOS:HR-1 Mice
Pawel T. Szymanski, Safwat A. Ahmed, Sherief Khalifa, Harukuni Tokuda, Eiichiro Ichiishi, Akira Iida, Nobutaka Suzuki and
Hesham Fahmy
Carmichaeline A: A New C20-diterpenoid Alkaloid from Aconitum carmichaeli
Shu-hua Li, Jun-ruXiong, Yuan-qin Zhang, Qing-xiang Xiang and Feng-zheng Chen
Steroidal Saponins from Dracaena marginata
Abdelmalek Rezgui, Anne-Claire Mitaine-Offer, David Pertuit, Tomofumi Miyamoto, Chiaki Tanaka, Stéphanie Delemasure,
Patrick Dutartre and Marie-Aleth Lacaille-Dubois
Zephgrabetaine: A New Betaine-type Amaryllidaceae Alkaloid from Zephyranthes grandiflora
Deepali Katoch, Dharmesh Kumar, Upendra Sharma, Neeraj Kumar, Yogendra S. Padwad, Brij Lal and Bikram Singh
Antioxidant and Anti-inflammatory Compounds in the Popular Landscape Plant Berberis thunbergii var. atropurpurea
Chuan-Rui Zhang, Robert E. Schutzki and Muraleedharan G. Nair
Two New Amides from Streptomyces michiganensis
Jinghua Xue, Liangxiong Xu, Zi-Hua Jiang and Xiaoyi Wei
Determination of Bioactive Compounds in the Juice of Pummelo (Citrus grandis Osbeck)
Marina Russo, Ivana Bonaccorsi, Germana Torre, Antonella Cotroneo, Paola Dugo and Luigi Mondello
Antiplasmodial Activity of Compounds from the Surface Exudates of Senecio roseiflorus
Leonidah Omosa Kerubo, Jacob Ogweno Midiwo, Solomon Derese, Moses K. Langat, Hosea M. Akala, Norman C. Waters,
Martin Peter and Matthias Heydenreich
Anti-inflammatory, Antioxidant and Cytotoxicity Activities of Methanolic Extract and Prenylated Flavanones Isolated from
Leaves of Eysehardtia platycarpa
Valeri Domínguez-Villegas, Vanessa Domínguez-Villegas, María Luisa García, Ana Calpena, Beatriz Clares-Naveros and
María Luisa Garduño-Ramírez
Phenolic Glycosides from Lindera obtusiloba and their Anti-allergic Inflammatory Activities
Hyun Gyu Choi, Hwa Dong Lee, Sang Hyun Kim, MinKyun Na, Jeong Ah Kim and Seung Ho Lee
Antiproliferative Effects of an Analog of Curcumin in Hep-2 cells: A Comparative Study with Curcumin
Mohankumar Kumaravel, Pajaniradje Sankar, Periyasamy Latha, Chellakan S Benson and Rajagopalan Rukkumani
Antiproliferative Activity of epi-Cercosporin in Human Solid Tumor Cell Lines
Ángel Trigos, César Espinoza, Maricela Martínez, Olivia Márquez, Leticia G. León, José M. Padrón, Manuel Norte and José J. Fernández
New Anthraquinone Derivatives from Geosmithia lavendula
Lourin G. Malak, Daoud W. Bishay, Afaf M. Abdel-Baky, Ahmed M. Moharram, Stephen J. Cutler and Samir A. Ross
Pancreatic Lipase Inhibitory Activity of Cassiamin A, a Bianthraquinone from Cassia siamea
Dilip Kumar, Aniket Karmase, Sneha Jagtap, Ruchi Shekhar and Kamlesh K. Bhutani
Antifeedant Activity of Spin-Labeled Derivatives of Deoxypodophyllotoxin against Brontispa longissima Larvae
Gang Feng, Jing Zhang, Liu Yang, Ying-Qian Liu, Zhi-Wei Zhang, Xuan Tian, Qi-An Jin and Zheng-Qiang Peng
Determination of Organic Acids in Salicornia herbacea by Solid-phase Extraction Combined with Liquid Chromatography
Dandan Han, Minglei Tian, Dong Wha Park and Kyung Ho Row
Hypoglycemic Effect of Bumelia sartorum Polyphenolic Rich Extracts
Halliny S. Ruela, Katia C. C. Sabino, Ivana C. R. Leal, Ana M. Landeira-Fernandez, Michelle R. A. de Almeida,
Talita S. M. Rocha and Ricardo M. Kuster
Protoanemonin Content Variation between Clematis spp.: Leaf, Stem and Root
Fangming Jin, Christian Narkowicz and Glenn A Jacobson
Methanolic Extract of Nigella sativa Seed Inhibits SiHa Human Cervical Cancer Cell Proliferation Through Apoptosis
Tarique N. Hasan, Gowhar Shafi, Naveed A Syed, Muhammad A Alfawaz, Mohammed A. Alsaif, Anjana Munshi, Kai Y. Lei and
Ali A. Alshatwi
Glucosinolate Biosynthesis in Hairy Root Cultures of Broccoli (Brassica oleracea var. italica)
Sun-Ju Kim, Woo Tae Park, Md. Romij Uddin, Yeon Bok Kim, Sang-Yong Nam, Kwang Hyun Jho and Sang Un Park
Characterization of Volatile Components of Zingiber roseum Essential Oil Using Capillary GC on Modified Cyclodextrins
VPPalayam S Pragadheesh, Anju Yadav, Manju Singh and Chandan S Chanotiya
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