Composition and antimicrobial activity of Anemopsis californica leaf oil

Article abstract of DOI:10.1021/jf0511244

Isolation and characterization of leaf volatiles in Anemopsis californica (Nutt.) Hook. and Am. (A. californica) was performed using steam distillation, solid-phase microextraction, and supercritical fluid extraction. Thirty-eight compounds were detected and identified by gas chromatography; elemicin was the major component of the leaf volatiles. While the composition of the leaf volatiles varied with method of extraction, α-pinene, sabinene, β-phellandrene, 1,8-cineole, piperitone, methyl eugenol, (E)-caryophyllene, and elemicin were usually present in readily detectable amounts. Greenhouse-reared clones of a wild population of A. californica had an identical leaf volatile composition with the parent plants. Steam-distilled oil had antimicrobial properties against 3 (Staphylococcus aureus, Streptococcus pneumoniae, and Geotrichim candidum) of 11 microbial species tested. Some of this bioactivity could be accounted for by the α-pinene in the oil.

Full text of DOI:10.1021/jf0511244

8694 J. Agric. Food Chem. 2005, 53, 8694−8698
Composition and Antimicrobial Activity of Anemopsis
californica Leaf Oil
ANDREA L. MEDINA,† MARY E. LUCERO,§ F. OMAR HOLGUIN,† RICK E. ESTELL,§
JEFF J. POSAKONY,‡ JULIAN SIMON,‡ AND MARY A. O’CONNELL*,†
Department of Agronomy and Horticulture, MSC 3Q, and USDA-ARS Jornada Experimental Range,
MSC 3JER, New Mexico State University, Las Cruces, New Mexico 88003-8003, and Clinical
Research Division, Fred Hutchinson Cancer Research Center, P. O. Box 19024, D2-100 Seattle,
Washington 98109-1024
Isolation and characterization of leaf volatiles in Anemopsis californica (Nutt.) Hook. and Arn. (A.
californica) was performed using steam distillation, solid-phase microextraction, and supercritical fluid
extraction. Thirty-eight compounds were detected and identified by gas chromatography; elemicin
was the major component of the leaf volatiles. While the composition of the leaf volatiles varied with
method of extraction, R-pinene, sabinene, â-phellandrene, 1,8-cineole, piperitone, methyl eugenol,
(E)-caryophyllene, and elemicin were usually present in readily detectable amounts. Greenhouse-
reared clones of a wild population of A. californica had an identical leaf volatile composition with the
parent plants. Steam-distilled oil had antimicrobial properties against 3 (Staphylococcus aureus,
Streptococcus pneumoniae, and Geotrichim candidum) of 11 microbial species tested. Some of this
bioactivity could be accounted for by the R-pinene in the oil.
KEYWORDS: Anemopsis californica, Yerba mansa, steam distillation, SPME, SFE, antimicrobial, elemicin,
methyl eugenol
INTRODUCTION
diuretic, and to provide pain relief. Salves and poultices are
used to prevent infection of burns and reduce swelling of bruises
and are included in sitz baths and douches (2, 3). Efforts are
underway to develop genetic selections of A. californica and
to design management plans for this plant as a high-value crop
for small farms in the state (5). We needed to establish a reliable
and rapid method of extraction for this plant to compare the
essential oil composition in A. californica grown in a variety
of locations and under different environmental conditions. Two
rapid methods of extraction, solid-phase microextraction (SPME)
and super critical fluid extraction (SFE), have been used with
varying results to characterize the volatile or essential oil
composition of plant organs (6-8). These methods were
compared with classic steam distillation to determine if the
essential oil composition of A. californica leaves could be
accurately described using either SPME or SFE. Additionally,
we investigated the antimicrobial activity of A. californica leaf
oil using bioassays against 11 microorganisms. To the best of
our knowledge, this is the first publication describing the
essential oil composition of A. californica leaves.
Anemopsis is one of five genera belonging to the Saururaceae
family. The genus contains a single species, A. californica
(Nutt.) Hook. and Arn. ()Houttuynia californica Benth. and
Hook.), commonly known as yerba mansa. Native to the
southwestern United States and northern Mexico, A. californica
leaf and root preparations have been used medicinally to treat
pain, inflammation, and infection (1-3). Sanvordeker and
Chaubal (4) previously identified 12 volatiles isolated from the
roots and rhizomes of A. californica collected in California. The
most abundant compound in their extracts was methyl eugenol
(57%). Other components detected in the root oil at greater than
1% (v/v) amounts were as follows: thymol, piperitone, thymol
methyl ether, R-pinene, 1,8-cineole, methylchavicol, and p-
cymene; leaf volatile oils were not examined.
A. californica is collected in New Mexico and used by local
healers to treat a variety of ailments. Fresh or dry leaf or root
samples are prepared as tinctures, decoctions, and teas for
internal use; wilted leaves or root powder are used externally
(2). Teas prepared from this plant are used to treat coughs,
nausea, kidney problems, and menstrual cramps, to act as a
EXPERIMENTAL PROCEDURES
* To whom correspondence should be addressed. Telephone: (505) 646-
5172. Fax: (505) 646-6041. E-mail: moconnel@nmsu.edu.
† Department of Agronomy and Horticulture, New Mexico State
University.
Plant Cultivation. A. californica from Dona Ana County, NM
(elevation, 1182 m) was harvested and propagated by root division in
August 1999. A voucher specimen was placed in the Range Science
Herbarium at New Mexico State University in Las Cruces, NM
(Collection number: Medina 6). Plants were greenhouse-cultivated in
§ USDA-ARS Jornada Experimental Range, New Mexico State Univer-
sity.
‡ Fred Hutchinson Cancer Research Center.
10.1021/jf0511244 CCC: $30.25
© 2005 American Chemical Society
Published on Web 10/07/2005

Anemopsis Essential Oil
J. Agric. Food Chem., Vol. 53, No. 22, 2005 8695
Metro Mix 360 (Greenhouse & Garden Supply Inc., Albuquerque, NM),
fertilized with Osmocote 14-14-14, and watered daily using drip
irrigation. All analyses were conducted on ground plant material using
fully expanded leaves collected as indicated from either greenhouse-
grown plants or plants from the Dona Ana county collection site; leaf
material from three to five plants were pooled in a composite sample.
For SPME and steam distillation, the leaf material was frozen before
grinding; for SFE, the leaf material was dried at room temperature
before grinding in a mortar and pestle.
Extraction Methods. Steam distillations were carried out in a
Likens-Nickerson apparatus as previously described (9). Briefly, 20 g
of ground leaf tissue was placed in a round-bottom flask with ∼100
mL of distilled water; 12-15 mL of pentane was added to the U-tube
of the Likens-Nickerson apparatus. A water bath under the pear-shaped
flask was heated to boil the pentane, 60-70 °C; a heating mantle under
the round-bottom flask was used to boil/reflux for 4-5 h. Pentane
fractions were frozen to remove residual water rather than drying over
anhydrous magnesium sulfate and filtering. This minimized the loss
of oil and shortened the process. Duplicate steam distillations using
20 g of ground tissue were carried out as previously described (9),
using pentane and water for 4-5 h.
SPME analyses were prepared by placing 0.5 g of ground tissue
into 4 mL screw-top vials sealed with poly(tetrafluoroethylene) (PTFE)/
silicon septa (Supelco). The vials were equilibrated at 30 °C for 2 h
and then exposed to a 100 µm PDMS fiber (Supelco), 1 cm deep into
the vial for 10 min. The fiber was immediately injected into the
appropriate gas chromatograph inlet to a depth of 3 cm. The fiber
remained in the injector for 5 min to remove residual volatiles, and
blank runs were performed after each sample.
For SFE extraction, 0.5 g of ground leaf tissue was loaded in thimbles
for extraction in ISCO SFX3560. The thimble was pressurized with
CO2 to a density of 0.72 g/mL (5150 psi, 100 °C), for 1 min (static
extraction setting on instrument), and then the solubilized compounds
were flushed from the thimble with 7 min dynamic extraction at a flow
rate of 2.0 mL/min. The extraction is vented into a tube with 10 mL of
methanol to trap the essential oil components as the CO2 is bubbled
off. The essential oil components in the methanol were quantified and
identified by GC/MS (gas chromatography/mass spectrometry) or GC/
FID (gas chromatography/flame ionization detection).
Gas Chromatography/Mass Spectrometry. Extracts were analyzed
by GC/MS using a Varian model 3400 GC with a DB-5 column (30 m
× 0.25 mm fused silica capillary, 0.25 µm film thickness), coupled to
a Finnigan ion trap mass spectrometer (EI, 70 eV) or a Shimadzu
GC8APF equipped with a flame ionization detector and a split/splitless
injector. Helium carrier gas flowed at 1 mL/min, and injector and
transfer line temperatures were 220 and 260 °C, respectively. The initial
column temperature was 60 °C, with a linear gradient of 3 °C/min
programmed into each 65 min run. Comparisons of mass spectra and
Kova´ts retention indices (10) with literature data (11) or authentic
standards were used to identify the peaks. Reference standards were
obtained from Sigma-Aldrich, St. Louis, MO (borneol, camphene,
camphor, (E)-caryophyllene, caryophyllene oxide, 1,8-cineole, p-
cymene, linalool, methyl eugenol, myrcene, R-phellandrene, R-pinene,
â-pinene, R-terpineol, terpinolene, thymol, tricyclene) and from Pfaltz
& Bauer, Waterbury, CT (piperitone, sabinene). Elemicin was synthe-
sized as described below. Two methods of quantitation of the essential
oil components were employed, percent peak area or mass of analyte
per gram dry weight of leaf. Percent peak areas were used for
quantitation when we did not have calibration curves for all of the
analytes of interest. Percent peak areas were calculated by dividing
the ion counts for a particular peak (detected by FID or MS) by the
total ion counts for the entire chromatogram and expressing this value
as a percent. Calibration curves with authentic standards were used to
quantify the abundance of R-pinene, 1,8-cineole, thymol, methyl
eugenol, piperitone, and elemicin. In this case the peak area detected
by FID or MS for each analyte shot at six or seven different
concentrations between 0 and 1000 mg/L were plotted. The regression
of this line was used to interpolate the abundance of individual essential
oil components from the total ion count measured in their respective
peaks. Dry matter percent of greenhouse tissue was determined using
AOAC procedures (12).
Figure 1. Synthesis of elemicin from 4-allyl-2,6-dimethoxyphenol.
Bioassays. Antimicrobial bioassays were conducted using the
following organisms: Candida keyfr ATCC 44691, Geotrichim can-
didum (G. candidum) ATCC 48112, Streptococcus pneumoniae (Strep.
pneumoniae) ATCC 6303, Staphylococcus aureus (Staph. aureus)
ATCC 27661, Enterobacter aerogenes (E. aerogenes) ATCC 13048,
Enterobacter clocae (E. clocae) ATCC 13047, Shigella flexneri (Shig.
flexneri) ATCC 29903, Klebsiella pneumoniae (K. pneumoniae) ATCC
132, Salmonella typhimurium (Sal. typhimurium) ATCC 14028, Chro-
mobacterium Violaceum (C. Violaceum) ATCC 12472, and Neissera
subflaVa (N. subflaVa) ATCC 14799. Triplicate serial dilutions of A.
californica leaf essential oil in nutrient broth ranging from 0.001 to
0.1% (v/v) were prepared in a 96-well microtiter plate. Controls included
nutrient broth, uninoculated serial dilutions of oil, and nutrient broth
inoculated with microorganisms minus essential oil. Cultures were
incubated at 37 °C in a BioTek PowerwaveX Select-I spectrometer
and agitated for 55 min before each reading. Optical density (600 nm)
was recorded hourly for 48 h. Assays with R-pinene, elemicin, methyl
eugenol, piperitone, 1,8-cineole, and sabinene were performed as above,
at concentrations from 1 × 10-5 to 0.01% (v/v).
Synthesis of Elemicin. 4-Allyl-2,6-dimethoxyphenol (technical
grade, 90%; 4.12 mL, 23.2 mmol, Sigma-Aldrich) and K2CO3 (9.61 g)
were combined in acetone (125 mL). Iodomethane (4.3 mL, 46.4 mmol,
Sigma-Aldrich) was then added, and the mixture was refluxed for 6 h
(Figure 1), at which time GC/MS analysis of an aliquot indicated the
reaction was complete. Then, the K2CO3 was filtered off and was rinsed
with EtOAc (100 mL). The solvent was removed, and the mixture was
loaded on a plug of silica (Merck, grade 9385, 230-400 mesh) which
was then eluted with 1:1 EtOAc:hexane (150 mL total). The solvent
was removed from the eluate, and the residue was distilled under
vacuum (20 mmHg). Distillate fractions were analyzed by GC/MS for
purity, and those with g90% purity, which were collected between
145 and 157 °C, were combined. The proton NMR spectrum of elemicin
was also obtained (Bruker Avance 500 MHz).
RESULTS AND DISCUSSION
Synthesis of Elemicin. Elemicin is an abundant component
of the essential oils of A. californica and Myristica fragrans
(M. fragrans) but not commercially available. Therefore, to
generate standard curves of this compound, it was necessary to
synthesize elemicin (Figure 1). The synthesis of elemicin from
3.75 g of 4-allyl-2,6-dimethoxyphenol (a commercially available
standard) yielded 2.3 g (11 mmol, 47%) of pure (>93%)
elemicin. Sample purity was determined by spectrometry (GC/
MS). The NMR spectrum of the elemicin product confirmed
1
the identity predicted by GC/MS data. H NMR (acetone-d6):
δ 3.32 (d of multiplets, J ) 4 Hz, 2H), 3.67 (s, 3H), 3.79 (s,
6H), 5.01 (d of multiplets, J ) 9 Hz, 1H), 5.09 (d of multiplets,
J ) 17 Hz), 5.9-6.0 (m, 1H), 6.50 (s, 2H).
Essential Oil and SPME Analysis. Distilled oil was prepared
from two independent samples, and SPME was performed on
two independent samples. All of the values for recovery and
the abundance of essential oil components in these two extracts
are expressed as the range of the value obtained for each of the
duplicate samples, or as the midpoint between these values.
Dry matter accounted for 43.0% of the leaf fresh weight, and
the leaf essential oil obtained by steam distillation accounted
for 0.67-0.97% of the leaf dry matter. Table 1 lists 38
compounds detected in either the steam distillate or the SPME
sample of leaf tissue. Many of these compounds (21/38, 55%)

8696 J. Agric. Food Chem., Vol. 53, No. 22, 2005
Medina et al.
distillate, compounds comprising more than 5% of the FID peak
areas were elemicin (53.1%), piperitone (11.1%), and methyl
eugenol (6.9%). In SPME, a remarkably lower abundance of
elemicin was observed; piperitone (16.2%), elemicin (13.2%),
R-pinene (11.7%), 1,8-cineole (10.1%), â-phellandrene (8.7%),
(E)-caryophyllene (8.3%), methyl eugenol (5.1%), and sabinene
(5.1%) exceeded 5% of the total peak area on the FID
chromatograms.
Table 1. Volatile Compounds Identified in A. californica Leaves:
Comparison of Peak Area Percentages Detected for Steam-Distilled
Oil and SPME Samples on the GC/FID
% peak area (n
oil SPME
1.1
NDd
trace
0.0 0.3
10.3 13.1
) 2)
compounda
stdb
KIc
1
2
3
4
5
6
7
8
2-(E)-hexenal
2,4-(E,E)-hexadienal
tricyclene
-pinene
camphene
sabinene
−
−
+
+
+
+
+
+
+
+
−
+
−
−
−
+
+
−
+
+
+
−
−
+
+
−
−
−
+
+
−
−
+
−
−
−
+
+
853
909
928
940
955
977
981
992
0.3−
ND
ND
−
−
Yields for elemicin were remarkably high in the steam-
distilled oil, and notably different from the SPME headspace
composition. The initial SPME elemicin recovery with an
equilibration temperature of 30 °C was much lower (13.2%)
than the elemicin content in the oil (53.1%). To determine the
role of extraction temperature in elemicin recovery, additional
vials of leaf tissue were equilibrated at 45, 55, and 65 °C prior
to SPME extraction. The resulting chromatograms revealed
elemicin peak area percents of 22, 34, and 49%, respectively.
Apparently, the low recovery of elemicin by SPME is due to
its low volatility at 30 °C.
R
0.4
0.0
0.0
0.3
0.1
0.2
0.0
0.9
1.5
0.2
1.1
−
3.4
0.1
0.1
1.5
0.3
0.5
0.2
2.3
3.5
0.3
2.1
−
−
−
−
−
−
−
−
−
−
0.5
4.4
0.0
0.8
0.2
1.5
8.6
−
0.5
5.7
0.3
1.0
0.2
2.2
8.7
−
−
−
−
−
−
â
-pinene
myrcene
-phellandrene
p-cymene
-phellandrene
1,8-cineole
(Z)- -ocimene
(E)- -ocimene
9
R
1006
1027
1032
1035
1041
1052
1069
1089
1099
1143
1145
1168
1190
1196
1236
1254
1292
1339
1377
1392
1403
1420
1437
1440
1454
1458
1485
1488
1552
1582
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
32
34
35
36
37
38
â
10.0−10.1
â
â
0.6−
0.6
2.3
2.0−
cis-sabinene hydrate
terpinolene
linalool
cis-pinene hydrate
camphor
borneol
ND
ND
trace
trace
0.8
ND
ND
−0.8
0.9−1.0
trace
trace
The A. californica root oil described by Sanvordeker and
Chaubal (4) had three components present at >5%: methyl
eugenol (57.0%), thymol (13.8%), and piperitone (8.0%). The
leaf oil we describe has similar levels of piperitone, but much
lower levels of thymol and methyl eugenol. Curiously, elemicin,
which was not detected by Sanvordeker and Chaubal (4) in roots,
comprised 53% of the leaf oil. Elemicin differs structurally from
methyl eugenol by a single methoxy group. The compositional
differences observed between studies are not surprising con-
sidering differences in tissue (root versus leaf, genetic variations,
growth environments, and phenology). Studies examining roots
of A. californica growing in New Mexico are underway.
0.3
0.4
0.7
−
−
−
0.3
0.5
0.8
0.3−
0.3−
1.2−
0.3
0.3
1.3
R
-terpineol
methylchavicol
thymol methyl ether
piperitone
ND
10.4
Trace
16.0
trace
0.1 0.1
trace
−
0.2
0.1
11.7
−
16.4
thymol
0.1−
δ
-elemene
-copaene
-elemene
methyl eugenol
(E)-caryophyllene
0.0−
−
R
ND
â
0.1
6.5
4.2
−
−
−
0.3
7.3
5.0
0.1
5.0
8.2
0.1
1.7
1.4
0.3
−
−
−
−
−
−
−
0.1
5.2
8.3
0.1
1.8
1.4
0.3
trans-
-guaiene
R
-bergamotene
ND
R
1.0
0.8
0.2
0.0
1.2
52.5
ND
−
1.3
1.1
0.5
0.1
1.5
SPME is especially useful for identifying compounds found
at the trace level in essential oil (Table 1). Ten compounds are
listed in Table 1 as not detected in the oil-GC/FID profiles that
were detected at trace or low levels in the SPME-GC/FID
profiles. Variability between samples was lower in SPME
extractions than in the steam-distilled oils. Examples of lower
reproducibility in the oil extractions as compared with SPME
can be seen in Table 1 for R-pinene, â-pinene, â-phellandrene,
1,8-cineole, piperitone, and methyl eugenol. The range of
extracted compounds, small tissue requirements, and the sim-
plicity of the technique make SPME an attractive method for
qualitative analysis of plant volatiles.
R
-humulene
−
−
−
−
(E)-
â
-farnesene
germacrene D
cis- -guaiene
trace
1.2 1.3
13.1 13.3
trace
â
−
elemicin
caryophyllene oxide
−
53.7
−
a Compounds listed in order of elution from GC. b Std, compounds identified
by comparison with reference standard. c KI, Kova´ts index. d ND, not detected.
SFE Analysis. While the SPME samples were useful for the
characterization of the leaf volatiles, there was no extract
generated by this method to be used in tests for biological
activity. In an effort to develop both an efficient method for
chemical analysis as well as a source of material for bioassay,
leaves from both greenhouse and wild plants were extracted
using a supercritical fluid extractor. In this method we performed
these extractions on three independent leaf samples.
Table 2 reports the abundances of leaf volatiles as a percent
of total peak area for each of the three methods of extraction.
A single value is reported for each volatile for each method. In
the case of the oil and SPME samples, the value represents the
midpoint of the two samples; for SFE it is the average of three
samples. Only those compounds detected at 5% or greater in
samples generated by any of the three extraction methods are
listed in this table. All but one of these compounds were
identified by comparison with authentic reference standards. The
presence of â-phellandrene is tentative, on the basis of the KI
and mass spectra match of this peak to values for â-phelland-
rene.
Figure 2. Selected components in essential oil from leaves of A.
californica.
were identified on the basis of comparison with reference
standards; the remaining 45% were tentatively identified on the
basis of KI and mass spectra matches. Reference compounds
were not readily available. The structures of several of the major
components are shown in Figure 2. There were only four peaks
that we were not able to identify; these compounds represented
<1% of the total peak area. In the oil prepared by steam

Anemopsis Essential Oil
J. Agric. Food Chem., Vol. 53, No. 22, 2005 8697
Table 2. Comparison of Abundances of Selected Essential Oil
Components Detected by the Three Different Extraction Protocols on
Leaf Tissues of A. Californica Cultured in the Greenhousea
avg % peak area
compoundb
-pinene
sabinene
-phellandrene
1,8-cineole
piperitone
methyl eugenol
(E)-caryophyllene
elemicin
stdc
SPME (n
)
2)
oil (n
)
2)
SFE (n ) 3)
R
+
+
−
+
+
+
+
+
11.7
5.0
8.6
10.1
16.2
5.1
1.9
0.1
1.6
2.5
11.5
6.9
â
0.3
4.5
7.0
2.1
69.9
8.3
13.2
4.6
53.1
Figure 4. The effects of A. californica leaf essential oil and
R
-pinene on
, media and bacteria; , 0.1%
-pinene.
a Only those compounds detected at
g5% by one or more extraction methods
Staph. aureus growing in nutrient broth:
(v/v) leaf essential oil; , 0.005% (v/v)
]
2
are listed. b Compounds listed in order of elution from GC. c Std, compounds
identified by comparison with reference standard.
9
R
-pinene; b, 0.01% (v/v) R
20 g of tissue and 6-8 h for preparation and extraction. SFE
requires 0.5 g of tissue and 1.5 h for preparation and extraction.
We conclude that SFE is an effective method for the extraction
of specific compounds in A. californica tissue, requiring minimal
time and tissue for preparation and extraction and yielding
extracted fractions that can be assayed for bioactivity.
Bioassays. All inhibition observed in bioassays was apparent
in the first 8 h. Growth of microorganisms as affected by
essential oil or pure compounds was determined relative to
growth of control microorganisms. For the purposes of this
study, inhibition is defined as e50% of control growth, as
determined by optical density at 600 nm. Leaf oil bioassays
were performed against several microorganisms, including gram-
positive and gram-negative bacteria, yeast, and fungi. The
growth of Staph. aureus, Strep. pneumoniae, and G. candidum
was inhibited by A. californica leaf oil at 0.1%.
Figure 3. Comparison of greenhouse (open bars) and wild-grown (filled
bars) A. californica leaf SFE extracts. Triplicate SFE extractions were
characterized using GC/MS, and the abundances of the major essential
oil components, quantified. Error bars represent the standard deviation
To determine whether the most abundant compounds in the
oil were responsible for some of the antimicrobial effects
observed, bioassays against Staph. aureus were repeated using
single volatile oil compounds (R-pinene, sabinene, 1,8-cineole,
piperitone, methyl eugenol, and elemicin) in place of the leaf
oil. Pure R-pinene at 0.01% (v/v) resulted in a complete kill of
the bacteria (Figure 4). None of the other constituents inhibited
the growth of Staph. aureus (data not shown). It is probable
that R-pinene in the leaf essential oil was only partially
responsible for the inhibition of Staph. aureus. A. californica
leaf essential oil at 0.1% (v/v) contains 0.002% (v/v) R-pinene.
This concentration of leaf oil inhibited 73% of Staph. aureus
growth. However, R-pinene at 0.005% (v/v) did not inhibit
Staph. aureus growth by 70% (Figure 4). If R-pinene in the
leaf oil was solely responsible for the antimicrobial effect, we
would expect inhibition to occur at this concentration. Instead
this dose (0.005%) of R-pinene only inhibited the growth of
the bacteria by ∼10%. These results suggest that other
compounds in the leaf essential oil are responsible for either
direct inhibition of Staph. aureus or additive or synergistic
enhancement of the inhibitive effect of R-pinene. No single
microorganism has been specifically linked to any of the
ailments historically treated with A. californica. Yet, the
symptoms resulting from infection by the microorganisms tested
in this study (13-15) are similar to symptoms traditionally
treated with A. californica (1-3).
around the mean (n
) 3).
Of the three methods, the SFE extracted the fewest com-
pounds, and SPME, the most. The SFE recovered detectable
levels of all of the compounds present in the steam-distilled oil
at 5% or higher. There were differences in the abundance of
compounds recovered by each of the three extraction methods.
Elemicin represented the most abundant, if not dominant,
compound in the steam-distilled oil and the SFE sample. Only
trace levels of R-pinene and â-phellandrene were recovered in
SFE samples, yet these compounds were present at close to 2%
of the steam-distilled oil. The SPME and steam-distilled
preparations were performed on frozen leaves, while the SFE
was performed on air-dried leaves. This difference in pre-
treatment might explain the lower levels of recovery of
compounds with lower boiling points in the SFE sample, i.e.,
R-pinene, sabinene, and â-phellandrene. For bay leaves, the
method of preparation (drying, freezing, or fresh tissue) had a
significant impact on the recovery of specific volatile compo-
nents (7).
There was little variability in leaf volatile composition in
leaves from plants grown in the greenhouse and their clonal
parent grown on the Rio Grande riverbank in Dona Ana county
(Figure 3). The SFE method was reliable and produced low
variability within the triplicate samples of either the field-grown
or greenhouse-grown plant material. This extraction method is
amenable to experiments comparing leaf or root volatile
compositions within and between different populations or
different environmental conditions. Steam distillation requires
ACKNOWLEDGMENT
We thank Yuan-Feng Wang for assistance with GC analysis.

8698 J. Agric. Food Chem., Vol. 53, No. 22, 2005
Medina et al.
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Received for review May 16, 2005. Revised manuscript received August
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JF0511244