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Aromas from Quebec. I. Composition of the essential oil of the rhizomes of
Acorus calamus L.
François-Xavier GARNEAU* and Guy COLLIN
Corporation LASEVE, Université du Québec à Chicoutimi (Québec),Saguenay, Canada G7H 2B1
André BÉLANGER,
3 Centre de recherche et de développement en horticulture, 400, boul. Gouin, Saint-Jean-sur-Richelieu (Québec),
Canada J3B 3E6
Serge LAVOIE, Hélène GAGNON, Nadia SAVARD and André PICHETTE
Laboratoire LASEVE, Université du Québec à Chicoutimi (Québec), Canada G7H 2B1
* address for correspondence
1
Abstract
The chemical composition of the essential oil obtained from the rhizomes of Acorus calamus
(sweet flag) collected in the Grondines region, Province of Quebec, was determined by GC/FID
and GC/MS analyses. Several components were isolated by liquid chromatography and were
identified by various NMR experiments such as: 1H-NMR, 13C-NMR, HSQC, HMBC, and
NOESY. The major compounds were identified as preisocalamendiol, acorenone, shyobunone,
and cryptoacorone. The complete stereochemical structure of cryptoacorone was elucidated.
Key Word Index
Acorus calamus, Araceae, essential oil composition, preisocalamendiol, acorenone, shyobunone,
epiacorone, stereochemistry, cryptoacorone.
3
INTRODUCTION
The essential oils of different anatomical structures of Acorus calamus L. Araceae, such as the
rhizomes (1) and the leaves (2) were described in several papers. Other papers reported the
chemical composition of the essential oils of plants growing in different countries such as Japan
(3), Bangladesh (4) and Turkey (5). Bélanger et al also published a preliminary study on the oil
composition of plants from Quebec (6). Reviews of the literature appeared elsewhere (7) as well
as a short description of commercial samples (8). In this study, we determined the structure of the
major components using spectroscopic methods. Of particular interest was the structure of
cryptoacorone. Vrkoc et al (9) proposed the stereochemical formula of cryptoacorone based on
the Hudson-Klyne rule, the octant rule and dipole moment measurements. They determined the
configuration of the chiral centers except for the configuration of the methyl group on the
cyclopentanone ring. In this paper, we confirmed these configurations and unambiguously
determined the relative configuration of the methyl group in the five-member ring using various
NMR experiments.
EXPERIMENTAL
Extraction of essential oil: Rhizomes of A. calamus were collected from plants cultivated in the
Grondines region, Quebec. The rhizomes (40.86 kg) of the plant were subjected to steam
distillation in a 400- litre Stainless Steel Still for 12 h giving 428 ml of a pale yellow oil in a yield
of 1.05% (v/w).
4
Oil Analysis: GC/FID analyses were carried out on a Hewlett-Packard 5890 gas chromatograph
fitted with both an apolar DB-5 capillary column and a polar Supelcowax 10 column (both 30 m
× 0.25 mm; film thickness 0.25 µm). GC/MS analyses were performed on an HP 5972 mass
spectrometer at 70 eV coupled to an HP 5890 GC equipped with a DB-5 column (same as above).
Temperature program for both GC-FID and GC-MS analyses was 40 °C for 2 min, then 2 °C/min
to 210 °C and held constant for 33 min. Identification of the components was done by
comparison of their retention indices and mass spectra with those of the literature (10, 11, 12).
Quantitative data were obtained electronically from GC-FID area percentages.
Refraction indices and relative density were measured according to standard methods NF ISO
279 and 280. The optical rotation was measured on a Autopol IV polarimeter from Rudolph
Research Analytical. HPLC-MS were carried out on an Agilent 1100 LC-MS system equipped
with a UV-VIS diode array detector (DAD) and an atmospheric pressure chemical ionisation
mass selective detector (APCI-MSD). Analytical HPLC separations were achieved using a
Zorbax Eclipse XDB-C18 5 µm column (4.6×150 mm) at a constant temperature of 20 °C. The
mobile phase was 55 % methanol and 45 % water and was delivered at a flow-rate of 1 mL/min.
UV-VIS detector was set at 250 nm with a 100 nm bandwidth. The APCI source of the MS
system worked with a nebulizing nitrogen gas pressure of 40 psi at 10 L/min and at a temperature
of 350 °C. Capillary voltage was set at 4.0 kV with a current of 4 µA and the fragmentor was set
at 70 V. Positive ions in the 100-700 m/z range were registered in the conventional scanning
mode. Preparative HPLC separations were achieved on an Agilent 1100 LC equipped with a
multiple wavelength detector and using a Zorbax ODS-C18 7 µm (21.2 × 250 mm) column. High
Resolution MS analyses were done with electrospray ionisations (ESI) on an Applied
5
Biosystems/MDS Sciex QSTAR XL QqTOF MS system (serial #T0970304, Toronto, ON,
Canada). Infrared spectra were acquired on KBr using a Perkin-Elmer apparatus. 1H-NMR, 13C-
NMR and 2D NMR spectra were recorded in deuterated chloroform on a Bruker Avance 400
spectrometer (5 mm QNP with Z-gradient probe) operating at 400.13 MHz (1H) or 100.61 MHz
(13C). Chemical shifts are expressed in parts per million (ppm) and were referenced with residual
chloroform (δH = 7.26 and δC = 77.0 ppm). Coupling constants (J) are expressed in hertz and
splitting patterns are designated as follows: s (singlet), d (doublet), t (triplet), q (quardruplet) and
m (multiplet). Purity of the isolated compounds was evaluated with the area of the GC peaks.
Isolation. Compound 1 (20.1 mg, 75.4% pure) was isolated from the essential oil by preparative
TLC using hexane/Et2O 25:1 as eluent. Thereafter, 5.0 g of the essential oil were
chromatographed on silica gel using a gradient of hexane (100%) to hexane/Et2O (10:1). Nine
fractions (F1-F9) were collected and evaporated. 80.0 mg of F8 (1.14 g) was submitted to
preparative HPLC and three compounds were isolated: 2 (TR = 56.5 min, 5.8 mg, 92.5% pure), 3
(TR = 24.7 min, 28.6 mg, 98.0% pure) and 4 (TR = 27.2 min, 20.1 mg, 49.6% pure). All
compounds were submitted to NMR for structure elucidation.
Cryptoacorone [3]: amorphous solid; [α]D = +120.6 (c=0.87, CHCl3); HREIMS m/z 236.1773
[M+] (calcd for C15H24O2, 236.1776); EIMS, see Figure 3; IR (KBr) ν 2961, 2929, 2873, 1734,
1707, 1459, 1169 cm−
1; 1H and 13C, see table II.
6
RESULTS AND DISCUSSION
The pale yellow essential oil had the following physical properties, n: 1.4983,
[α]D = + 36.5 ± 0.1° (net), d20 : 0.964. The chemical composition is shown in Table I. The most
important components are monooxygenated sesquiterpenes (ca. 70 %). Preisocalamendiol [1]
(18 %), acorenone (14.2 %), shyobunone (10.8 %), and cryptoacorone [3] (7.5 %) are the main
products. From the oil relative density, its rotation index value, and the low percentage of β-
asarone, the plant corresponds to a triploid (European) variety (8).
Because many compounds could not be unambiguously identified, some where isolated using a
variety of chromatographic techniques (TLC, CC, HPLC). Thus, preisocalamendiol [1],
isocalamendiol [2], and epiacorone [4] (Fig.1) were identified by 1D and 2D NMR spectral
analyses and by comparison of the MS and NMR spectra published in the literature (13, 14 and
15 respectively). The mass spectrum of isocalamendiol [2] closely resembles the MS published
recently (16), the only exception being the m/z =159 peak. This peak is the base peak in ref. (16)
and counts for less than 5 % in our spectrum.
The molecular formula of compound 3 C15H24O2, an amorphous solid, was determined by HRMS
(m/z 236.1773 obs. 236.1776 calc.) and was supported by 13C and DEPT-135 spectra, which
revealed 4 methyls, 4 methylenes, 4 methines and 3 quaternary carbons, including two carbonyls
at δC 212.8 and 217.6. Since epiacorone [4] was already identified and that its 1H and 13C spectra
were similar to those of compound 3, the latter was suspected to be another acorone stereoisomer.
This was confirmed by the systematic analysis of COSY, TOCSY, HSQC and HMBC spectra
7
(table II). The relative stereochemistry was determined with the NOESY spectrum. Figure 2
presents the most important NOESY correlations, which clearly show that methyls 15 and 13
reside on the same side as methylene 6. Concerning the stereochemistry of methine 8, no clear
NOESY correlation could be used. However, the S stereochemistry would lead to the same
structure as epiacorone [4], which was already identified. The uncertainty regarding the
stereochemistry of the methyl group at C-4 in Vkroc’s study was cleared up by means of the
NOESY correlation which ascertained the β configuration (9). So the structure of compound 3
was determined to be (1S,4S,5S,8R)-4,8-dimethyl-1-(2-propyl)-spiro[4.5]decane-2,8-dione or
cryptoacorone. The complete NMR data of this compound is reported for the first time. The mass
spectrum of cryptoacorone [3] is shown in Figure 3 and closely resembles that of epiacorone [2].
ACKNOWLEDGEMENTS
We would like to thank Professor Bernd Keller, Mass Spectrometry Laboratory., Dept. of
chemistry, Queen’s University, Kingston, Ontario for the measurement of the exact mass of
cryptoacorone.
REFERENCES
1. M. N. Todorova, I. V. Ognyanov and S. Shatar, Chemical composition of essential oil
from Mongolian Acorus calamus L. rhizomes. J. Essent. Oil Res., 7, 191-193 (1995).
2. P. R. Venskutonis and A. Dagilyte, Composition of essential oil of sweet flag (Acorus
calamus L.) leaves at different growing phases. J. Essent. Oil Res., 15, 313-318 (2003).
8
3. M. Niwa, A. Nishiyama, M. Iguchi and S. Yamamura, Sesquiterpenes from Acorus
calamus L. Bull. Chem. Soc. Japan, 48, 2930-2934 (1975).
4. I. Bonaccorsi, A. Cotroneo, J.U. Chowdhury and M. Yusuf, Studies on essential oils
bearing plants of Bangladesh. Part VII. Composition of the rhizomes oil of Acorus calamus
L.(sweet flag). Essenze, Deriv. Agrum., 67, 392-402 (1997).
5. M. Özcan, A. Akgül and J.-C. Chalchat, Volatile constituents of the esssential oil of
Acorus calamus L. grown in Konya province (Turkey). J. Essent. Oil Res., 14, 366-368
(2002).
6. A. Bélanger, L. Dextrase, H. Goudmand, F.-X. Garneau and G. Collin, Essential oil
composition of Acorus calamus from Quebec. Riv. Ital. EPPOS, Spec. Num. 15ièmes Journées
Int. Huiles Ess., Digne-les-Bains, France, 529-534 (1997).
7. a) B. Lawrence, Progress in essential oils. Perfum. & Flavor., 11, 52-54 (1986);
b) B. Lawrence, Progress in essential oils. Perfum. & Flavor., 22, 65-67 (March/April 1997).
8. C. M. Bertea, C.M. Azzolin, S. Bossi, G. Doglia and M. E. Maffei. Identification of an
EcoRI restriction site for a rapid and precise determination of
β
-asarone-free Acorus
calamus cytotypes. Phytochem., 66, 507-514 (2005).
9. J. Vrkoč, J. Jonáš, V. Herout and Šorm, F.; On terpenes. CLVII. Steric structure of
acorone, isoacorone, and cryptoacorone. Collection Czechoslov. Chem. Commun., 29,
539-550 (1964).
10. R.P. Adams, Identification of Essential Oil Components by Gas Chromatography/
Quadrupole Mass Spectrometry. Allured Publishing Corp., Carol Stream, IL (2001).
11. D. H. Hochmuth, MassFinder 3, 2004, Hamburg, Germany (www.massfinder.com).
9
12. D. Joulain and W. A. König, The atlas of spectral data of sesquiterpene hydrocarbons,
E. B. Verlag Hamburg (1998).
13. C. Zdero, F. Bohlmann, J.C. Solomon, R. M. King and H. Robinson, Ent-Clerodanes
and other constituents from Bolivian Baccharis species. Phytochem., 28, 531-542 (1989).
14. M. Iguchi and A. Nishiyama, Isolation and Structure of Isocalamendiol. Tetrahedron
Letters, 3729 (1969).
15. K. Nawamaki and M. Kuroyanagi, Sesquiterpenoids from Acorus calamus as
germination inhibitors. Phytochem., 43, 1175-1182 (1996).
16. M. Gonny, P. Bradesi and J. Casanova, Identification of the components of the
essential oil from wild Corsican Daucus carota L. using 13C-NMR spectroscopy. Flav. &
Fragr. J., 19, 424-433 (2004).
10
Table I. Composition of the essential oil of the rhizomes of Acorus calamus L.
Compound Retention index Area
%
Mode of
identification**
DB5 Supelcowax
α-pinene 940 1020 0.1 a,b,c
camphene 953 1065 0.2 a,b,c
β-pinene 978 1107 0.2 a,b,c
myrcene 992 1169 tr. a,b,c
limonene 1032 1194 tr. a,b,c
camphor 1149 1506* 0.1 a,b,c
decanal 1204 1506* 0.1 a,b,c
octyl acetate 1215 1485 0.1 a,b,c
2,3-dimethoxytoluene 1244 1801 0.2 a,b,c
bornyl acetate 1293 1573 0.5 a,b,c
nonyl acetate 1315 1589 tr. a,b,c
α-funebrene 1380 1558 0.2 a, b
β-elemene 1390 1582 0.3 a,b,c
7-epi-α-cedrene 1396 1539 0.1 b,d
(Z)-isoeugenol 1403 2275 0.2 a,b,c
methyl eugenol 1403 2029* tr. a,b,c
β-funebrene 1408* 1571 1.6 b
α-cedrene 1408* 1555 0.5 c
decyl acetate 1412 1698 0.1 a, b
β-cedrene 1415 1580* 1.0 a,b,c
α-cedrene, isomer†1429 1503 0.1 a, b
(E)-α-bergamotene 1437 1580* 0.8 a,b,c
prezizaene isomer 1444 1610 0.8 a,b,c
(E)-isoeugenol 1444 2334 0.1 a,b,c
prezizaene 1449 1617 1.7 a,b,c
zizaene 1453 0.15 d
acoradiene†1458 0.4 a, b
trans-β-farnesene 1463* 1671 1.1 a,b,c
11
Compound Retention index Area
%
Mode of
identification**
DB5 Supelcowax
(Z)-methyl isoeugenol 1463* 2239 0.6 a.b
acora-3(10),14-diene 1469 1664 0.4 b
β-acoradiene 1476* 1668* 0.4 b
4,5-di-epi-aristolochene 1476* tr. b
α-neocallitropsene 1478 0.1 b
germacrene D 1484* 1701* 0.4 a,b,c
γ-curcumene 1484* 1686 0.2 a,b,c
ar-curcumene 1488 1768 0.6 a,b,c
5-epi-aristolochene 1491 1716 0.5 b
6-epi-shyobunone 1497 1840 3.1 c
hinesene 1499* 0.2 a
bicyclogermacrene 1499* 1722 0.2 a,b,c
α-muurolene 1503 1718 0.2 a,b,c
isogermacrene A 1505 1712 0.2 b,c
cuparene 1507 1801 0.2 a,b,c
shyobunone 1518 1887 13.3 c
δ-cadinene 1525 1749* 0.5 a,b,c
β-sesquiphellandrene 1528 1763 2.1 a,b,c
isoshyobunone 1532 1867 1.3 c
(E)-nerolidol 1566 2042* 1.5 a,b,c
spathulenol 1575* 2118* 0.7 a,b,c
germacren-D-4-ol 1575* 2036 0.1 a,b,c
vulgarone A 1584 2042* 0.3 a
Preisocalamendiol [1] 1600 1999 18.0 e
sesquithuriferol 1608 2115 0.3 a
unknown A1625 2067 1.6
unknown B1628 2016 0.6
unknown C1641* 2042* 2.1
tau-muurolol 1645 2171 0.1 a,b,c
trans-isoelemicin 1650* 2389 tr a.c
12
Compound Retention index Area
%
Mode of
identification**
DB5 Supelcowax
α-cadinol 1654 2212 0.8 a,b,c
unknown D1659 2118* 1
4-epi-acorenone 1679 2131 0.7 a
acorenone 1688 2158 14.2 a
6α-hydroxygermacra-1(10),4-diene 1703 2304 0.2 b.c
khusiol 1709 2295 0.1 a.c
acora-7(11),9-dien-2-one†1723 2239 0.6 b
Isocalamendiol [2] 1741* 2450* 3.1 c,e
unknown E1741* 2357 0.5
calamendiol†1746* 2430 0.5 c
eudesma-3,11-dien-2-one†1782 2450* 0.1 b
unknown F1792 2450* 0.5
Cryptoacorone [3] 1797 2524 7.5 a,e
epi-acorone [4] 1801 2524 1 a,e
acorone, isomer 1 1807 2524 0.9 a
acorone, isomer 2 1819 2519 2.1 a
unknown G1839 0.3
Total 94.6
*at least two products are co-eluted; ** a) Adams (10), b) MassFinder (11), c) LASEVE data bank. d) Joulain (12),
e) these compounds were isolated and analyzed by various spectroscopic techniques; † tentatively identified
Unidentified peaks: A. 220[M]+(9), 192(100), 81(81), 149(76), 41(72), 69(71), 55(62), 107(53), 95(53), 177(51); B.
222[M]+(30), 138(100), 151(67), 111(59), 41(56), 95(51), 55(45), 81(44), 109(41), 69(39), 67(37); C. 222[M]+(28),
138(100), 111(78), 151(68), 95(65), 41(62), 81(55), 109(52), 69(51), 55(51); D. 159(69), 147(100), 121(85), 81(75),
69(75), 41(65), 109(50), 79(48), 91(46), 43(44); E. 236[M]+(6), 193(100), 137(55), 43(51), 41(45), 166(44), 69(40),
95(37), 55(34), 81(33); F. 218[M]+(42), 121(100), 91(78), 108(66), 133(64), 79(62), 41(59), 147(53), 107(51),
93(50); G. 234[M]+(9), 135(100), 82(68), 136(62), 122(42), 121(42), 41(42), 110(41), 164(40), 109(35);
13
Table II. 1H and 13C NMR data of Cryptoacorone [3].
Position δC (multa)δH (multb)
1 65.7 (d) 1.95 (t, J = 1.6 Hz)
2 217.6 (s) -
3α45.7 (t) 2.34 (m)
3β1.88 (m)
4 40.2 (d) 1.92 (m)
5 49.7 (s) -
6α41.9 (t) 2.45 (dd, J = 15.1, 2.2 Hz)
6β2.21 (dd, J = 15.1, 1.0 Hz)
7 212.8 (s) -
8 44.2 (d) 2.36 (m)
9α30.8 (t) 1.66 (m)
9β2.11 (m)
10α37.5 (t) 2.01 (m)
10β1.7 (m)
11 27.9 (d) 1.69 (m)
12 25.2 (q) 1.17 (d, J = 6.9 Hz)
13 18.4 (q) 0.92 (d, J = 6.7 Hz)
14 15.3 (q) 1.11 (d, J = 6.6 Hz)
15 15.0 (q) 1.07 (d, J = 6.4 Hz)
a Multiplicities were determined by DEPT. b Multiplicities and coupling constants in Hz are in parentheses.
14
O
O
O
O
7
8
9
10
5
6
43
2
1
O
14
15
O
11 13
12
(5) Acorone
(4) Epiacorone(3) Cryptoacorone
OOH
HO
H
(1) Preisocalamendiol (2) Isocalamendiol
Figure 1. Structures of isolated sesquiterpenes and acorone.
15
5
6
7
89
10
H
H
H
H
H
H
O
H
Me
14
12
3
4
Me
15
H
11
Me
13
Me
12
H
O
Figure 2: Important NOESY correlations of cryptoacorone [3].
16
40 60 80 100 120 140 160 180 200 220 240
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
m/z-->
Abundance
Scan 7450 (67.046 min): NSAC01.D
41
5555
69
81
95
109 123
138
147
151
166
179
193
203
221
223
236
Figure 3. Mass spectrum of cryptoacorone [3].
17
