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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2783
Geranium macrorrhizum L. (Geraniaceae) Essential Oil: A Potent Agent
Against Bacillus subtilis
by Niko S. Radulović* a ), Milan S. Dekić a ) b ), Zorica Z. Stojanović-Radić c ), and Suad K. Zoranić d )
a
) Department of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 33,
18000 Niš, Serbia (phone: þ 381-18223430; fax: þ 381-18533014; e-mail: [email protected])
b
) Department of Bio-Chemical and Medical Sciences, State University of Novi Pazar,
Vuka Karadžića bb, 36300 Novi Pazar, Serbia
c
) Department of Biology and Ecology, Faculty of Science and Mathematics, University of Niš,
Višegradska 33, 18000 Niš, Serbia
d
) Department of Chemistry, Faculty of Science and Mathematics, University of Priština, Knjaza Miloša
17, 38220 K. Mitrovica, Serbia
The volatile hydrodistilled compounds from aerial parts and rhizomes of the ethnopharmacologically highly valued plant species Geranium macrorrhizum L. were screened for their antimicrobial
activity in disc-diffusion and microdilution assays. The assays pointed out to a very high and selective
activity of the oils against Bacillus subtilis with minimum inhibitory concentrations (MIC) of 0.4 – 1.0 mg/
ml. This prompted us to perform detailed compositional analyses of the oils. GC and GC/MS analyses
allowed the identification of 283 constituents. The oils consisted mainly of sesquiterpenoids, the main
ones being germacrone (49.7% in the oil from aerial parts) and d-guaiene (49.2% in rhizome oil).
Significant qualitative and quantitative compositional differences in the oils from the two plant parts
were observed. Further antimicrobial testing enabled us to determine that germacrone, the major
constituent of the oil from aerial parts, was not the sole agent responsible for the observed activity.
Introduction. – In the last few decades, numerous studies were carried out on a large
number of well-known aromatic, spicy, medicinal, and other plants, with the aim to
develop new, natural formulations and additives for food, cosmetic, and other
applications, while scientific data on various plants, particularly on those that are less
widely used for culinary and medicinal proposes, are still rather scarce. The assessment
of such data remains an interesting and useful task, particularly for finding new sources
for functional foods and nutrients. Herbal remedies have been used for centuries in
traditional medicine around the world. A great number of new drugs discovered in the
last few decades originate from natural sources [1]. In recent years, complementary and
alternative medicine enjoy increasing popularity worldwide. Natural products have
been increasingly used for the prevention and treatment of various conditions [2].
The genus Geranium L. (Geraniaceae) comprises ca. 250 species, distributed mainly
in the temperate region of the northern hemisphere. According to Janković [3], 19
Geranium species can be found in the Serbian flora. A number of the species belonging
to this genus (known as hardy geraniums) are broadly used as ornamental, medicinal,
and melliferous plants. The most economically important species of this genus for their
aromatic and healing properties is G. macrorrhizum, a common species of the Balkan
2010 Verlag Helvetica Chimica Acta AG, Zrich
2784
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Peninsula. In Serbia, G. macrorrhizum (wild geranium or zdravac, in Serbian, the
meaning of which is related to the English term health) occurs sporadically on
calcareous soil and shade locations in mountainous terrain [3]. Due to decorative and
aromatic properties, this plant species is cultivated and frequently grown in gardens. It
is highly valued in Serbian tradition and traditional medicine of other Balkan peoples
for the treatment of stomach disorders, as an aphrodisiac and emblematic plant, and as
a symbol for good health [4].
Previous phytochemical investigations of this species resulted in the identification
of flavonoids, terpenoids, and tannins [4f] [5]. The extracts of G. macrorrhizum have
been reported to possess a broad spectrum of antimicrobial, hypotensive, spasmolytic,
astringent, cardiotonic, antioxidant, capillary, and sedative activity [5b] [5c] [6].
Essential oil of G. macrorrhizum is highly valued in perfumery due to excellent
fixative properties and could be used in fougeres, chypres, crepe de Chines, Oriental
bases, colognes, and fantasy fragrances. It is also used as a food-flavoring agent [7].
Recently, having in mind the increased demand for naturalness of foodstuff,
Miliauskas et al. [6a] [8] investigated several medicinal and aromatic plants with the
aim of finding and using plant extracts as new natural antioxidants in food preparations.
They found that the leaf extract of G. macrorrhizum possesses a strong radicalscavenging capacity, similar to or higher than that of the extracts isolated from sage
(Salvia officinalis) and rosemary (Rosmarinus officinalis), which are industrially
applied in food products and widely used in cosmetic products as natural antioxidants,
and concluded that the application of G. macrorrhizum extract in this sense was also
possible in hydrophilic food products.
The essential oil of G. macrorrhizum and related (solvent) extracts, appeared to
attract little interest to date mainly due to the limited availability of plant material
derived from wild-growing populations of G. macrorrhizum that are restricted in
occurrence to a relatively small geographical area, and hence the limitation caused by
relatively high prices of the commercial product (oil). In the last several years,
worldwide availability attracted attention of both scientists and consumers for the
possible use in many fields of application. The commercial essential oil obtained from
the aerial parts of G. macrorrhizum is produced solely in Bulgaria and limited to
several hundreds of kilograms. In recent years, the availability of the wild-growing G.
macrorrhizum became limited for industrial usage due to the new biodiversity
protection laws, which forced the development of large-scale cultivation for essentialoil production [4g], which is reflected on the price and increased interest for the
potential use of this plant species in food, and cosmetic and other applications.
No previous reports dealing with the phytochemical analysis of G. macrorrhizum
rhizome volatiles could be found in scientific literature. The volatiles from aerial parts
attracted the attention of chemists during the 1970s and 1980s, and resulted in the
isolation and/or identification of only several essential-oil constituents [4f] [5e – i].
Hence, the aim of this work was to perform detailed compositional analyses of the
volatiles isolated from aerial parts and rhizomes of G. macrorrhizum originating from
Serbia. Furthermore, having in mind the great ethnomedicinal application of this plant
species, and, with the final aim to possibly correlate the composition with biological
activity, we also investigated the antimicrobial effect of the mentioned essential oils and
one of their major constituents against a panel of standard microorganisms and clinical
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2785
isolates, including three fungal strains which are very common molds in human habitats
and are also believed to be responsible for human respiratory allergic diseases [9].
Results and Discussion. – Antimicrobial Activities of the Essential Oils. The
essential oils of G. macrorrhizum were evaluated for antimicrobial activity (discdiffusion and microdilution assays) against pathogenic strains of Gram-positive
(Staphylococcus aureus, Bacillus subtilis, Clostridium sporogenes), and Gram-negative
(Escherichia coli (two strains) and Klebsiella pneumoniae) bacteria. The oils were
active against all of the tested bacteria, while the yeast Candida albicans was not
susceptible to the rhizome oil. Antifungal activity against the tested molds (Aspergillus
fumigatus, A. restrictus, and Penicillium chrysogenum) was weak, with high MIC values.
The rhizome oil showed significantly lower activity compared to the aerial parts oil,
where in the microdilution assay, lower concentrations (compared to the rhizome oil)
of the latter inhibited both bacterial and fungal cultures (Tables 1 and 2).
Gram-positive and Gram-negative bacteria were equally susceptible to the tested
oils in both assays. The results for the pure rhizome oil of the disc-diffusion method
indicated the highest susceptibility of B. subtilis. The most resistant strains to the
rhizome oil were E. coli and C. sporogenes. Low antifungal activity of the rhizome oil
was evident from the values of the inhibition zones and, in almost all cases, with the
highest observed MIC values. Again, B. subtilis was the most susceptible strain towards
the aerial parts oil, while the most resistant one was E. coli. In the disc-diffusion assay
against fungi, the aerial parts oil was inactive only in the case of A. restrictus, while C.
albicans was susceptible only to the oil obtained from the aerial parts.
The obtained MIC values for bacteria mostly correlate to those from the discdiffusion assays, both showing that the most susceptible strain was B. subtilis with the
best result of 0.4 mg/ml obtained for the rhizome oil, while the most resistant one was E.
coli. The oil from the aerial parts exhibited higher activity than the rhizome oil against
both fungal and bacterial strains. However, the two methods (dilution and disc
diffusion) gave somewhat different results in the case of fungal strains. Specifically, the
tested concentrations of the oils, in the dilution assay, were not high enough to cause
growth inhibition of C. albicans with regard to all tested samples, while the discdiffusion assay revealed a susceptibility of this strain to the aerial parts oil.
In accordance with the results of the disc-diffusion assays, where the rhizome oil
had no effect against the fungi, the MIC values also suggested strong resistance of these
fungi, with the highest tested concentrations having both inhibitory and microbicidal
effect. An activity (MIC) observed at 0.5 mg/ml and lower values suggests a strong
antimicrobial effect of the substances tested, while one should consider the activity
associated with MIC values in the range of 0.6 – 1.50 mg/ml as a moderate one [10].
Everything above this last MIC value is of little interest. Based on these delimitations,
our assays showed very high activity against B. subtilis with respect to both rhizome and
aerial-part oils, while, against E. coli, S. aureus, and S. aureus (clinical isolate), only the
oil from aerial parts showed moderate activity.
Chemical Composition of the Essential Oils. The results of antimicrobial assays
prompted us to perform detailed compositional analyses of the oils. Table 3 lists the
identified volatile constituents of G. macrorrhizum by means of detailed GC and GC/
MS analyses, which allowed the identification of 283 components comprising ca. 90% of
n.a.
n.a.
n.a.
n.a.
Fungal strains
C. albicans
A. restrictus
P. chrysogenum
A. fumigatus
n.a.
n.a.
n.a.
n.a.
10
13
14
8
17
10
23
50%
v/v ) )
b d
n.a.
n.a.
n.a.
n.a.
8
8
12
8
10
8
15
25%
n.a.
n.a.
n.a.
n.a.
7
7
10
7
n.a.
7
9
12.5%
n.a.
n.a.
n.a.
n.a.
7
7
n.a.
n.a.
n.a.
n.a.
7
6.25%
20
n.a.
8
22
12
26
25
32
25
15
33
Pure oil )
b
b d
17
n.a.
8
20
11
25
22
19
12
14
21
50%
v/v ) )
Aerial parts oil
14
n.a.
n.a.
12
11
13
14
n.a.
13
8
20
25%
12
n.a.
n.a.
10
10
8
12
n.a.
9
7
17
12.5%
9
n.a.
n.a.
8
8
7
10
n.a.
8
n.a.
9
6.25%
n.a.
12
12
10
34
26
n.a.
25
20
29
34
33.3%
w/w ) )
b d
n.a.
9
10
n.a.
20
20
n.a.
18
16
19
22
20%
Germacrone
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
EtOH b )
20
10
16
15
17
29
20
16
30
15
48
Control c )
a
) Disc diameter 6.0 mm. b ) 15 ml of the tested samples. c ) 30 mg per disc; amoxycillin against bacterial strains, nystatin against fungal strains. d ) EtOH
Solution at the given concentration. e ) n.a. ¼ No activity observed.
13
13
18
17
24
14
38
Bacterial strains
E. coli (clinical isolate)
E. coli
K. pneumoniae
S. aureus
S. aureus (clinical isolate)
C. sporogenes
B. subtilis
Pure oil )
b
Rhizome oil
Table 1. Zones of Growth Inhibition (in mm, including the diameter of the disc) a ) of the Essential Oils, Germacrone, and Their Dilutions
2786
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2787
Table 2. Minimum Inhibitory Concentrations ( MICs [mg/ml]) and Minimum Bactericidal Concentrations ( MBCs [mg/ml]) of the Essential Oils and Germacrone
Rhizome oil
MIC
Bacterial strains
E. coli (clinical isolate)
K. pneumoniae
E. coli
S. aureus
S. aureus (clinical isolate)
C. sporogenes
B. subtilis
Fungal strains
P. chrysogenum
A. restrictus
A. fumigatus
C. albicans
5
1.25
2.5
2.5
0.625
2.5
0.0004
10
10
5
> 10
MBC
10
5
2.5
2.5
1.25
10
0.001
10
10
10
> 10
Aerial parts oil
Germacrone
MIC
MIC
MBC
1.25
>5
1.25
2.5
5
5
1.25
>5
>5
5
>5
>5
5
2.5
>5
>5
2.5
>5
>5
>5
>5
>5
2.5
0.625
0.312
0.039
0.312
0.625
0.001
10
10
5
> 10
MBC
>5
5
0.312
1.25
1.25
0.625
0.001
> 10
10
> 10
> 10
the isolated oils. Sesquiterpenoids predominate in both oils. Aerial parts produced an
oil rich with oxygenated sesquiterpenoids (54.4%), while the sesquiterpene hydrocarbon fraction accounted for 68.8% of total rhizome oil.
The aerial parts of G. macrorrhizum yielded a yellowish semi-solid oil with a strong,
but pleasant odor. Chemical composition was in agreement with the data on previously
isolated and/or identified constituents [4f] [5e – g]. The oil was dominated by
germacrone (49.7%), with a considerable amount of germacrene B (11.3%), gcurcumene (4.1%), trans-b-elemenone (1.6%), and g-elemene (1.3%) among the
identified sesquiterpenoids. The presence of a high amount of germacrane-type
sesquiterpenoids (61.6%; cf. Table 1) in the aerial-part essential oil should be noted.
One should be careful when considering the relative amount of elemane compounds,
since it is generally accepted that these are formed during a GC run as a consequence of
thermal [3,3]-sigmatropic rearrangements of germacrane precursors [11]. It might be
possible that these compounds (e.g., trans-b-elemenone and g-elemene) are not native
plant metabolites [12].
Hydrodistillation of the rhizomes gave a green-yellow oil with a strong aromatic
scent. In contrast to the aerial-part oil, the underground parts of the plant produced
an oil in which d-guaiene (49.2%) and other sesquiterpenoids with the guaiane
skeleton predominated (61.1% of total oil). Other terpenoids present in appreciable amount were the following sesquiterpenoids: germacrone (11.5%), a-guaiene
(8.7%), d-selinene (1.7%), guaia-6,10(14)-dien-4b-ol (1.5%), and eudesm-11-en-4a-ol
(1.5%).
Additionally, the essential oil from the aerial parts was utilized as the source of
germacrone for the further testing, and the ketone was isolated in pure form according
to a previously published procedure [13]. This gave the opportunity of corroborating
the identity of germacrone by means of additional spectral analysis (IR, and 1H- and
13
C-NMR).
2788
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Table 3. Percentage Composition of the Essential Oils Isolated from the Aerial Parts and Rhizomes of G.
macrorrhizum
No. RIcalc. a ) Component
1
2
763
793
3
4
5
800
801
822
6
7
832
832
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
834
849
852
862
864
889
902
914
926
929
936
937
953
964
968
977
982
986
992
992
1000
1003
1009
1011
1014
1019
1023
1027
1031
1033
1034
1035
1036
1044
1046
1047
1060
Butanoic acid
3-Methylbut-2-enal
( ¼ Prenal)
Octane
Hexanal
3-Methylbutanoic acid
( ¼ Isovaleric acid)
2-Methylpent-2-enal e )
2-Methylbutanoic acid
( ¼ Active valeric acid)
Furfural
Furyl alcohol
( E)-Hex-3-en-1-ol
Hexan-1-ol
Pentanoic acid
2,5-Diethylfuran
Heptanal
2-Acetylfuran
Tricyclene
a-Thujene
4-Methylpentanoic acid
a-Pinene
Camphene
Benzaldehyde
Hexanoic acid
Sabinene
b-Pinene
6-Methylhept-5-en-2-one
b-Myrcene
2-Pentylfuran
D2-Carene
Octanal
a-Phellandrene
Hexyl acetate
D3-Carene
a-Terpinene
p-Methylanisole
p-Cymene
Limonene
b-Phellandrene
1,8-Cineole
Benzyl alcohol
( E)-b-Ocimene
3-Methylbutyl butanoate
2-Phenylacetaldehyde
( Z)-b-Ocimene
g-Terpinene
Compound class b ) Percentage [%] c )
Method of
d
Aerial parts Rhizomes identification )
FAD
TER/CDC
–
t
t
–
RI, MS, Co-GC
RI, MS
FAD
FAD
FAD/TER
t
t
t
–
t
0.1 0.01
RI, MS, Co-GC
RI, MS, Co-GC
RI, MS, Co-GC
FAD
FAD/TER
t
–
–
t
RI, MS
RI, MS, Co-GC
OTH
OTH
FAD
FAD
FAD
FAD
FAD
FAD
TER(tri)
TER(thu)
FAD
TER(pin)
TER(ica)
OTH
FAD
TER(thu)
TER(pin)
CDC
TER(acy)
FAD
TER(car)
FAD
TER(men)
FAD
TER(car)
TER(men)
OTH
TER(men)
TER(men)
TER(men)
TER(men)
OTH
TER(acy)
FAD
OTH
TER(acy)
TER(men)
t
–
t
t
t
0.1 0.02
t
t
–
0.1 0.04
t
0.2 0.02
0.1 0.02
t
t
0.1 0.00
0.1 0.01
t
0.2 0.05
t
t
t
t
0.1 0.02
0.1 0.01
0.3 0.01
t
0.4 0.02
0.4 0.04
t
t
t
0.2 0.05
t
–
1.5 0.12
1.4 0.05
t
t
–
t
t
–
t
t
t
–
–
t
0.1 0.02
t
t
–
t
t
–
t
t
t
–
–
–
t
t
t
t
t
–
t
–
–
t
–
t
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
MS,
MS,
MS
MS,
MS,
MS
MS,
MS,
MS,
MS,
MS
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS
MS,
MS
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2789
Table 3 (cont.)
No. RIcalc. a ) Component
Compound class b ) Percentage [%] c )
45
46
47
48
1068
1069
1072
1075
FAD
FAD
OTH
TER(acy)
–
t
t
0.1 0.01
t
–
–
0.3 0.02
49
50
51
1078
1090
1092
FAD
TER(acy)
t
t
t
0.5 0.04
–
RI, MS
0.2 0.01 RI, MS, Co-GC
52
53
54
55
56
57
58
59
60
1092
1093
1093
1099
1100
1100
1100
1101
1105
FAD
TER(men)
TER(men)
TER(acy)
FAD
TER(pin)
FAD
TER(acy)/CDC
FAD
t
1.6 0.07
t
t
t
–
t
0.9 0.11
t
–
–
t
–
–
0.1 0.02
–
0.1 0.03
–
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
MS,
MS,
MS
MS
MS,
MS
MS
MS,
MS,
61
62
63
64
1105
1106
1108
1108
FAD
TER(acy)
FAD
CDC
0.1 0.00
t
t
t
t
–
–
t
RI,
RI,
RI,
RI,
MS, Co-GC
MS
MS
MS
65
66
1112
1116
TER(acy)
FAD
t
t
–
–
RI, MS
RI, MS
67
68
69
70
71
72
73
74
75
1117
1122
1130
1134
1142
1142
1144
1148
1148
OTH
CDC
TER(ica)
0.1 0.01
t
t
t
t
0.1 0.03
t
t
t
t
t
0.1 0.02
0.2 0.02
–
–
0.1 0.04
–
–
RI, MS, Co-GC
RI, MS
RI, MS
76
77
78
79
80
81
82
83
84
85
86
1149
1150
1152
1153
1154
1161
1166
1168
1170
1173
1180
t
t
t
t
0.1 0.00
–
–
–
0.1 0.05
t
t
–
0.1 0.02
0.3 0.01
–
–
t
t
t
1.1 0.82
1.6 0.45
–
RI, MS
RI, MS,
RI, MS,
RI, MS
RI, MS,
MS
RI, MS,
RI, MS,
RI, MS,
Heptanoic acid
Octan-1-ol
2-Methylbenzaldehyde
cis-Linalool oxide
(furanoid)
Unidentified component f )
Undec-1-ene
trans-Linalool oxide
(furanoid)
Nonan-2-one
Terpinolene
p-Cymenene
Rose furan
Undecane
a-Pinene epoxide ( Isomer I )
Isopentyl 2-methylbutanoate
Linalool
3-Methylbutyl 3methylbutanoate
Nonanal
Hotrienol
Pentyl 3-methylbutanoate
( E )-6-methylhepta-3,5dien-2-one
cis-Rose oxide
3-Methylbut-3-en-1-yl 3methylbutanoate
b-Phenylethanol
a-Cyclocitral
a-Campholenal
Unidentified component f )
Pentyl 2-methylbutanoate
p-Mentha-1,4,8-triene
trans-Pinocarveol
4,8-Epoxyterpinolene
3-Methylbut-2-en-1-yl 3methylbutanoate
Isopulegol
Camphor
trans-Verbenol
Camphene hydrate
Citronellal
g-Campholenol
trans-Pinocamphone
Pinocarvone
Borneol
Unidentified component f )
Methyl 2-phenylacetate
Method of
d
Aerial parts Rhizomes identification )
FAD
TER(men)
TER(pin)
TER(men)
FAD
TER(men)
TER(pin)
TER(pin)
TER(ica)
TER(acy)/CDC
TER(ica)
TER(pin)
TER(pin)
TER(cam)
OTH
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
MS, Co-GC
MS, Co-GC
MS
MS, Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
MS
MS
MS, Co-GC
MS
MS
RI, MS
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
2790
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Table 3 (cont.)
No. RIcalc. a ) Component
87 1181
88
89
90
91
92
93
94
95
96
97
98
1182
1182
1189
1189
1190
1191
1191
1193
1195
1196
1198
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
1200
1201
1202
1204
1206
1207
1211
1215
1220
1222
1225
1228
1230
1234
1236
1241
1241
1243
1247
1249
1255
1273
1276
1279
1289
1291
1293
1294
1294
1299
1300
1300
1302
1304
p-Mentha-1,8-dien-4-ol
( ¼ Limonen-4-ol)
Terpinen-4-ol
Unidentified component f )
p-Methylacetophenone
p-Cymen-8-ol
Butyl hexanoate
Dodec-1-ene
( E )-Ocimenol
Decan-2-one
a-Terpineol
Methyl salicylate
2,6-Dimethylocta-3,7-diene2,6-diol ( ¼ Hodiendiol I ) e )
Dodecane
Myrtenol
Estragole
Safranal
a-Campholenol
Decanal
Octyl acetate
Verbenone
p-Menth-1-en-9-al
trans-Carveol
b-Cyclocitral
Citronellol
Benzothiazole
cis-Carveol
Thymol methyl ether
cis-p-Menth-2-en-7-ol
Hexyl 3-methylbutanoate
Neral
trans-p-Menth-2-en-7-ol
3-Methylbutyl hexanoate
( E )-Geraniol
Geranial
Citronellyl formate
Perilla aldehyde
Isobornyl acetate
Tridec-1-ene
Limonen-10-ol
Undecan-2-one
Thymol
Dihydroedulan II
Tridecane
Benzyl 2-methylpropanoate
Perilla alcohol
Carvacrol
Compound class b ) Percentage [%] c )
Method of
d
Aerial parts Rhizomes identification )
TER(men)
0.3 0.01
t
RI, MS
TER(men)
–
1.4 0.22
–
0.2 0.01
–
–
–
–
0.3 0.01
–
–
RI, MS, Co-GC
OTH
TER(men)
FAD
FAD
TER(acy)
FAD
TER(men)
OTH
TER(acy)
0.1 0.04
–
t
0.1 0.01
t
t
0.1 0.02
t
0.8 0.06
t
t
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
MS
MS
MS, Co-GC
MS
MS
MS
MS, Co-GC
MS, Co-GC
MS
FAD
TER(pin)
OTH
CDC
TER(ica)
FAD
FAD
TER(pin)
TER(men)
TER(pin)
CDC
TER(acy)/CDC
OTH
TER(men)
TER(men)
TER(men)
FAD
TER(acy)/CDC
TER(men)
FAD
TER(acy)/CDC
TER(acy)/CDC
TER(acy)/CDC
TER(men)
TER(cam)
FAD
TER(men)
FAD
TER(men)
CDC
FAD
OTH
TER(men)
TER(men)
t
0.1 0.04
t
t
t
t
t
t
t
t
–
0.3 0.01
t
t
t
–
t
t
t
t
0.1 0.04
t
t
t
t
t
–
0.1 0.00
t
t
t
t
t
t
–
0.3 0.10
–
–
t
–
–
0.1 0.03
–
0.2 0.08
t
–
–
t
–
0.1 0.01
–
–
0.9 0.13
–
–
–
–
–
–
–
0.1 0.02
–
0.1 0.03
–
–
–
0.2 0.05
t
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
MS,
MS,
MS,
MS
MS
MS,
MS,
MS,
MS
MS,
MS
MS,
MS
MS
MS,
MS
MS
MS,
MS
MS
MS,
MS,
MS
MS
MS,
MS
MS
MS
MS,
MS
MS,
MS
MS
MS,
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2791
Table 3 (cont.)
No. RIcalc. a ) Component
Compound class b ) Percentage [%] c )
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
1307
1312
1317
1331
1332
1342
1347
1354
1354
1361
1374
1376
1376
1377
1378
1381
1386
FAD
TER(acy)/CDC
OTH
OTH
TER(men)
TER(ele)
OTH
TER(cub)
TER(acy)/CDC
OTH
TER(sat)
TER(far)
OTH
TER(cop)
OTH
TER(cop)
FAD
t
t
t
t
–
t
t
t
t
t
t
t
t
t
t
–
t
–
–
–
–
t
0.1 0.00
–
–
–
0.1 0.03
0.2 0.03
–
–
0.7 0.06
–
t
–
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
MS
MS
MS
MS,
MS
MS
MS,
MS
MS,
MS,
MS
MS
MS,
MS
MS,
MS
MS
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
1388
1389
1391
1393
1395
1396
1396
1409
1410
1419
1424
1426
1435
1438
1440
1443
1444
1449
CDC
FAD
TER(bou)
TER(bou)
FAD
OTH
TER(ele)
FAD
TER(aco)
TER(ced)
TER(cop)
TER(car)
TER(cop)
TER(ele)
OTH
TER(gua)
OTH
OTH
t
t
0.1 0.00
t
t
t
0.6 0.02
t
0.3 0.03
t
t
0.4 0.05
t
1.3 0.08
t
0.1 0.01
t
t
–
–
t
–
–
–
0.1 0.00
–
t
–
–
0.5 0.05
t
0.1 0.00
–
8.7 0.44
–
–
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
MS
MS,
MS
MS
MS
MS,
MS
MS
MS
MS
MS
MS,
MS
MS
MS,
MS
MS,
MS
168
169
170
171
1450
1450
1454
1456
TER(ger)
TER(gym)
CDC/TER
TER(gua)
t
t
t
–
–
–
–
0.3 0.02
RI,
RI,
RI,
RI,
MS
MS
MS
MS
172
173
174
175
176
177
1458
1460
1462
1462
1465
1470
TER(far)/CDC
TER(hum)
TER(eud)
TER(far)/CDC
TER(crc)
TER(aco)
t
0.1 0.02
t
t
t
t
–
0.5 0.05
–
–
–
–
RI,
RI,
RI,
RI,
RI,
RI,
MS
MS, Co-GC
MS
MS
MS
MS
Undecanal
Citronellic acid
p-Vinylguaiacol
Isobutyl benzoate
p-Mentha-1,4-dien-7-ol
d-Elemene
Benzyl butanoate
a-Cubebene
Citronellyl acetate
Eugenol
Cyclosativene
Farnesane e )
Butyl benzoate
a-Ylangene
Methyl 4-methoxybenzoate
a-Copaene
( Z )-Hex-3-en-1-yl ( Z )-hex3-enoate
trans-b-Damascenone
Benzyl 2-methylbutanoate
b-Bourbonene
1,5-Diepi-a-bourbonene
Dodecan-2-one
Benzyl 3-methylbutanoate
b-Elemene
Dodecanal
Italicene
a-Cedrene
b-Ylangene
b-Caryophyllene
b-Copaene
g-Elemene
3-Methylbutyl benzoate
a-Guaiene
2-Phenylethyl butanoate
3-Methylbut-3-en-1-yl
benzoate
Isogermacrene D
b-Barbatene
( E )-Geranyl acetone e )
4(aH ),10(aH)-Guaia1(5),6-diene
( E )-b-Farnesene
a-Humulene
Selina-4(15),7-diene
Homofarnesane e )
Cabreuva oxide B
a-Acoradiene
Method of
d
Aerial parts Rhizomes identification )
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
2792
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Table 3 (cont.)
No. RIcalc. a ) Component
Compound class b ) Percentage [%] c )
178
179
180
181
182
183
184
185
186
187
188
189
190
1470
1473
1475
1475
1477
1479
1481
1482
1482
1486
1487
1487
1490
TER(cad)
TER(aco)
TER(ere)
FAD
OTH
TER(cad)
TER(eud)
TER(cad)
TER(bis)
TER(cad)
TER(bis)
TER(ger)
OTH
t
t
t
0.2 0.04
t
t
t
–
4.1 0.18
–
0.6 0.05
0.6 0.04
t
t
–
–
–
–
–
–
0.9 0.03
–
0.2 0.03
–
0.3 0.03
–
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
MS
MS
MS
MS, Co-GC
MS, Co-GC
MS
MS
MS
MS
MS
MS
MS, Co-GC
MS
191
192
193
194
1491
1491
1493
1495
TER(eud)
OTH
TER(eud)
OTH
t
t
0.4 0.02
t
–
–
0.7 0.04
–
RI,
RI,
RI,
RI,
MS
MS
MS, Co-GC
MS, Co-GC
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
1495
1495
1497
1500
1498
1502
1505
1506
1510
1510
1511
1512
1515
1519
1521
1521
1523
FAD
TER(far)/CDC
TERe(ud)
FAD
TER(bis)
TER(eud)
TER(cad)
TER(gua)
TER(far)/CDC
FAD
TER(bis)
TER(gua)
TER(bis)
TER(bis)
TER(aco)
TER(cad)
TER(spi)
t
t
0.2 0.01
t
t
0.3 0.02
t
–
t
t
t
1.3 0.15
0.6 0.04
t
t
0.2 0.02
t
–
–
1.7 0.08
–
–
0.7 0.03
–
0.9 0.05
–
–
–
49.2 1.54
–
–
–
0.5 0.06
–
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
MS
MS
MS
MS,
MS
MS,
MS
MS
MS
MS
MS,
MS,
MS
MS
MS
MS
MS
212
213
214
215
216
217
218
219
220
221
222
1528
1529
1530
1532
1536
1537
1540
1541
1544
1547
1548
TER(acy)/CDC
TER(cad)
TER(cad)
TER(cad)
TER(bis)
TER(cad)
TER(aco)
TER(eud)
TER(cad)
OTH
TER(eud)
t
0.3 0.01
–
–
0.2 0.01
t
t
t
0.4 0.05
t
0.5 0.06
–
0.5 0.02
0.1 0.01
t
–
–
–
–
t
–
–
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
MS, Co-GC
MS, Co-GC
MS
MS
MS
MS
MS
MS
MS
MS, Co-GC
MS
cis-Muurola-4(14),5-diene
b-Acoradiene
4,5-Diepiaristolochene
Dodecan-1-ol
Pentyl benzoate
trans-Cadina-1(6),4-diene
Selina-4,11-diene
g-Muurolene
g-Curcumene
a-Amorphene
a-Curcumene
Germacrene D
2-Phenylethyl 2-methylbutanoate
cis-Eudesma-6,11-diene
Isoamyl phenylacetate
b-Selinene
2-Phenylethyl 3methylbutanoate
Tridecan-2-one
(3Z,6E )-a-Farnesene
d-Selinene
Pentadecane
a-Zingiberene
a-Selinene
a-Muurolene
Aciphyllene
( E,E)-a-Farnesene
Tridecanal
b-Bisabolene
d-Guaiene ( ¼ a-Bulnesene)
b-Curcumene
( Z )-g-Bisabolene
10-Epiitalicene ether
g-Cadinene
Spirovetiva-1(10),7(11)diene
Citronellyl butanoate
d-Cadinene
trans-Calamenene
Zonarene
( E )-g-Bisabolene
trans-Cadina-1,4-diene
Italicene ether
Selina-4(15),7(11)-diene
a-Cadinene
Benzyl hexanoate
Selina-3,7(11)-diene
Method of
d
Aerial parts Rhizomes identification )
Co-GC
Co-GC
Co-GC
Co-GC
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2793
Table 3 (cont.)
No. RIcalc. a ) Component
223 1549
224 1553
225 1556
226
227
228
229
1558
1564
1565
1567
230 1570
231 1570
232 1574
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
1576
1580
1590
1591
1600
1600
1600
1605
1610
1613
1615
1620
1627
1629
1635
1635
1639
1645
1649
1652
1661
1661
1663
1673
1676
1678
1682
1690
1691
1700
1701
264 1702
265 1715
266 1718
a-Calacorene
b-Vetivenene
Eremophila1(10),11-dien-9b-ol
Cadina-1(10),6,8-triene
Germacrene B e )
( E )-Nerolidol
(4bH,5aH)-cisEudesm-6-en-11-ol
b-Calacorene
b-Bulnesene
( Z )-Hex-3-en-1-yl
benzoate
Tridecan-1-ol
Hexyl benzoate
Acora-3,5-dien-11-ol e )
Caryophyllene oxide
Hexadecane
Globulol
Salvial-4(14)-en-1-one
Unidentified component f )
trans-b-Elemenone
Tetradecanal
Humulene epoxide II
Selin-6-en-4-ol e )
Selin-6-en-4-ol e )
a-Corocalene
Amorpha-4,7-dien-11-ol
Guaia-6,10(14)-dien-4b-ol
g-Eudesmol
2-Phenylethyl hexanoate
epi-a-Muurolol
d-Cadinol ( ¼ a-Muurolol)
a-Cadinol
Eudesm-11-en-4a-ol
Unidentified component f )
Eudesm-7(11)-en-4a-ol
Unidentified component f )
Tetradecan-1-ol
Cadalene
epi-a-Bisabolol
Heptadec-1-ene
Heptadecane
( E,E)-Germacra-3,7(11),9trien-6-one ( ¼ Germacrone)
Juniper camphor
Pentadecanal
(2E,6Z )-Farnesal
Compound class b ) Percentage [%] c )
Method of
d
Aerial parts Rhizomes identification )
TER(cad)
TER(ere)
TER(ere)
t
t
1.1 0.20
TER(cad)
TER(ger)
TER(far)
TER(eud)
t
11.3 0.88
t
t
TER(cad)
TER(gua)
OTH
FAD
OTH
TER(aco)
TER(car)
FAD
TER(gua)
TER(ida)
TER(ele)
FAD
TER(hum)
TER(eud)
TER(eud)
TER(cad)
TER(cad)
TER(gua)
TER(eud)
OTH
TER(cad)
TER(cad)
TER(cad)
TER(eud)
TER(eud)
FAD
TER(cad)
TER(bis)
FAD
FAD
TER(ger)
TER(eud)
FAD
TER(far)/CDC
–
–
t
t
t
t
0.6 0.8
t
t
t
0.3 0.05
1.6 0.24
t
t
t
–
–
1.1 0.12
–
0.3 0.02
t
–
–
–
t
2.1 0.32
t
1.2 0.08
0.4 0.02
t
t
t
t
49.7 1.88
t
t
t
0.6 0.02 RI, MS
–
RI, MS
–
RI, MS
–
1.1 0.06
–
–
MS
RI, MS
RI, MS, Co-GC
RI, MS
0.2 0.03 RI, MS
t
RI, MS
–
RI, MS
–
–
–
–
–
0.5 0.06
–
0.5 0.04
0.5 0.11
–
–
–
0.4 0.03
t
–
1.5 0.20
0.2 0.02
–
0.5 0.02
0.3 0.04
t
1.5 0.11
–
–
1.0 0.09
0.2 0.00
t
–
–
–
11.5 0.51
–
t
–
RI, MS,
RI, MS,
RI, MS
RI, MS,
RI, MS,
RI, MS
RI, MS
Co-GC
Co-GC
Co-GC
Co-GC
RI, MS
RI, MS, Co-GC
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS, Co-GC
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS
RI, MS, Co-GC
RI, MS
RI, MS
RI, MS
RI, MS, Co-GC
RI, MS, Co-GC,
NMR
RI, MS
RI, MS
RI, MS
2794
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Table 3 (cont.)
No. RIcalc. a ) Component
Compound class b ) Percentage [%] c )
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
TER(acy)/CDC
1724
1742
1750
1769
1769
1780
1782
1800
1809
1817
1846
1860
1875
1900
1920
1953
1961
2000
2023
2076
2100
2109
2118
2200
2300
2800
Citronellyl hexanoate
Unidentified component f )
Eudesma-3,7(11)-dien-8-one
Unidentified component f )
Benzyl benzoate
Pentadecan-1-ol
Phenanthrene
Octadecane
Tetradecyl acetate
Hexadecanal
Hexahydrofarnesyl acetone
2-Phenylethyl benzoate
Benzyl salicylate
Nonadecane
2-Phenylethyl 2-phenylacetate
Pimara-8(14),15-diene
Hexadecanoic acid
Eicosane
13-Epimanool oxide
Octadecan-1-ol
Heneicosane
g-Hexadecanolactone
(E )-Phytol
Docosane
Tricosane
Octacosane
Grouped Components
Terpenoids ( TER )
Monoterpenoids
Monoterpene hydrocarbons
Oxygenated monoterpenes
Tricyclic type (tri)
Thujane type (thu)
Isocamphane type (ica)
Pinane type (pin)
Acyclic type (acy)
Carane type (car)
Menthane type (men)
Camphane/bornane type
(cam)
Sesquiterpenoids
Sesquiterpene hydrocarbons
Oxygenated sesquiterpenes
Simple farnesane type (far)
Cyclofarnesane type (crc)
Elemane type (ele)
Cubebane type (cub)
Sativane type (sat)
Method of
d
Aerial parts Rhizomes identification )
TER(eud)
OTH
FAD
OTH
FAD
FAD
FAD
CDC
OTH
OTH
FAD
OTH
TER
FAD
FAD
TER
FAD
FAD
FAD
TER/CDC
FAD
FAD
FAD
t
2.9 0.23
t
1.7 0.15
t
t
t
0.2 0.03
t
t
t
t
t
t
t
t
t
t
–
t
–
t
t
t
t
–
0.9 0.09
–
0.2 0.04
–
t
–
–
–
t
t
t
–
–
–
–
t
t
–
t
0.1 0.00
t
–
t
t
t
88.2
9.9
6.8
3.1
–
0.2
0.1
0.4
3.5
0.1
5.5
0.1
90.7
5.0
0.1
4.9
t
–
0.2
1.2
0.6
t
1.9
1.1
78.3
23.9
54.4
t
t
3.5
t
t
85.7
68.8
16.9
–
–
0.8
–
0.2
RI, MS, Co-GC
RI, MS
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
RI,
MS,
MS
MS,
MS,
MS,
MS,
MS
MS,
MS,
MS,
MS
MS
MS,
MS,
MS
MS,
MS,
MS
MS
MS,
MS,
MS,
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
Co-GC
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2795
Table 3 (cont.)
No. RIcalc. a ) Component
Compound class b ) Percentage [%] c )
Method of
d
Aerial parts Rhizomes identification )
Bourbonane type (bou)
Cedrane type (ced)
Caryophyllane type (car)
Germacrane type (ger)
Gymnomitrane type (gym)
Guaiane type (gua)
Humulane type (hum)
Eudesmane type (eud)
Acorane type (aco)
Cadinane type (cad)
Eremophilane type (ere)
Bisabolane type (bis)
Spirovetivane type (spi)
Isodaucane type (ida)
Diterpenoids
Fatty acid derivatives ( FAD)
Carotenoid derived
compounds (CDC )
Others (OTH ) g )
Identified
Unidentified ( > 0.1%)
0.1
t
1.0
61.6
t
1.4
0.1
1.7
0.3
2.0
1.1
5.5
t
t
t
1.2
t
t
–
0.5
12.9
–
61.1
0.5
5.2
t
3.8
–
–
–
–
–
0.4
t
0.1
89.5
8.2
0.1
91.2
6.3
Total
97.7
97.5
a
) RIcalc. ¼ Experimentally determined linear retention indices on HP-5MS column. b ) For compoundclass abbreviations, cf. Grouped Components at the end of the Table. c ) Values are means of triplicate
analyses; t ¼ trace amounts (< 0.05%); – ¼ not detected. d ) RI ¼ Retention indices matching with
literature data [24]; MS ¼ mass spectra matching; Co-GC ¼ co-injection with a pure reference
compound; NMR ¼ characterization by 1H- and 13C-NMR spectroscopy. e ) Correct stereoisomer not
determined. f ) MS (70 eV, 2308; in m/z (rel %)): RI 1078: 93 (7), 92 (80), 91 (100), 77 (4), 65 (9), 59 (6),
51 (4), 43 (13), 41 (4), 39 (7); RI 1134: 93 (8), 92 (81), 91 (100), 79 (10), 77 (9), 65 (9), 59 (11), 51 (6),
43 (18), 39 (8); RI 1173: 109 (100), 108 (45), 93 (16), 83 (13), 81 (35), 79 (18), 67 (40), 55 (16), 41 (29),
39 (18); RI 1182: 109 (73), 108 (51), 93 (27), 85 (100), 81 (33), 79 (31), 67 (47), 55 (21), 41 (40), 39
(25); RI 1605: 220 (50), 187 (48), 161 (72), 121 (51), 119 (100), 107 (92), 105 (87), 93 (50), 91 (86), 43
(59); RI 1663: 121 (32), 107 (20), 95 (22), 93 (23), 79 (22), 70 (100), 68 (20), 67 (29), 42 (29), 41 (34);
RI 1676: 136 (70), 135 (78), 121 (25), 108 (20), 107 (100), 105 (22), 91 (33), 79 (20), 67 (49), 41 (27); RI
1742: 218 (42), 121 (36), 109 (49), 96 (43), 93 (42), 91 (47), 79 (39), 68 (100), 67 (58), 41 (45); RI 1769:
138 (72), 137 (67), 123 (70), 119 (58), 109 (86), 105 (64), 91 (72), 81 (72), 67 (64), 41 (100).
g
) Unclassified constituents, compounds of possible anthropogenic origin.
Antimicrobial Activity of Germacrone and Discussion of Active Components. The
major constituent, germacrone, of the essential oil of the aerial and underground parts
of the plant has been tested along with the oils for their antifungal and antibacterial
effect in the hope of finding a relation between the observed activity and the
composition of the oils. In the assays performed in this study, germacrone showed a
different pattern of the antimicrobial effect compared with that of the tested oils. The
most obvious difference, as visible from the disc-diffusion method results, was the
2796
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
complete lack of activity against K. pneumoniae at the appropriate dose, which was
susceptible to both aerial and rhizome oils. Antifungal activity was, as expected, very
low and similar to that of the oils. Larger inhibition zones, when compared with the oil
zones, were observed for germacrone. The most susceptible organisms to germacrone
were B. subtilis and E. coli.
Another very important aspect worth noting is that germacrone, according to the
determined MIC values, is neither the sole nor even the principle carrier of the
antimicrobial activity of the two oils. The activity of germacrone, clearly evident from
the differing MIC values of the oils and the ketone against B. subtilis, S. aureus, and C.
perfringens, seems to contribute but not fully explain the obtained results.
Contrary to the results of the dilution assay, the disc-diffusion assay gives a
completely different picture with regard to the activity of germacrone. Here,
germacrone showed in several cases higher activity than the aerial-part oil in the
highest tested amount dose. This discrepancy might be explained in the differing
solubility/diffusion capability of germacrone or the other active principles involved.
One should not exclude a possibility of a synergistic or antagonistic interaction among
the oil components.
This result disagrees, when considering its antimicrobial effect, with the commonly
accepted notion that germacrone is the main responsible biologically active constituent
of this and other plant species [14]. For example, germacrone was found to be one of
the main constituents in the essential oil of Asarum caulescense, used in Chinese folk
medicine to treat colds, coughs, and asthma. This plant showed antimicrobial activity
against Gram-positive and -negative bacteria, as well as against fungal strains [15].
Also, it was found that the geometrical isomer of germacrone isolated from the corals
of the genus Gorgonia exhibited activity against E. cloaceae and K. pneumoniae [16].
However, most of the other referenced works state germacrone as the active biological
principle only according to an educated guess relating the activity to the main
constituent present [17].
Tsai and co-workers investigated ca. 110 different essential oils from Chinese herbs
for natural compounds with anti-inflammatory activity. Among them, patchouli
essential oil (Pogostemon cablin) showed a significant inhibitory activity against i)
platelet aggregation induced by platelet-activating factor (PAF), an inflammatory
phospholipid mediator produced by various cells and involved in allergic disease,
inflammation, asthma, rhinitis, shock, and cardiovascular disease, and ii) arachidonic
acid-induced secondary platelet aggregation. They concluded that the a-bulnesene
(syn. d-guaiene), isolated from the essential oil of P. cablin was responsible for the
mentioned activity and has potential as an anti-PAF agent [18]. Based on these findings,
the current rhizome oil of G. macrorrhizum may exhibit a similar activity and acts as an
anti-platelet agent, where the anti-platelet sesquiterpenoid d-guaiene was present in a
significant amount. However, this needs further confirmation in future studies.
Biosynthetic Hypothesis. Significant qualitative and quantitative compositional
differences in the rhizome and aerial-part oils were observed. We tried to summarize
putative biosynthetic pathways mutually connecting the main essential-oil constituents
(Scheme) in order to obtain a more general picture encompassing the differences noted
in the oils from the two plant parts [19]. Both the rhizome and above-ground parts
share a common starting biogenetic point : germacryl cation. From this point, the
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2797
Scheme. Putative Biosynthetic (and artefactual) Pathways to the Main Sesquiterpenoids Found in G.
macrorrhizum (structures placed in boxes were identified in the oils under study)
biosynthesis in the two mentioned plant parts diverges (one being dominated in the
corresponding plant organs), with the production of the guaiane-type sesquiterpenoids
found in rhizome oil, a- and d-guaiene originating from germacrane A, which, along
with b-elemene (a Cope rearrangement product), and a- and b-selinene (acid-induced
cyclization products) [20], constitute 59.4% of rhizome oil, while, in the aerial parts,
germacrene B is being predominantly allylically oxidized to germacrone. In short,
germacrene A pathway was more active in rhizomes, and germacrene B pathway, along
with the corresponding artifactually formed elemane-type sesquiterpenoids (63.9%)
[12] [21], prevails in the aerial parts of G. macrorrhizum.
The data accumulated in this work regarding the rhizome volatiles are here
published for the first time and show a distinctive essential-oil (bio)chemistry of the
rhizome compared to the aerial parts. This once again stresses the importance of
detailed analyses of plant metabolites originating from different plant organs.
This work was supported by the Ministry of Science and Technological Development of Serbia
(project No. 172061).
2798
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Experimental Part
Plant Material. Plant material (aerial and underground parts) was collected in September 2007 from
wild-growing populations, on the northern slopes of the Vlasina Mountain (SE Serbia) and dried at r.t.
for two weeks. Voucher specimens were deposited with the Herbarium Collection of the Faculty of
Science and Mathematics, University of Niš, under the accession number VN100042.
Essential-Oil Isolation. Air-dried, to constant weight, plant material (5 batches of 1000 g of aerial
parts and 5 batches of 1500 g of underground parts) was subjected to hydrodistillation with ca. 3 l of dist.
H2O for 2.5 h using the original Clevenger-type apparatus [22]. The yields were 0.026 and 0.018% (w/w,
fresh weight basis) for the oils from the aerial parts and rhizomes, resp. The obtained oils were separated
by extraction with Et2O (Merck, Germany), dried (Na2SO4 ; Aldrich, USA), and immediately analyzed.
GC/MS Analyses. Analyses of the oils were carried out by GC and GC/MS. The GC/MS analyses
were performed on a Hewlett-Packard 6890N gas chromatograph equipped with a fused silica-gel cap.
column HP-5MS (5% phenylmethylsiloxane, 30 m 0.25 mm, film thickness 0.25 mm, Agilent Technologies, USA) and coupled with a 5975B mass-selective detector from the same company. The injector and
interface were operated at 2508 and 2808, resp. Oven temp. was raised from 70 to 2908 at a heating rate of
58 min 1 and then isothermally held for 10 min. As a carrier gas, He was used at 1.0 ml min 1. The sample,
1 ml of the oil soln. in Et2O (1 : 100), was injected in a pulsed split mode (the flow was 1.5 ml/min for the
first 0.5 min and then set to 1.0 ml/min throughout the remainder of the analysis; split ratio 40 : 1). Massselective detector was operated at the ionization energy of 70 eV, in the 35 – 500-amu range and scanning
speed of 0.33 s. GC (FID) analysis was carried out under the same exper. conditions using the same
column as described for the GC/MS. The percentage composition was calculated from the GC peak areas
without the use of correction factors. Qual. analysis of the essential-oil constituents was based on the
comparison of their linear retention indices (RIs) rel. to retention times of C7 – C28 n-alkanes on the HP5MS column [23] with those reported in the literature [24], and by comparison of their mass spectra with
those of authentic standards, as well as those from Wiley 6, NIST02, MassFinder 2.3, and a homemade MS
library with the spectra corresponding to pure substances and components of known essential oils, and
wherever possible, by co-injection with an authentic sample (the alkanes, some of the terpenoids, and
aromatic compounds).
Isolation of Germacrone. Germacrone was isolated by simple crystallization from the crude essential
oil (1.3 g) at 48. After filtration, obtained crystals were washed with abs. EtOH and then dried in air at r.t.
for 1 d to afford 0.55 g of white crystals (m.p. 54 – 568). The purity and identity was checked by GC/MS,
and 1H- and 13C-NMR analyses [25].
Test Microorganisms. The essential oils were tested against a panel of microorganisms including
Gram-positive Staphylococcus aureus ATCC 25923, S. aureus (clinical isolate), Clostridium sporogenes
ATCC 19404, Bacillus subtilis ATCC 6633, Gram-negative Escherichia coli ATCC 25922, E. coli
(clinical isolate), Klebsiella pneumoniae (clinical isolate), and the yeast Candida albicans ATCC 10231.
Fungal strains Aspergillus restrictus, A. fumigatus, and Penicillium chrysogenum were isolated from
mattress dust and identified by Dr. B. Ranković, Department of Biology, Faculty of Science, University of Kragujevac, Serbia. Cultures of isolated molds were maintained on potato dextrose agar
(PDA).
Antimicrobial Screening. The disc-diffusion method employed for the determination of antimicrobial
activities of the essential oils was according to the recommendations of the National Committee for
Clinical Laboratory Standards (NCCLS) [26]. Briefly, a suspension of the overnight culture of the tested
microorganisms (100 ml of 108 cells per ml) was spread on the solid media plates. Pure essential oils were
tested, as well as dilutions in EtOH at concentrations of 50, 25, 12.5, and 6.25% (v/v). Germacrone was
tested as the EtOH soln., 33.3 and 20% (w/w). Sterile filter paper discs (6 mm in diameter) were
impregnated with 15 ml of the oils, and the dilutions of the oils or the germacrone solns. were placed on
the inoculated plates. These plates, after staying at 48 for 2 h, were incubated at 378 for bacteria and 308
for 48 h for the fungal organisms. The diameters of the resulting inhibition zones were measured in mm.
Amoxicillin was used as a positive control for bacteria and nystatin for fungi, while the solvent (EtOH)
was used as the negative control. The presented results are the zones of inhibition including the diameter
(6 mm) of the paper disc. The experiments were conducted in quintuplicate.
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2799
Also the antimicrobial activity was evaluated using a broth microdilution method [27]. Minimum
inhibitory concentration (MIC) determination was performed by a serial dilution method in 96-well
microtitre plates. Bacterial species were cultured at 378 in Mueller Hinton agar for bacteria and
Sabouraud dextrose agar for fungi (308). After 18 h of cultivation, bacterial suspensions were made in
Mueller Hinton broth, and their turbidity was standardized to 0.5 McFarland. Optical density of every
suspension was confirmed on a spectrophotometer (UV/VIS 1610, Shimatzu). The final density of
bacterial and yeasts inoculum was 5 105. Suspensions of the molds were made in Sabouraud dextrose
broth, and their turbidity was confirmed by viable counting in a Thoma chamber. Final size of the fungal
inoculum was 1 104. Stock solns. of the essential oils were prepared in 10% aq. DMSO, and then serial
dilutions of the oils were made in a concentration range from 10 to 0.019 mg/ml (in the case of B. subtilis,
additional 10 dilutions were prepared). The inoculum was added to all wells, and the plates were
incubated at 378 during 24 h (bacteria) or at 308 for 48 h (fungal strains). The bacterial growth was
visualized by adding 20 ml of 0.5% 2,3,5-triphenyltetrazolium chloride (TTC) aq. soln. [10]. MIC was
defined as the lowest concentration of the oil that inhibited visible growth (red-colored pellet on the
bottom of the wells after the addition of TTC), while minimum bactericidal concentration (MBC) was
defined as the lowest oil concentration that killed 99.9% of bacterial cells. To determine MBC/MFC
(minimum fungicidal concentration), broth was taken from each well without visible growth and
inoculated in Mueller Hinton agar (MHA) for 24 h at 378 for bacteria or in Sabouraud dextrose agar
(SDA) for 48 h at 288 (molds) and 308 (yeasts). The experiments were performed in quintuplicate.
Statistical Analysis. To evaluate statistically any significant differences among mean values, a oneway ANOVA test was used. In all tests, the significance level at which we evaluated critical values
differences was 5%.
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