Essential Oil from Zanthoxylum hyemale

Abstract

The essential oils from aerial parts of young (sample A) and mature leaves (sample B), fruit (sample C), and flowers (sample D) of Zanthoxylum hyemale were obtained by hydrodistillation and analyzed by GC, GC/MS, and chiral phase gas chromatography (CPGC). Thirty-four compounds were identified from the essential oils, representing approximately 90.71, 91.19, 87.33, and 89.08 % of the oils, respectively. The major constituent of the young leaf essential oil was the sesquiterpene trans-nerolidol (51 %), while the main constituent of mature leaf (31 %) and flower oils (22 %) was an as yet unknown humulane-type sesquiterpenoid, which was characterized by spectral techniques (EI-MS and 1D-, 2D-NMR) as 3,7,10,10-tetramethylcycloundeca-3,7-dien-1-ol ( 1) and given the trivial name "hyemalol". In the fruit essential oil, the most abundant components were the monoterpenes beta-pinene (25 %) and alpha-pinene (10 %). The antimicrobial activity of the essential oils and some isolated compounds is also reported.

Full text

Introduction
Zanthoxylum hyemale A.St. Hill (Rutaceae),locally called ªcoen-
trilhoº, is a plant native to South America (Southern Brazil, Uru-
guay, Paraguay, and Argentine). In Brazil, Z. hyemale is generally
used in popular medicine as a potent antipyretic, antispasmodic,
astringent, and a tonic agent [1]. In continuation of our chemical
studies on plants of the Rutaceae family [2], [3], [4], [5], this work
reports the composition of essential oils obtained from the
leaves, flowers, and fruit of Zanthoxylum hyemale gathered in
Rio Grande do Sul state (Southern Brazil).
Materials and Methods
General
Optical rotations were measured on a Perkin Elmer-341 digital po-
larimeter. The HR-ESI-MS was recorded on a Finnigan MAT TQS
7000. 1H- and 13C -NMR spectra were recorded on a Bruker DPX
400 (400.1/100.6MHz) NMR spectrometer, in CDCl3with TMS as
internal standard. TLC were performed on precoated silica gel 60
F254 plates (Merck) and detection was achieved using UV light
(254 nm) and by spraying with 10% H2SO4, followed by heating.
Plant material
Zanthoxylum hyemale was collected in the town of Santana do
Livramento, Rio Grande do Sul state, Brazil and identified by
Essential Oil from Zanthoxylum hyemale
EuclØsio Simionatto1
Carla Porto1
Ionara I. Dalcol1
Ubiratan F da Silva2
Ademir F. Morel1
Affiliation
1Departamento de Química, Ncleo de Pesquisa de Produtos Naturais, Universidade Federal de Santa Maria
(UFSM), Santa Maria-RS, Brazil
2Instituto de Ciencias Exatas e Geocincias, Química, Universidade de Passo Fundo, UPF, Passo Fundo-RS,
Brazil
Correspondence
Prof. Dr. Ademir Farias Morel ´ Departamento de Química ´ Ncleo de Pesquisa de Produtos Naturais ´
Universidade Federal de Santa Maria ´ Campus Camobi ´ CEP 97105±900 ´ Santa Maria RS ´ Brazil ´
E-mail: afmorel@base.ufsm.br
Received October 11, 2004 ´ Accepted January 31, 2005
Bibliography
Planta Med 2005; 71: 759±763 ´  Georg Thieme Verlag KG Stuttgart ´ New York
DOI 10.1055/s-2005-864184 ´ Published online July 29, 2005
ISSN 0032-0943
Abstract
The essential oils from aerial parts of young (sample A) and ma-
ture leaves (sample B), fruit (sample C), and flowers (sample D)
of Zanthoxylum hyemale were obtained by hydrodistillation and
analyzed by GC, GC/MS, and chiral phase gas chromatography
(CPGC). Thirty-four compounds were identified from the essen-
tial oils, representing approximately 90.71, 91.19, 87.33, and
89.08% of the oils, respectively. The major constituent of the
young leaf essential oil was the sesquiterpene trans-nerolidol
(51%), while the main constituent of mature leaf (31%) and flow-
er oils (22%) was an as yet unknown humulane-type sesquiter-
penoid, which was characterized by spectral techniques (EI-MS
and 1D-, 2D-NMR) as 3,7,10,10-tetramethylcycloundeca-3,7-
dien-1-ol (1) and given the trivial name ªhyemalolº. In the fruit
essential oil, the most abundant components were the monoter-
penes
b
-pinene (25%) and
a
-pinene (10%). The antimicrobial ac-
tivity of the essential oils and some isolated compounds is also
reported.
Key words
Zanthoxylum hyemale ´ Rutaceae ´ essential oils ´ hyemalol ´ anti-
microbial activity
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Prof. Renato Zµcchia. Leaves and flowers were collected between
July and September, 2002 and fruits were collected in December,
2002 from the wild population of one location at Santana do Liv-
ramento. Voucher specimens (SMDB 5687±5690) have been de-
posited at the Herbarium of the Federal University of Santa Mar-
ia.
Chemical analysis
Fresh young (sample A) and adult leaves (sample B), fruit (sam-
ple C), and flowers (sample D) were subjected to hydrodistilla-
tion for 4 h using a modified Clevenger apparatus followed by ex-
traction of the essential oils from the distillate with diethyl ether.
After solvent removal, crude oil yields were 0.30%, 045 %, 0.25 %,
and 0.70% (v/w) for samples A, B, C, and D, respectively.
±Sample A: d20:: 0.89 g mL±1;n20: 1.4701; [
a
]25: ±9.4 (c0.016,
CHCl3);
±Sample B: d20: 0.86 g mL±1;n20: 1.5502; [
a
]25: ±19.4 (c0.013,
CHCl3);
±Sample C: d20: 0.83 g mL±1;n20: 1.4301; [
a
]25: ±14.9 (c0.012,
CHCl3);
±Sample D: d20: 0.85 g mL±1;n20: 1.5301; [
a
]25: ±11.6 (c0.017,
CHCl3).
The oils were analyzed by GC and GC-MS. GC analyses were per-
formed using a Varian CP-3800 gas chromatograph with a data
handling system and FID and SE-54 fused-silica column (25
m 0.25 mm i. d., film thickness 0.25
m
m). Operating conditions
were as follows: injector and detector temperatures, 220 and
2808C, respectively; carrier gas, H2; oven temperature program
from 50 8C to 2508Cat48C/min. GC-MS analyses were performed
using a Varian model 3800 Saturn system operating in the EI
mode at 70 eV equipped with a CP-SIL cross-linked capillary col-
umn (30 m0.25 mm). The identity of the oil components was
established from their GC retention times, by comparison of their
MS with those reported by Adams [6], by computer matching
with the Wiley 5 mass spectra library [7], and by co-injection
with standards available in our laboratories whenever possible.
Chiral monoterpene constituents (
a
-pinene,
b
-pinene, and limo-
nene) and the sesquiterpene nerolidol of Z. rhoifolium oils were
identified by peak enrichment on enantioselective capillary GC
with two fused capillary columns, 25 m 0.25 mm, coated with
heptakis-(6-O-methyl-2,3-di-O-pentyl)-
b
-cyclodextrin and octa-
kis-(3-O-methyl-2,6-di-O-butyryl)-
g
-cyclodextrin, each diluted
with polysiloxane OV-1701. A Varian-3800 apparatus equipped
with a flame ionization detector (FID) was used with hydrogen
as the carrier gas. All runs were performed with the temperature
program 358C for 15 min, and from 358C to1808Cat38C/min.
Part of the resulting oils of mature (50 mg) and young leaves (50
mg) and fruit (100 mg) were further subjected to preparative TLC
(SiO2; hexane-EtOAc, 90:10) and afforded hyemalol (1, 7 mg),
nerolidol (2, 20 mg), and cadinol (3, 10 mg), respectively. The au-
thenticity of nerolidol and cadinol was determined by direct
comparison with authentic samples.
Hyemalol (1): Oil; [
a
]D:±106(c0.0033, CHCl3); EI-MS: m/z = 222
[M]+, 205, 180, 149, 135, 109, 95; HR-ESI-MS: m/z =[M +
H]+223.2060 (calcd. for: C15H27O + H: 223.2062); 1H-NMR
(CDCl3,400.1 MHz):
d
= 4.86 (1H, m, H-4), 4.85 (1H, m, H-8),
3.40 (1H, m, H-1), 2.11 ± 2.25 (2H, m, H-5), 2.06± 2.14 (2H, m, H-
6), 2.05 ±2.13 (2H, m, H-2), 1.91± 1.99 (2H, m, H-9), 1.06± 1.96
(2H, m, H-11), 1.63 (3H, s, H-12), 1.46 (3H, s, H-13), 1.07 (3H, s,
H-14), 0.90 (3H, s, H-15); 13C-NMR (CDCl3, 100.6 MHz):
d
= 133.6 (C-7), 132.1 (C-3), 126.8 (C-4), 124.6 (C-8), 70.1 (C-1),
49.6 (C-2), 47.0 (C-11), 39.3 (C-6), 39.1 (C-9), 33.5 (C10), 31.2 (C-
15), 27.3 (C-14), 25.5 (C-5).
Determination of absolute configuration of 1 by Horeau's
method
Approximately 6
m
mol of racemic
a
-phenylbutyric anhydride
and 30
m
L of dry pyridine were added to approx. 5
m
mol of 1and
kept at room temperature for 1 h. After standing with 10
m
Lof
water for 30 min, usual work-up gave a mixture of the enantio-
mers of 2-phenylbutyric acid. A solution of diazomethane in di-
ethyl ether (50
m
L) was added until a permanent yellow color
was attained. The solution was reduced to approximately half of
its volume under an N2stream and used for gas chromatography.
Applying Horeau's modified rule for chiral GC [8], the excess en-
antiomer (±)-(R) observed indicated the (R) configuration for C-1
in compound 1.
Antimicrobial bioassay
The antibacterial activity of samples A ± D was assayed using the
minimal inhibitory concentration (MIC) determination. A collec-
tion of nine microorganisms was used, including three Gram-posi-
tive bacteria: Staphylococcus aureus (ATCC 6538p), Staphylococcus
epidermidis (ATCC 12228), Bacillus subitilis (ATCC 6633); and four
Gram-negative bacteria: Klebsiella pneumoniae (ATCC 10 031),
Eschericchia. coli (ATCC 11103), Pseudomonas aeruginosa (ATCC
27873), Salmonella setubal (ATCC 19196); and two yeasts:
Candida albicans (ATCC 10231), and Saccharomyces cerevisae
(ATCC 2601). Standard microorganism strains were obtained
from American Type Culture Collection (ATCC), and standard anti-
biotics chloramphenicol and nistatine were used in order to con-
trol microbial test sensitivity [9]. The minimal inhibitory concen-
tration (MIC) was determined in 96-well culture plates by a micro-
dilution method using a microorganism suspension with a density
of 105CFU/mL in casein soy broth (CSB) incubated for 24h at 378C
for bacteria, and Sabouraud broth (SB) incubated for 72 hours at
258C for yeasts. The cultures that did not grow were used to inocu-
late solid medium plates (Muller Hinton agar and Sabouraud agar)
in order to determine the minimal lethal concentration (MLC).
Proper blanks were assayed simultaneously, and samples were
tested intriplicate. Technical data have been described previously
[10], [11], [12].
Bioautographic bioassay
The antimicrobial activity of isolated compounds 1,2, and 3was
assayed using the bioautography technique [13], [14], [15] and
the collection of microorganisms described above. Compound
quantities ranging from 50
m
g to 1.56
m
g were applied to pre-
coated TLC plates. Muller Hinton agar and Sabouraud agar media
inoculated with mocroorganisms suspended in saline solution
(105CFU/mL) were distributed over TLC plates. Bacteria and
yeast plates were incubated for 24 h at 37 8C, and 72 h at 25 8C,
respectively. Standard antibiotics, chloramphenicol and nista-
tine, were used in order to control microbial test sensitivity [9].
The results were stained with an aqueous solution of 2,3,5-tri-
phenyltetrazolium chloride (TTC, 1 mg/mL). The appearance of
Simionatto E et al. Ess ential Oil from¼ Planta Med 2005; 71: 759 ± 763
Original Paper
760
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inhibition zones was used to determine the lowest sample
amount capable of inhibiting microbial growth. Samples were
tested in triplicate.
Results and Discussion
As shown in Table 1, the qualitative and quantitative oil composi-
tions displayed significant differences. Young (sample A) and
mature leaf (sample B), and flower (sample D) oils were rich in
sesquiterpenes, mainly nerolidol in sample A, and an unknown
sesquiterpene, named hyemalol (1), in samples B and D. In the
fruit oil (sample C), monoterpenes
a
-pinene and
b
-pinene predo-
minated. Interestingly, an inversion of the quantities of
a
-pinene
isomers in flower, leaf, and fruit oils was observed. In flower and
leaf oils, the major isomer is (±)-
a
-pinene (85% and 71%, respec-
tively), while in fruit oil, the major isomer is (+)-
a
-pinene (71%).
Hyemalol (1) was obtained as a colorless oil. The molecular for-
mula of C15H16O for 1was established by means of HR-ESI-MS,
which gave a pseudomolecular ion peak at m/z = 223.2060 [M +
H]+, in combination with 13C-NMR. The 1H-NMR spectrum of 1
showed five pairs of methylene hydrogens at
d
= 2.25±2.11
(H2±5, m), 2.13± 2.05 (H2± 2, m), 2.14± 2.06 (H2±6, m), 1.99±
1.91 (H2± 9, m), and 1.96± 1.06 (H2±11, m), three methynic hy-
drogens, two of which represented olefinic hydrogens at
d
= 4.86 (H-4, m) and 4.85 (H-8, m), and one oxymethine hydro-
gen at
d
= 3.41 (H-1, m), as well as four methyl groups at
d
= 0.90 (H-14, s), 1.07, (H-15, s), 1.46 (H-13, s), and 1.63 (H-12,
s). The 13C-NMR spectrum of 1showed fifteen signals. DEPT
Table 1Percentual composition of Zanthoxylum hyemale essential oils
CompoundsaKIbKIcSamples Identification
ABCD
01 (+)-
a
-Pinene 939 1 016 0.84 ± 7.4 2.85 GC,MS, Co
02 (±)-
a
-Pinene 939 1 016 2.06 17.3 2.6 16.15 GC,MS, Co
03 (+)-
b
-Pinene 987 1 124 0.1 ± 0.16 0.3 GC,MS, Co
04 (±)-
b
-Pinene 987 1 124 5.5 9.12 24.9 10.2 GC,MS, Co
05 (+)-Limonene 1 025 1 210 ± 1.50 1.0 3.45 GC,MS, Co
06 (±)-Limonene 1 025 1 210 ± 1.18 0.89 0.9 GC,MS, Co
07 Menth-2-en-1-ol 1 140 1595 ± ± 0.46 ± GC,MS
08 trans-
b
-Terpineol 1 163 1 586 ± ± 1.15 ± GC, MS
09 neo-Isopulegol 1 168 ± ± ± 0.45 ± GC, MS
10
a
-Terpineol 1 185 1 609 ± ± 0.67 ± GC,MS, Co
11 3-Decanone 1 190 1 580 ± ± 0.35 ± GC,MS
12 Carvacrol ethyl ether 1 204 1 572 ± ± 0,36 ± GC,MS, Co
13 2-Undecanone 1 289 1 623 ± 5.60 2.14 2.90 GC,MS, Co
14
d
-Elemene 1 334 1 483 0.45 0.1 0.24 0.20 GC,MS
15
b
-Elemene 1414 1 595 2.69 0.74 1.84 1.0 GC,MS
16 E-
b
-Caryophyllene 1449 1 586 1.05 2.0 1.58 2.36 GC,MS
17
a
-Acoradiene 1463 ± 1.04 ± 0.39 ± GC,MS
18
d
-Himachalene 1476 1 657 0.13 0.1 2.44 ± GC,MS
19 Humulene 1 480 1 674 9.68 3.63 2.27 3.6 GC,MS
20 Germacrene-D 1 493 1 706 4.74 5.24 3.08 5.8 GC,MS, Co
21
b
-Bisabolene 1 509 1 724 3.55 0.27 0.58 ± GC,MS
22
d
-Cadinene 1513 1 767 0.64 0.41 0.27 ± GC,MS
23 Bicyclogermagrene 1 520 1 718 1.55 0.22 1.08 6.60 GC,MS
24 Elemol 1 547 ± 0.14 0.1 0.37 ± GC,MS
25 Cadinene 1 551 1 744 0.17 0.34 0.33 2.61 GC,MS
26
b
-Calacoralene 1563 1 912 ± ± 0.94 ± GC,MS
27 (±)-E-Nerolidol 1566 2 087 51.5 ± 1.44 0.28 GC,MS, Co
28 Caryophyllene alcohol 1 571 1 954 ± 1.28 3.39 ± GC,MS
29 Caryophyllene oxide 1 580 1 986 ± 0.47 0.65 ± GC,MS, Co
30 epi-
a
-Muurolol 1 640 2 187 0.93 2.90 4.5 0.7 GC,MS
31 epi-
a
-Cadinol 1 642 2 185 0.74 0.85 1.23 2.2 GC,MS
32 Cadinol 1 653 2 145 2.31 5.72 9.58 4.5 GC,MS, NMR
33 Hyemalol 1664 2 205 0.66 31.8 7.0 22.31 GC,MS, NMR
34 epi-
a
-Bisabolol 1 687 2 235 0.24 0.32 1.47 0.17 GC,MS
Total 90.71 91.19 87.33 89.08
aCompounds listed in order of elution from a SE-54 column.
bRI:Kovats Indices determined on an apolar SE-54 column.
cRI:Kovats Indices determined on a polar PEG-20M column.
Co: peak identifications are based on standard comparison with relative retention time.
Simionatto E et al. Ess ential Oil fr om ¼ Planta Med 2005; 71: 759 ± 763
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135o, HMQC, and HMBC experiments showed the presence of five
methylenes at
d
= 25.1 (C-5), 39.1 (C-9), 39.3 (C-6), 47.0 (C-11),
and 49.6 (C-2), three methines at
d
= 70.1 (C-1), 124.6 (C-7),
and 126.8 (C-4), four methyls at
d
= 15.8 (C-13), 18.3 (C-12),
27.7 (C-14), and 31.2 (C-15), and three non-hydrogenated car-
bons at
d
= 47.0, 132.1, and 133.6. After all hydrogen resonances
had been assigned to those of their directly bonded carbon atoms
via HMQC measurements and after analysis of 1H-1H COSY and
HMBC experiments, it was possible to deduce four molecular
fragments in 1(a,b,c, and d). Thus, from the COSY spectrum of
1, three spin systems could be deduced. The first spin system
shows coupling between H-1 and H2±2, and between H-1 and
H2±11 (fragment a). Furthermore, couplings were observed be-
tween H-4 and H2± 5, which, in turn, coupled with H2±6, leading
to the second spin system (fragment b). The third spin system
shows connectivity between H-8 and H-9 (fragment c). Further-
more, HMBC cross-peaks observed between the two geminal
methyl hydrogens and the two methylene carbons, C-9 and C-
11, define the position of C-10, allowing fragment dto be joined
to fragments aand c. The partial fragments a,b,c, and dwere
then combined with the aid of the HMBC spectrum to give the
structure of 1, as shown in Fig.1. According to the above data,
the structure of 1was elucidated as 3,7,10,10-tetramethylcy-
cloundeca-3,7-dien-1-ol, a new natural sesquiterpene derived
from a humulane-skeleton. In addition, the absolute configu-
ration of the secondary alcohol function (C-1) was determined
as (R), based on Horeau's method.
The antimicrobial activity of the oils was evaluated by determin-
ing the minimal inhibitory concentration (MIC). The results are
given in Table 2. The oils were active against all microorganisms
tested, while the best MIC value observed was 0.67 mg/mL for
sample C (flower essential oil) against K. pneumoniae. Because
of the low quantity and the low solubility of the isolated com-
pounds hyemalol (1), nerolidol (2), and cadinol (3), they were
evaluated against the same strains by the bioautography method
in a TLC bioassay. (±)-trans-Nerolidol exhibited antibacterial ac-
tivity against E. coli (1.56
m
g), while cadinol was active against K.
pneumoniae (3.12
m
g) and S. cerevisiae (6.25
m
g). Hyemalol (1)
only showed weak activity against the tested microrganisms.
Chloramphenicol for bacteria (0.4± 0.7
m
g) and nistatine for
yeasts (2.0±2.4
m
g) were used as positive control.
Acknowledgements
This work was supported by FAPERGS (FundacË o de Amparo à
Pesquisa do estado do Rio Grande do Sul), CAPES (FundacË o Co-
ordenacË o de AperfeicË oamento de pessoal de Nível Superior),
and CNPq (Conselho Nacional de Desenvolvimento Cientíifco e
Tecnológico).
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¨
) and HMBC (-
®
1Hto13C) correla-
tions (right).
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