Qualitative and quantitative analysis of the active components of the essential oil from Brickellia veronicaefolia by nuclear magnetic resonance spectroscopy

Article abstract of DOI:10.1021/np060180b

The composition of the spasmolytic essential oil of the medicinal species Brickellia veronicaefolia was established by NMR spectroscopy in addition to GC-MS analysis and HPLC studies. Seven major compounds, representing ca. 86% of the oil, were identified as benzyl 2,6-dimethoxybenzoate (1), 2-hydroxybenzyl 2′-methoxybenzoate (2), chamazulene (3), β-caryophyllene (4), germacrene D (5), bicyclogermacrene (6), and β-eudesmol (7). A sensitive and accurate analytical ¹H NMR method has been developed for the quantification of the major compounds in the essential oil of B. veronicaefolia. The method was validated using benzyl 2,6-dimethoxybenzoate (1) and β-caryophyllene (4), two of the active principles in the oil, and successfully applied to the determination of these pharmacologically active compounds in three different batches of the oil collected in different geographical regions and/or seasons.

Full text of DOI:10.1021/np060180b

1
172
J. Nat. Prod. 2006, 69, 1172-1176
Qualitative and Quantitative Analysis of the Active Components of the Essential Oil from
Brickellia Weronicaefolia by Nuclear Magnetic Resonance Spectroscopy#
†
†
†
,§
,†
Blanca Rivero-Cruz, Isabel Rivero-Cruz, Juan M. Rodr ´ı guez, Carlos M. Cerda-Garc ´ı a-Rojas,* and Rachel Mata*
Departamento de Farmacia, Facultad de Qu ´ı mica, UniVersidad Nacional Aut o´ noma de M e´ xico, Ciudad UniVersitaria, M e´ xico D.F., 04510,
M e´ xico, and Departamento de Qu ´ı mica, Centro de InVestigaci o´ n y de Estudios AVanzados del Instituto Polit e´ cnico Nacional, M e´ xico D.F.,
0
7000 M e´ xico
ReceiVed April 24, 2006
The composition of the spasmolytic essential oil of the medicinal species Brickellia Veronicaefolia was established by
NMR spectroscopy in addition to GC-MS analysis and HPLC studies. Seven major compounds, representing ca. 86%
of the oil, were identified as benzyl 2,6-dimethoxybenzoate (1), 2-hydroxybenzyl 2′-methoxybenzoate (2), chamazulene
(
3), â-caryophyllene (4), germacrene D (5), bicyclogermacrene (6), and â-eudesmol (7). A sensitive and accurate analytical
1
H NMR method has been developed for the quantification of the major compounds in the essential oil of B. Veronicaefolia.
The method was validated using benzyl 2,6-dimethoxybenzoate (1) and â-caryophyllene (4), two of the active principles
in the oil, and successfully applied to the determination of these pharmacologically active compounds in three different
batches of the oil collected in different geographical regions and/or seasons.
Brickellia Veronicaefolia (Kunth) Gray (Asteraceae) is a bitter-
tasting shrub that grows in the oak and pine woodlands of Mexico
from Coahuila to Oaxaca. The commercial drug consists of the dried
aerial parts of the plant, and it is extensively commercialized in
Mexico for the treatment of gastrointestinal discomforts including
stomachache, biliary colic, dyspepsia, arthritis, local inflammations,
1
and infectious diseases. In addition, some herbal preparations
containing B. Veronicaefolia are widely used to cure gastritis.
Early phytochemical studies of the aerial parts of this species
resulted in the isolation and characterization of several 6-methoxy-
flavonols and labdane-type diterpenes.2 In addition, the anti-
oxidant and hypoglycemic activities of the extract and a flavonoid,
centauridin, isolated from the plant have been described.6 More
-5
,7
recently we demonstrated the antispasmodic properties of a CH
2
-
Cl -MeOH (1:1) extract prepared from the aerial parts of the plant.
2
Bioassay-guided fractionation of the active extract led to the
isolation of smooth muscle relaxant salicylic acid and benzyl
benzoate derivatives, including benzyl 2,6-dimethoxybenzoate (1)
and 2-hydroxybenzyl 2′-methoxybenzoate (2) (Figure 1), as well
8
as substantial amounts of chamazulene (3). The isolates were char-
acterized from physical data and induced a concentration-dependent
inhibition of the spontaneous contractions of the guinea-pig ileum
with IC50’s ranging from 1.49 to 4.96 µM.
Despite the continued popularity of B. Veronicaefolia, there are
no procedures for quality control and/or standardization of its crude
9
drug and herbal preparations. Therefore, the present study was
undertaken in order to develop a suitable analytical method to quan-
tify the most important active principles of the oil of this Mexican
species. The ultimate goal would be the establishment of quality
control procedures for the crude drug and herbal preparations of
this widely commercialized Mexican plant. Two different strategies
were initially envisaged: a classical GC procedure and a more
1
contemporary approach based on H NMR spectroscopy. The GC
method was considered because it is well-known that many phar-
macologically active components in herbal medicines are volatile.
GC analysis of the volatile oils has a number of advantages, in
Figure 1. Structures of the main components (1-7) present in
the essential oil of B. Veronicaefolia.
#
particular, its high sensitivity of detection and the fact that the
components can be readily identified using GC-MS analysis. In
addition, the extraction of the volatile oil is relatively straightforward
and can be standardized. Finally, this type of analysis renders a
reasonable “fingerprint”, which can be used to identify the plant.
On the other hand, NMR spectroscopy has proved to be useful
for quantification of individual components in crude extracts,
Taken in part from the Ph.D. thesis of B. Rivero submitted to Posgrado
en Ciencias Qu ´ı micas, UNAM, 2006.
*
Corresponding authors. Tel: (525)-55-622-5289 or (525)-55-061-3800.
Fax: (525)-55-622-5329 or (525)-55-747-7113. E-mail: rachel@
servidor.unam.mx or ccerda@cinvestav.mx.
1
0
†
Universidad Nacional Aut o´ noma de M e´ xico.
§
Centro de Investigaci o´ n y de Estudios Avanzados del Instituto Poli-
t e´ cnico Nacional.
1
0.1021/np060180b CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/27/2006

ActiVe Components of Brickellia Veronicaefolia
Journal of Natural Products, 2006, Vol. 69, No. 8 1173
Figure 2. 1H NMR spectra (300 MHz in CDCl
3
) of B. Veronicaefolia essential oil collected in three different locations. The δ 7.10-4.20
region shows key signals for quantitative analysis of 1, benzyl 2,6-dimethoxybenzoate; 2, 2-hydroxybenzyl 2′-methoxybenzoate; 3,
chamazulene; 4, â-caryophyllene; 5, germacrene D; 6, bicyclogermacrene; and 7, â-eudesmol. 1,4-Dimethoxybenzene was used as internal
standard (IS).
1
2
essential oils, or dietary preparations without the need of fraction-
ation or isolation procedures.11-13 This methodology is particularly
valuable when the studied compounds show at least one well-
resolved signal in the region from 3.0 to 10.0 ppm. Quantitative
NMR spectroscopy offers a comprehensive validation of reference
compounds of natural products that can compete with or even
surpass chromatographic validation based on molecular analysis.
1
In particular, H NMR spectroscopy allows the precise determina-
tion of the sample content as well as the amount and nature of
nonactive and/or marker compounds, providing useful fingerprints
for the identification of herbal drugs, which can be applied in the
certification and quality control of reference compounds. Further-

1
174 Journal of Natural Products, 2006, Vol. 69, No. 8
RiVero-Cruz et al.
more, due to the sophisticated design of contemporary NMR instru-
ments, calibrated and standardized analytical conditions are readily
accessed.
Results and Discussion
The active volatile oil of B. Veronicaefolia (batch II) was
qualitatively analyzed by GC-MS and comprised benzyl benzoates
and sesquiterpenoids. Seven major compounds, representing ca.
8
6% of the oil, were identified, i.e., benzyl 2,6-dimethoxybenzoate
8
14
15
(1), 2-hydroxybenzyl 2′-methoxybenzoate (2), chamazulene (3),
16
17
18
â-caryophyllene (4), germacrene D (5), bicyclogermacrene (6),
19
and â-eudesmol (7). HPLC separation of the oil yielded compound
, 2, 3, and 5. The isolates were characterized by comparison of
its physical data with literature data and those of authentic
1
samples.8
,15,17
The presence of compounds 1, 2, and 7 with known
spasmolytic properties seems to account for the antispasmodic
activity of the oil.8 The presence of 3 could be related to the
attributed anti-inflammatory properties of the plant.21
,20
The oil from three additional samples (batches I, IIa, and III),
collected in different geographic regions and seasons, was analyzed
using the same GC-MS methodology. In all batches the qualitative
composition remains constant and the dominant components were
4-6. However, the proportions of the analyzed constituents,
including the active substances and reference standards in combina-
tion with the essential oil, show variation, which precludes the
validation of the procedure with accuracy, linearity, and precision
according to international guidelines and pharmacopoeia require-
ments. All attempts probably failed because of variations in the
stability and volatility of the sesquiterpenes under the analysis
conditions, as observed in recent studies particularly for germacrene
D, bicyclogermacrene, and â-caryophyllene,22 which account for
Figure 3. Plot of the area of H-3/H-5 of benzyl 2,6-dimethoxy-
benzoate (a) and H-15 of â-caryophyllene (b) versus the amounts
of each compound.
60% of the oil components of B. Veronicaefolia. Therefore, in our
case, the classical GC-MS procedure was not adequate for a rigorous
quantitative analysis of the essential oil.
comparison with the area corresponding to the signal of the standard.
The accuracy of the method was checked by adding to the oil (50.0
mg) known amounts of 1 (0.05 to 0.5 mg) and 4 (1.3 to 13.0 mg).
The peak areas corresponding to each compound increased pro-
portionally to the added concentration of the added standards (1
and 4). Although calibration curves were not needed for quantifica-
tion of individual components because the integration of the peaks
is always proportional to the amount of compounds, the calibration
curves for 1 and 4 were determined in the range 0.05-0.5 and
Considering that NMR spectroscopy has also proved to be useful
for quantification of single compounds in crude extracts, essential
oils, or dietary preparations without the need of fractionation or
isolation procedures,1 we proceeded to develop a procedure to
1,12
1
estimate the amount of compounds 1-7 using H NMR. Further-
more, the method was validated, according to standard procedures,23
using compounds 1 and 4. Compounds 1 and 4 were chosen for
the validation process because of their accessibility as standards.
Thus, 1 was synthesized and 4 commercially purchased. Repeated
1.30-13.0 mg/mL in order to evaluate the linearity of this method.
Each calibration curve is shown in Figure 3. The linearity param-
1
H NMR analysis of the oils prepared from different batches
2
eters (r ) for 1 and 4 were higher than 0.997 and 0.998, respectively.
revealed that the NMR profiles stayed unchanged. The results of
the validation procedure indicated that the method was selective,
linear, accurate, and precise in the range of concentrations tested.
For optimal analysis conditions, the characteristic NMR signals
The repeatability of the method was demonstrated by determining
the levels of 1 and 4 in three batches whose NMR profile remained
unchanged during at least three consecutive days.
12
Once the procedure was validated, we analyzed the most widely
used OTC preparation of B. Veronicaefolia. The NMR and TLC
analyses of the essential oil prepared from this herbal mixture did
not contain the compounds identified and/or quantified in the crude
drug of B. Veronicaefolia (Table 1). The results of such analyses
are very important because the lack of appropriate regulations in
Mexico have resulted in many commercialized phytomedicines
commonly consumed by people being misidentified or containing
adulterants or less effective substitutes. The statistical analysis by
ANOVA followed by a Tukey’s test indicated that there is a
significant difference among the means of three different batches.
Therefore, the NMR quantification developed in this study can also
be applicable for seasonal and geographic variation studies of
B. Veronicaefolia.
of the individual components need to be identified. Therefore,
the assignment of all NMR signals for the seven components was
1
first carried out. As observed in Figure 2, the H NMR spectra of
the three analyzed oils clearly revealed characteristic and well-
defined key resonances for each component. In the low-field region
(
Figure 2), between δ
in 1 (δ 6.54, d, J ) 8.8 Hz) and the one ascribable to H-3′ (δ
.42, dd, J ) 8.4, 1.0 Hz) in 2 were used to quantify these two
components. For chamazulene (3), the key resonance appeared at
6.97, d, J ) 10.8 Hz (H-5). In the δ 5.90-4.30 region the
olefinic signals selected for the analysis of 4-7 resonated at δ
.95 (br s, H-15a), 5.78 (d, J ) 15.9 Hz, H-5), 4.33 (d, J ) 11.5
Hz, H-5), and 4.48 (m, H-14), respectively. The signal of the internal
standard (1,4-dimethoxybenzene) appeared at δ 6.84 as a singlet
H
7.10 and 6.20, the signals due to H-3/H-5
H
H
6
δ
H
H
H
4
H
and was not overlapped by the signals of the essential constituents.
This standard was selected because of its stability and solubility
In summary, quantitative NMR spectroscopy has proven to be
an excellent analytical tool for both qualitative and quantitative
analysis of the essential oil of B. Veronicaefolia, offering several
advantages over the classical chromatographic methods. In addition,
the developed method complied with the international requirements
for the quality control of herbal pharmaceutical products.
1
and because it displays only two sharp singlets in the H NMR
spectrum.12
1
The integration of the key resonances in the H NMR spectrum
allowed quantification of the different constituents of the oil by

ActiVe Components of Brickellia Veronicaefolia
Journal of Natural Products, 2006, Vol. 69, No. 8 1175
Table 1. Contents of Benzyl 2,6-Dimethoxybenzoate (1), 2-Hydroxybenzyl 2′-Methoxybenzoate (2), Chamazulene (3),
1
â-Caryophyllene (4), Germacrene D (5), Bicyclegermacrene (6), and â-Eudesmol (7) Quantified by H NMR Spectrum of the Essential
Oils (mg/g ( SD)
sample
1
2
3
4
5
6
7
batch I
0.47 ( 0.01
0.24 ( 0.01
0.28 ( 0.01
0.08 ( 0.01
3.51 ( 0.01
2.40 ( 0.05
2.19 ( 0.04
0.67 ( 0.01
0.85 ( 0.01
0.63 ( 0.04
0.52 ( 0.03
0.05 ( 0.02
3.27 ( 0.12
6.52 ( 0.03
1.91 ( 0.12
10.78 ( 0.12
7.63 ( 0.26
11.44 ( 0.21
3.31 ( 0.25
4.49 ( 0.16
4.42 ( 0.17
6.30 ( 0.3
2.86 ( 0.19
1.30 ( 0.06
2.06 ( 0.02
4.91 ( 0.20
1.29 ( 0.06
0.04 ( 0.01
batch II
batch IIa
batch III
Experimental Section
of CDCl
a 45 deg pulse (9.0 µs), a relaxation delay of 1 s, a spectral width of
505 Hz, and an acquisition time of 3.636 s in 64K data points. A
total of 256 scans were recorded for each sample, yielding an analysis
time of ca. 21 min per sample. The digital resolution was kept below
3
. The spectra were acquired using the standard s2pul sequence,
General Experimental Procedures. Melting points were determined
on a Fisher-Johns apparatus and are uncorrected. IR spectra were
obtained using KBr disks on a Perkin-Elmer FT 1605 spectrophotom-
eter. Open column chromatography was carried out on silica gel 60
4
0
.4 Hz. NMR files were Fourier transformed with the Mestre-C 4.0
(70-230 mesh, Merck). Analytical TLC was performed on precoated
program (Mestrelab Research, Santiago de Compostela, Spain) to
determine the peak integrals. Phase adjustment and baseline correction
were applied prior to integration of the δ 7.10-4.20 region, where
diagnostic resonances were found for the seven analyzed substances.
1
silica gel 60 F254 plates (Merck). The 300 MHz H NMR spectra were
recorded at 295 K on a Varian Mercury 300 equipped with a direct
detection 5 mm probe using tetramethylsilane (TMS) as the zero
reference point. EI-MS were registered on a JEOL SX 102 mass
spectrometer.
1
H NMR Data of Compounds 1-7 in CDCl . 1: δ 7.44 (2H, br d,
3
J ) 7.5 Hz, H-2′ and H-6′), 7.35 (2H, m, H-3′ and H-5′), 7.34 (1H, m,
H-4′), 7.27 (1H, t, J ) 8.3 Hz, H-4), 6.54 (2H, d, J ) 8.3 Hz, H-3 and
H-5), 5.38 (2H, s, H-7′), 3.80 (6H, s, OCH -2,6). 2: δ 11.42 (1H, s,
Plant Material. Two different batches of the aerial parts of B.
Veronicaefolia were collected in the States of Michoac a´ n (batch I) and
Mexico (batches II and IIa). A third sample, batch III, was purchased
at the Mercado de Sonora in M e´ xico City. Batch I was collected on
May 20, 2004. Batches II and IIa were cropped on December 10, 2003,
and June 25, 2005, respectively. Finally, batch III was acquired on
April 16, 2004. Voucher specimens of B. Veronicaefolia (BCR-1M,
BCR-2M, BCR-2MA, and BCR-3M, respectively) were deposited in
the National Herbarium (MEXU), Instituto de Biologia, UNAM.
Essential Oils from the Aerial Parts. The essential oils of the aerial
parts of each batch (I-III) were prepared by hydrodistillation from
3
OH), 7.47 (1H, br d, J ) 8.0 Hz, H-6), 7.38 (2H, br t, J ) 7.2 Hz, H-4
and H-4′), 7.32 (2H, t, J ) 7.3 Hz, H-5 and H-5′), 6.59 (1H, dd, J )
8.4, 1.0 Hz, H-3), 6.42 (1H, d, J ) 8.4 Hz, H-3′), 5.41 (2H, s, H-7′),
3.84 (3H, s, OCH ). 3: δ 8.16 (1H, br d, J ) 1.8 Hz, H-8), 7.61 (1H,
3
br d, J ) 3.6 Hz, H-2), 7.38 (1H, dd, J ) 10.2, 2.1 Hz, H-6), 7.21
(1H, d, J ) 3.9 Hz, H-3), 6.98 (1H, d, J ) 10.8 Hz, H-5), 2.84 (2H,
q, J ) 7.8 Hz, H-11), 2.83 (3H, s, H-9), 2.65 (3H, s, H-10), 1.34 (3H,
t, J ) 7.6 Hz, H-12). 4: δ 5.40-5.20 (1H, m, H-5), 4.95 (1H, br s,
H-15a), 4.88-4.83 (1H, 2 br s due to conformational mixture, H-15b),
1.62 (3H, br s, H-12), 1.00 (3H, br s, H-14), 0.98 (3H, br s, H-13). 5:
δ: 5.78 (1H, d, J ) 15.9 Hz, H-5), 5.25 (1H, dd, J ) 16.0, 9.9 Hz,
H-6), 5.13 (1H, m, H-1), 4.79 (1H, m, H-15), 4.74 (1H, m, H-15′),
1.66 (3H, d, J ) 1.5 Hz), 0.86 (3H, d, J ) 7.2 Hz, H-13), 0.81 (3H,
d, J ) 6.9 Hz, H-12). 6: δ 4.80 (1H, m, H-1), 4.33 (1H, d, J ) 11.5
Hz, H-5), 1.65 (3H, s, H-15), 1.46 (3H, s, H-14), 1.07 (3H, s, H-13),
1.0 (3H, s, H-12). 7: δ 4.67 (1H, dd J ) 1.8 Hz, H-14a), 4.48 (1H, m,
J ) 1.8 Hz, H-14b), 2.20 (1H, ddd, J ) 14, 10, 5 Hz, H-3a), 1.90 (1H,
ddd, J ) 14, 4, 2 Hz, H-3b), 1.69 (1H, dd, J ) 10, 4 Hz, H-5), 1.57
(2H, m, H-6), 1.55 (2H, m, H-8), 1.52 (2H, m, H-2), 1.45 (2H, m,
H-9), 1.35 (2H, m, H-1), 1.31 (1H, m, H-7), 1.2 (6H, s, H-12 and 13),
0.62 (3H, s, H-15).
1
50 g of plant material, according to a procedure previously described.9
The yields were 0.35, 0.27, 0.39, and 0.43 g, respectively.
Isolation of Compounds 1-3 and 5. The essential oil from batch
II (100 mg) was subjected to chromatographic separation by HPLC.
The HPLC system consisted of a Waters instrument equipped with
Waters 996 UV photodiode array detector (900) set at 264 nm, using
a µPorasil column (19 × 300 mm). Control of the equipment, data
acquisition, processing, and management of chromatographic informa-
tion were performed by the Millennium 2000 software program
(
Waters). The mobile phase was hexane and the flow rate 6 mL/min.
This process allowed the isolation of 1 (t ) 15.1, 4 mg), 2 (t ) 14.0,
mg), 3 (t ) 6.2, 15 mg), and 5 (t ) 13.8, 10 mg) identical to
authentic samples.
R
R
6
R
R
Specificity, Linearity, Accuracy, and Reproducibility of the NMR
Analytical Method. The specificity of the method was established for
each test substance by demonstrating the lack of interference among
them, as well as from the internal standard and the solvent. The linearity
of the method was evaluated by measuring the relationship between
the NMR detector response and the sample concentration employing a
regression analysis of the response data. Five samples containing 50.0
mg of the oil, 1.0 mg of 1,4-dimethoxybenzene, and increasing amounts
(0.050, 0.125, 0.250, 0.375, and 0.500 mg) of benzyl 2,6-dimethoxy-
benzoate (1) and (1.30, 3.25, 6.50, 9.75, and 13.00 mg) of 4 (99.9%,
GC-MS Analysis of the Oils. The GC-MS measurements were
conducted on a Hewlett-Packard 5989A spectrometer. The GC column
was HP-5MS (30 m × 0.31 mm i.d.). The typical linear temperature
programming was from 70 to 250 °C, at a rate of 20 °C/min, and the
carrier gas was He (1 mL/min). Solutions of samples were prepared in
CHCl (1 mg/mL) and injected (1 µL) using the split injection method.
3
Major constituents of the essential oils were identified by matching
their 70 eV mass spectra with those of the reference library. In the
case of benzyl 2,6-dimethoxybenzoate (1), 2-hydroxybenzyl 2′-meth-
oxybenzoate (2), chamazulene (3), and â-caryophyllene (4) the identities
3
Sigma St. Louis, MO) in 0.8 mL of CDCl were prepared; after each
1
were established by comparison with authentic samples. The t
retention times) for 1-7 in the case of batch II were 6.58, 6.37, 5.18,
.00, 4.24, 4.31, and 4.90 min, respectively.
Preparation of Compound 1. To a solution of benzyl alcohol (4.8
mmol, 527 mg) and 2,6-dimethoxybenzoyl chloride (1.2 mmol, 240
mg) in 10 mL of acetonitrile was added dropwise Et N (675 µL). The
reaction mixture was stirred at room temperature during 3 h, poured
into cold H O, and extracted with CH Cl
(15 mL × 3). The organic
phase was dried over Na SO and concentrated in vacuo. The crude
reaction mixture was purified by open column chromatography on silica
gel 60 (70-230 mesh, Merck, ∼100 g) eluting with hexane-CH Cl
1:1) to yield 510 mg of pure compound identical to the natural product.
Analytical TLC was performed on precoated silica gel 60 F254 plates
R
’s
addition of 1 and 4, the H NMR spectrum of the resulting sample was
recorded in triplicate. Altogether 15 spectra were registered. All
validation parameters were obtained by analyzing the three oil batches
(I-III) in triplicate.
(
4
Acknowledgment. This work was supported by grants from
CONACyT (C01-018) and DGAPA-UNAM (IN 212005). B.R. ac-
knowledges the scholarship awarded by CONACyT to carry out
graduate studies.
3
2
2
2
2
4
References and Notes
2
2
(
(1) Mart ´ı nez, M. Las Plantas Medicinales de M e´ xico; Editorial Botas:
M e´ xico, D. F., 1989; pp 249-250.
(
(
(
2) Roberts, M. F.; Timmermann, B. N.; Mabry, T. J.; Brown, R.; Matlin,
(Merck).
S. A. Phytochemistry 1984, 23, 163-165.
NMR Measurements. Samples were prepared with 50.0 mg of the
essential oil, 0.8 mL of CDCl , and 100 µL of the internal standard
solution and transferred to 5 mm NMR tubes. The internal standard
solution was prepared dissolving 10 mg of pure 1,4-dimethoxybenzene
3) Calder o´ n, J. S.; Quijano, L.; Cristia, M.; G o´ mez, F.; R ´ı os, T.
Phytochemistry 1983, 22, 1783-1785.
3
4) Inhuma, M.; Roberts, M. F.; Matlin, S. A.; Stacey, V. E.; Timmer-
mann, B. N.; Mabry, T. J.; Brown, R. Phytochemistry 1985, 24,
1367-1368.
(99.9%), purchased from Sigma-Aldrich Co. (St. Louis, MO), in 1 mL

1
176 Journal of Natural Products, 2006, Vol. 69, No. 8
RiVero-Cruz et al.
(
(
(
(
(
5) Roberts, M. F.; Timmermann, B. N.; Mabry, T. J. Phytochemistry
(15) Bertelli, D. J.; Crabtree, J. H. Tetrahedron 1968, 24, 79-89.
1980, 19, 127-129.
(16) Clericuzio, M.; Alagona, G.; Ghio, C.; Toma, L. J. Org. Chem. 2000,
65, 6010-6916.
6) P e´ rez, R. M.; Cervantes, H.; Zavala, M. A.; S a´ nchez, J.; P e´ rez, S.;
P e´ rez, C. Phytomedicine 2000, 7, 25-29.
(17) Mori, M.; Okada, K.; Shimazaki, K.; Chuman, T.; Kuwahara, S.;
Kitahara, T.; Mori, K. J. Chem. Soc., Perkin Trans. 1 1990, 1769-
1777.
(18) McMurry, J. E.; Bosch, G. K. J. Org. Chem. 1987, 52, 4885-4893.
(19) Raharivelomanana, P.; Bianchini, J. P.; Cambon, A.; Azzaro, M.;
Faure, R. Magn. Reson. Chem. 1995, 33, 233-235.
(20) Morita, M.; Nakanishi, H.; Morita, H.; Mihashi, S.; Itokawa, H. Chem.
Pharm. Bull. 1996, 44, 1603-1606.
(21) Rekka, E.; Chrysselis, M.; Siskou, I.; Kourounakis, A. Chem. Pharm.
Bull. 2002, 50, 904-907.
7) P e´ rez-Guti e´ rrez, R. M.; P e´ rez-Gonz a´ lez, C.; Zavala-S a´ nchez, M. A.;
P e´ rez-Guti e´ rrez, S. Salud P u´ blica de M e´ xico 1998, 40, 354-358.
8) Rivero-Cruz, B.; Rojas, A.; Rodr ´ı guez-Sotres, R.; Cerda-Garc ´ı a-Rojas,
C. M.; Mata, R. Planta Med. 2005, 71, 320-325.
9) Quality control methods for medicinal plant materials; Geneva, World
Health Organization, 1998.
(
(
10) Liang, Y. Z.; Xie, P.; Chan, K. J. Chromatogr. B 2004, 812, 53-70.
11) Bilia, A. R.; Bergonzi, M. C.; Lazari, D.; Vincieri, F. F. J. Agric.
Food Chem. 2002, 50, 5016-5025.
(
(
(
12) Pauli, G. F.; Jaki, B. U.; Lankin, D. C. J. Nat. Prod. 2005, 68, 133-
(
22) Lee, G. J.; Lee, C. G.; Kwag, J. J.; Buglass, A. J.; Lee, G. H. J.
Chromatogr. A 2005, 1089, 227-234.
149.
13) Martin, M. L. In FlaVor Chemistry; Martin G. J., Ed.; Kluwer: New
(23) ICH Harmonised Tripartite Guideline, ICH Q2V, Text on Validation
on Analytical Procedures: Methodology, 1997.
York; 1999; pp 18-30.
14) Bohlmann, F.; Jakupovic, J.; Lonitz, M. Chem. Ber. 1977, 110, 301-
314.
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