NAVARRETE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO.
1, 2006 1
DIETARY SUPPLEMENTS
Quantitative Determination of Triterpenes from
Amphiptherygium adstringens by Liquid Chromatography and
Thin-Layer Chromatography and Morphological Analysis of
Cuachalalate Preparations
ANDRÉS NAVARRETE1, BHARATHI AVULA, VAISHALI C. JOSHI, and XIUHONG JI
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, The University of
Mississippi, University, MS 38677
PAUL HERSH
Instituto Nacional de Antropologia e Historia, Delegacion Morelos, Matamoros 14 Colonia Acapatzingo 62440,
Cuernavaca Morelos, México
IKHLAS A. KHAN
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, Department of
Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677
Amphiptherygium adstringens
(Anacardiaceae/Julianaceae), local name
"cuachalalate," is used in folk medicine for the
treatment of cholelithiasis, fevers, fresh wounds,
hypercholesterolemia, gastritis, gastric ulcers, and
cancer of the gastrointestinal tract. The
development of column high-performance liquid
chromatography–photodiode array detector
(LC-PDA) and high-performance thin-layer
chromatography (HPTLC)–densitometry methods
for the determination of masticadienonic acid and
3-hydroxymasticadienonic acid in cuachalalate
preparations is described in this paper. Good
separation of the compounds could be achieved by
both methods. Either might be preparable
depending on the requirements. The LC separation
was performed on a Phenomenex Synergi MAX-RP
80A reversed-phase column operated at 40°C with
detection at 215 nm. The plant materials were
extracted with methanol by sonication. The
triterpenes present in the plant material and
commercial extracts were separated with an
acetonitrile–water reagent alcohol isocratic
system. The limit of detection was 0.1–0.2 mg/mL.
The relative standard deviation values for the
determination of triterpenes in plant extracts were
less than 1.00%. This is the first report of an
analytical method developed for the quantitative
analysis of triterpenes from Amphiptherygium
adstringens by LC-PDA and HPTLC. The stem bark
Received July 1, 2005. Accepted by AP August 1, 2005.
1
Corresponding author's current address: Universidad Nacional
Autónoma de México, Facultad de Química, Departamento de Farmacia,
Ciudad Universitaria, Coyoacan 04510, México D.F., México; e-mail:
anavarrt@servidor.unam.mx
showed higher amounts of triterpenes, and low
amounts in root and stem root. The microscopic
description of the crude drug of cuachalalate was
also provided.
T
he stems bark of Amphipterygium adstringens
(Schltdl.) Schiede ex Standl. (Julianaceae), local name
“cuachalalate,” is the most important anti-ulcer remedy
in the Mexican traditional medicine (1). Extensive studies
have been published on the chemistry of this plant (2–10) and
have demonstrated 2 types of major compounds: triterpenoids
and long chain phenols. Young described the presence of
5-deoxyflavonoids in the leaf and heartwood of
A. adstringens (11). The gastroprotective effect of this
medicinal plant has been very well demonstrated in animal
models (1, 12). 3a-Hydroxymasticadienonic acid,
3-epioleanolic acid, and b-sitosterol have been identified as
the gastroprotective-active principles in the stem bark of
cuachalalate (13). Anti-inflammatory activity and cytotoxicity
effect against leukemia cells (L-12-10) has been reported for
3a-hydroxymasticadienonic acid and masticadienonic
acid (10, 14). A hexane extract of cuachalalate showed a
hypocholesterolemic effect in rats (6). Anacardic acids are
present in high amounts in cuachalalate (5, 6). These
compounds have shown various physiological activities, such
as inhibition of medicinally important enzymes and antitumor,
antimicrobial, anti-acne, molluscicide, and antifeedant
activity (15, 16).
On the other hand, the Mexican Herbal Pharmacopoeia
(Farmacopea Herbolaria de los Estados Unidos
Mexicanos; 17) includes a monograph for cuachalalate that
describes a thin-layer chromatography (TLC) method for the
qualitative identification of 3a-hydroxymasticadienonic acid
using hexane–ethyl acetate as the mobile phase and ceric
2 NAVARRETE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 1, 2006
Figure 1. Triterpene markers of A. adstringens:
masticadienonic acid (1); 3-hydroxymasticadienonic
acid (2); and oleanolic acid (3).
ammonium sulfate as the detection reagent, but it does not
describe a method for quantitative analysis nor does it give a
microscopic description for this crude drug. In order to ensure
the quality of herbal products, appropriate analytical methods
must be available that, ideally, should be based on markers
specific to each plant (18).
The crude drug and commercial extracts are exported from
Mexico to different countries, and, to date, there has been no
study dealing with quality control of these drugs prepared
from A. adstringens. This paper describes the development of
column high-performance liquid chromatography (LC) and
high-performance TLC methods for the quantitative
analysis of A. adstringens. The LC and TLC methods were
used for the examination of the masticadienonic acid and
3-hydroxymasticadienonic acid contents in stem bark, stem
root, and root of A. adstringens collected from different places
in Mexico and also in 2 commercial extracts. Microscopic
analysis of the stem bark, stem, and root was performed with
the aim to give elements to conform a pharmacopoeia
monograph for this medicinal plant.
Experimental
Plant Material and Commercial Extracts
Samples were collected in July 2004 from 3 places in
Mexico: Temalac, Municipio de Tenango del Rio (AAP-1:
root; AAP-2: stem root; and AAP-3: stem bark) and
Mezquitlan, Municipio de Copalillo (AAP-4: root; AAP-5:
stem root; and AAP-6: stem bark) both from the Estado de
Guerrero at south of Mexico, and 1 sample (AAP-7: stem
bark) from Estado de Colima west of Mexico. Voucher
specimens of all samples are deposited at the National Center
for Natural Products Research (NCNPR), University of
Mississippi. Commercial extracts (CE-1 and CE-2) were
obtained from 2 different companies in Mexico. Both were
hydroalcoholic fluid extracts.
Chemicals
The standard compounds (Figure 1) masticadienonic
acid and 3-hydroxymasticadienonic acid were available for
our previous studies (5, 13); their identity and purity were
confirmed by chromatographic (TLC and LC) methods and
comparison with published spectral data (infrared, one- and
2-dimensional nuclear magnetic resonance, and high
resolution electrospray ionization mass spectrometry).
Oleanolic acid, safranin O, ruthium red, and fast green FCF
were purchased from Sigma (St. Louis, MO). Acetonitrile,
reagent alcohol, acetic acid, hexane, acetone, formic acid, and
phosphoric acid were of LC grade and purchased from Fisher
Scientific (Fair Lawn, NJ). Water for the LC mobile phase was
purified in a Milli-Q system (Millipore, Bedford, MA).
Standard Solutions
Individual stock solutions of triterpenes were prepared at a
concentration of 0.1 mg/mL in methanol. The quantification
was performed using 5 levels of external standards. The
ranges obtained were 10–100 mg/mL depending on the
concentration of each stock solution. Table 1 shows the
calibration data and calculated limit of detection (LOD). The
calibration data for Analyte 3 was not established due to the
presence of this compound in very minute amounts.
Sample Preparation
Ground parts (300 mg, particle size <420 mm, mesh 40) of
the plant material were sonicated in 2.5 mL methanol for
20 min followed by centrifugation for 15 min at 900 rpm. The
Table 1. Calibration data [regression equation and
correlation coefficient (r2)] and limit of detection (LOD)
for Compounds 1 and 2 by LC
Regression
Analyte equation r2 LOD, mg/mL
1 Y = 1.52 ´ 104x 0.99999 0.100
2 Y = 1.43 ´ 104x 0.99997 0.200
 NAVARRETE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 1, 2006 3
supernatant was transferred to a 10 mL volumetric flask. The
procedure was repeated thrice and the respective supernatants
combined. The final volume was adjusted to 10 mL with
methanol. The commercial extract CE-2 was diluted 5 times
more than CE-1.
Prior to injection, an adequate volume (ca 2 mL) was
passed through a 0.45 mm nylon membrane filter. The first
1 mL was discarded, and the remaining volume was collected
in an LC sample vial. Each sample solution was injected in
triplicate.
LC
The LC system consisted of Waters Corp. (Milford, MA)
Model 6000A pumps, Model U6K injector, Model 680
automated gradient controller, Model 996 photodiode array
detector (PDAD), and computerized data station equipped
with Millennium software. Separation was achieved on a
Synergi MAX-RP 80A column (150 ´ 4.6 mm id, 4 mm
particle size; Phenomenex, Torrance, CA) operated at 40°C.
The column was equipped with a 2 cm LC-18 guard column
(5 mm particle size; Supelco, Bellefonte, PA). The mobile
phase consisted of 0.1% aqueous acetic acid–acetonitrile
containing 0.1% acetic acid–reagent alcohol (18 + 52 +
30, v/v) used isocratically for 20 min; the flow rate was
1.0 mL/min and the detection wavelength 215 nm. Each run
was followed by a 5 min wash with 100% acetonitrile and an
equilibration period of 15 min.
TLC
A Linomat 5 sample applicator with 100 mL syringe
(Camag, Wilmington, NC) was used to apply 2 mL test and
standard solution (1 mg/mL) as separate 4 mm bands 2.4 mm
apart and 8 mm from the bottom of a TLC plate (silica gel 60
F254; E. Merck, Darmstadt, Germany). The plate was
developed to a height 8 cm in the ascending direction in a
chamber previously saturated with the
hexane–acetone–formic acid–acetic acid (15 + 5 + 0.5 + 0.5,
v/v/v/v) mobile phase. The TLC plate was dried in a current of
cold air for 5 min. The absorption was determined at 200 nm
using Camag TLC Scanner 3. Then the plate was immersed in
the derivatization reagent (anisaldehyde–sulfuric acid) for 1 s
and heated at 100°C for 5 min. Plates were photographed
using a Camag Videodensitometer with a Reprostar 3 (white
light) and Videoscan evaluation software (version 1.02.00).
Microscopy
Amphipterygium adstringens bark (quills), root, and stem
samples were boiled in water. Transverse as well as
longitudinal sections of bark, stem, and root, ca 1 mm
thickness, were taken using a Leica Cryostat CM1850 (Meyer
Instruments Inc., Houston, TX). Bark sections were stained
using ruthium red. Root and stem sections were stained with
safranin O and passed though 50% ethyl alcohol. The sections
were counter stained with fast green and passed through
grades of ethyl alcohol (20, 40, 60, 80, and 99% of ethyl
alcohol in water v/v). Dehydrated sections were mounted on
glass slides in permount mounting media. Root and stem
samples were macerated and digested in 10% nitric acid with
2–3 crystals of sodium hydroxide (Fisher Scientific) and
heated at 40–60°C for 15–20 min. The macerated samples
were centrifuged and transferred to water. From water, root
and stem sections were dehydrated using the same procedure
as for bark. All samples were examined under a Nikon Eclipse
E600 microscope. Digital photomicrographs were taken using
Nikon camera attached to the microscope, and images were
processed using Adobe Photoshop software.
Results and Discussion
LC Method Validation
The separation of a mixture of Standards 1–3 is shown in
Figure 2. Several analytical parameters were determined to
validate the new LC method, and all requirements could be
fulfilled.
(a) Calibration graphs, LOD values, and limit of
quantification (LOQ) values.—Standards containing
methanolic mixtures of 1 and 2 were prepared in the range
from 1.0–100 mg/mL using 1 mg/mL stock solution. These
mixtures were analyzed as described in the LC section above.
Table 1 summarizes the data obtained by linear regression for
analytes 1 and 2 (the errors in the slopes and intercepts did not
reveal significant differences) and LOD. The calibration
graphs allowed the calculation of 1 and 2 concentration levels
in various plant samples and in commercial extracts. The LOD
and LOQ values obtained for a signal-to-noise (S/N) ratio of
3 and 10, respectively, using the calibration graphs were
0.1 and 0.5 mg/mL, and 0.2 and 0.2 mg/mL, for 1 and
2, respectively.
(b) Precision (repeatability and reproducibility).—
Repeatability (within-run precision) was examined for 1 and
2 by analyzing all plant material and commercial extracts
within a day. Individual standards (3 concentrations) were
used, and each sample was run 4 times (n = 4).
Reproducibility (between-run precision) was evaluated on 3
different days using calibration graphs. The calculated relative
standard deviation was below 3.0% for all experiments.
(c) Accuracy.—Accuracy was assessed at 3 different
concentration levels of 1 and 2 by replicate measurements
(n = 4). Standards of 1 and 2 were added to plant material
sample, processed under the Sample Preparation conditions
above, and analyzed using the proposed LC method. Table 2
shows the amounts spiked, recoveries, and relative standard
deviation (RSD) values found for 1 and 2 in samples.
(d) Selectivity.—To assess the selectivity, various samples
were analyzed under conditions described for sample
preparation. Detection and identification of 1 and 2 based on
the retention times and a comparison of UV spectra with
standards (19) was performed. The RSD of each sample was
lower than 1%. The UV spectrum of each peak in the
chromatogram was subsequently compared with the
standards. Impurities were investigated further by displaying
the spectra obtained at different points across the peak.
Impurities were not detected, and the method showed the
4 NAVARRETE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 1, 2006
Figure 2. A typical column liquid chromatogram of
pure triterpene standards (1–3), plant extract (AAP-1),
and commercial extracts (CE-1 and CE-2) with detection
at 215 nm.
ability to analyze 1 and 2 in various plant samples and
commercial products with good analytical results.
Composition of the mobile phase was crucial and when
using an acidic mobile phase the peak symmetry could be
significantly improved. The addition of acetic acid was
advantageous. A mixture of 0.1% aqueous acetic acid,
acetonitrile containing 0.1% acetic acid, and reagent alcohol
was finally chosen. Without the addition of acetic acid in the
mobile phase, the peak of Compound 1 merged with other
peaks. The use of reagent alcohol in the mobile phase resolved
all of the peaks completely, with total analysis time of 13 min.
A further improvement was to increase the separation
temperature to 40°C, as this adjustment led to a considerable
reduction of the separation time. Compounds 1–3 showed a
sufficient absorption at 215 nm, and all experiments were
monitored at this wavelength (Figure 2). Under these
chromatographic conditions, it was not possible to
quantify individually the isomers (3a- and
3b-hydroxymasticadienonic acid) of Compound 2 because
they showed the same retention time and, hence, quantified as
3-hydroxymasticadienonic acid.
LC Analysis of Triterpenes in Plant Samples and
Commercial Extracts
Figure 3 shows the variations in content of 2 major
triterpenes in 7 plant samples (AAPI-7). Compounds 1 and 2
were present in levels from 0.011–0.86% and 0.14–1.29%,
respectively, in plant samples. The highest concentration of
1 was present in stem-bark AAP-3 (0.86%), and the lowest
concentration was in AAP4 (0.01%); the highest
concentration of 2 was present in stem-root AAP-5 (1.29%)
and the lowest amount in AAP4 (0.14%). The ranges of 1 and
2 for the commercial extracts analyzed was between
0.76–3.35 and 0.87–1.13 mg/mL, respectively. Compound 3
was present in trace amounts in all the plant samples and
commercial extracts analyzed. As shown in Table 3, the
samples of stem-bark collected from 3 different locations in
Mexico showed small variation in the concentration of 1, and
the content of 2 was higher in AAP-3 collected in Tenango del
Rio, Mexico, than in the other 2 samples of stem-bark
collected in Copalillo and Estado de Colima. CE-1 and CE-2
were the fluid extracts of cuachalalate preparations. The
analysis of cuachalalate commercial fluid extracts (CE-1 and
CE-2) showed significant differences in their qualitative and
quantitative composition (Table 3). The content of 1 in CE-2
was 5 times more than in CE-1, whereas the content of 2 was
30% less in CE-2 than in CE-1. These differences may be due
to the heterogeneity of the plant materials used to prepare the
commercial extracts.
TLC–Densitometry
A TLC–densitometry method was developed to estimate
Compounds 1 and 2. The method was applicable in estimating
Compound 1 in crude drug and commercial extracts (Table 4).
The Rf values were 0.26 and 0.20 for 1 and 2, respectively
(Figure 4). The calibration graph for Compound 1 was found
to be linear over the range of 10–100 mg/mL, and the
correlation coefficient (r2) was 0.9999. The results were quite
satisfactory and found to match with the results of LC. Sample
AAP-3 (bark) showed the highest concentration (0.843%),
whereas Sample AAP-4 (root) showed the lowest
concentration (0.0103%) for Compound 1.
There was no significant difference in content of
Compound 1 from 3 different locations. The levels of 1 found
in this work are in agreement with those reported previously
by Olivera et al. (9).
Microscopic Characters of Amphiptherygium
adstringens
The drug consists of dried bark of stem and branches
(Figure 5A). The cuts consist of straight bits or quill. The outer
Table 2. Recovery study for Analytes 1 and 2 at
3 levels of concentration
Spiked, Recovery, Avg. recovery,
Sample Compounds mg/mL %a %b
AAP-3 1 1 101.65 (0.34) 102.19 (0.37)
2 1 98.34 (0.89) 99.12 (0.70)
1 25 102.94 (0.23)
2 25 99.97 (0.78)
1 75 101.99 (0.53)
2 75 101.45 (0.43)
a
% Recovery is expressed as the mean, and the RSD is shown in
parenthesis.
b
Average recovery = the average of 3 levels, 9 determinations.
 NAVARRETE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 1, 2006 5

phloem. Secretory ducts are oval, ranging from 50–60 mm in

Figure 3. Variations in the concentration of triterpenes
in various plant samples as determined by LC.
surface is grayish brown, matt to dull luster, finely fissured
with a few elongated lenticels; the lenticels are pale brown to
gray, and at times brown-colored resin is observed on the
outer surface. The inner bark is reddish brown, sometimes
blackish brown, finely striated longitudinally, with distinct
resin ducts seen all along the fissured bark as well as the inner
sides of the bark. No distinct odor was observed, and the taste
is astringent.
A transversal section of bark is shown in Figure 5B. The
bark has an outer epidermis followed by 3–4 layers of cork
cells forming periderm. Phellogen cells are rectangular, the
walls colorless. Some of the phelloderm parenchyma cells are
converted to stone cells. Stone cells (ST; Figure 5D) are in
groups of 3–4 or individual, varying in size and shape, usually
rectangular with thickened walls having numbers of slit
shaped pits.
Stratified cork is composed of several layers of tubular
cells containing tannin sac, brown in color, of varying
abundance. Secretory ducts (SD; Figure 5B) are observed at
regular interval in tangential bands all along the secondary
diameter. Secretory ducts have slightly raised canal walls.
Cortex parenchyma is thin-walled with no intercellular
space. A number of parenchyma cells are filled with irregular
shaped clustered crystals of calcium oxalate (CR; Figure 5C).
Individual cubical crystals were also observed in a few
samples. Crystals appear grayish when examined under a light
microscope. Secondary phloem has multiseriate medullar rays
(MR; Figure 5C), usually on the inner end of the bark.
Medullary rays are 4–5 cells wide. Long fibers (F; Figure 5C),
sometimes septate, are visible in the longitudinal section.
The wood anatomy of root and stem differ only in size.
The vessels (V; Figure 5E) are large in root, ranging
between 100–150 mm in diameter, and in stem between
70–100 mm. Biseriate rays were observed in both root
and stem. Ray (R; Figure 5E) width ranges from 17–22
mm in root and 10–14 mm in stem. Vessels (Figure 5F) are
short, with oblique end walls ranging between about
450–600 mm in length. Vessels have scalariform pitting.
The long fibers measure approximately 100–120 mm
with pits. The wood anatomy secondary xylem and
pollen for this plant have been studied (20), however, the
bark anatomy or secondary phloem has not been described
for this Mexican medicinal plant. So a detailed description
has been provided in this work. The phylogenetic
relationships of Julianaceae have been the subject of
controversy (11). Based on anatomical observations, there
is a group of researchers inclined to place the family
Julianaceae in Anacardiaceae. The secondary xylem of
Julianaceae suggests that it is closely related to
Anacardiaceae (20). Similar observations were made with
regard to bark anatomy and secondary phloem in
A. adstringens. Secretory ducts of A. adstringens showed
similarity with that of Rhus species, an Anacardiaceae
member (21). These anatomical and chemical
observations, such as presence of anacardic acids (5, 6),
give additional support to suggest that it is necessary to

Table 3. The percentages of triterpenes in 7 different plant samples and commercial extracts of Amphiptherygium
adstringens by LC
Sample Plant part Collection location 1, % 2, % 3

AAP-1 Root Temalac 0.344 (0.13)a 0.64 (0.11)a Trace

AAP-2 Stem-root Temalac 0.349 (0.18) 0.52 (0.14) Trace
AAP-3 Stem-bark Temalac 0.864 (0.09) 1.22 (0.23) Trace
AAP-4 Root Mazquitlan 0.011 (0.46) 0.14 (0.05) Trace
AAP-5 Stem-root Mazquitlan 0.746 (0.002) 1.29 (0.18) Trace
AAP-6 Stem-bark Mazquitlan 0.789 (0.07) 0.87 (0.09) Trace
AAP-7 Stem-bark Colima 0.688 (0.08) 0.71 (0.12) Trace
b b
CE-1 Stem-bark — 0.76 (0.12) 1.13 (0.21) Trace
CE-2 Stem-bark — 3.35b (0.33) 0.87b (0.17) Trace

a
Mean values (n = 3); RSD values are in parenthesis.
b
Content (mg/mL) of triterpenes in 2 commercial liquid extracts.
6 NAVARRETE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 1, 2006

Table 4. The percentages of triterpene in 7 different analysis of 1 and 2 in plant material and commercial
plant samples and commercial extracts of preparations, and it can also be helpful in assuring safety and
Amphiptherygium adstringens determined by TLC quality control of cuachalalate preparations. TLC–densitometry
Sample Plant part Compound 1, % can also be utilized for this purpose.

Acknowledgments
AAP-1 Root 0.340 (0.25)a
AAP-2 Stem-root 0.341 (0.23)
This research was funded in part by “Botanical Dietary
AAP-3 Stem-bark 0.843 (0.11) Supplements: Science-Base for Authentication,” U.S. Food
AAP-4 Root 0.010 (0.33) and Drug Administration Grant No. FD-U-002071-01.
AAP-5 Stem-root 0.743 (0.05) Research performed during sabbatical year of A.
AAP-6 Stem-bark 0.777 (0.15) Navarrete at NCNPR. A. Navarrete acknowledges the
sabbatical support from Direcci\n General de Asuntos del
AAP-7 Stem-bark 0.681 (0.05)
Personal AcadJmico (DGAPA), Universidad Nacional
CE-1 Stem-bark 0.65b (0.19)
Aut\noma de MJxico (UNAM), and the partial financial
CE-2 Stem-bark 3.33b (0.33) support from UNAM (DGAPA 203902) and Consejo
a
Nacional de Ciencia y TecnologRa (CONACYT 41231 and
Mean values (n = 3); RSD values are in parenthesis.
b C01-018).
Content (mg/mL) of triterpenes in 2 commercial liquid extracts.

consider A. adstringens as a member of Anacardiaceae

family rather than Julianaceae, in agreement with
Aguilar-Ortigoza et al. (22).

Conclusions

It can be concluded that because of the simple sample

preparation, short analysis time, and high reproducibility, the
LC method presented in this paper can be a useful tool for routine

Figure 4. Typical thin-layer chromatograms of pure
triterpene standards (1-2) and plant extracts (A = AAP-1;
B = AAP-2; C = AAP-3; D = AAP-4; E = AAP-5; F = AAP-6;
and AAP-7). Compounds 2a (3a-hydroxymasticadienonic
acid) and 2b (3b-hydroxymasticadienonic acid) are
isomers. 2 Is the mixture of 2a and 2b.
Figure 5. A: Bark; B: transversal section of bark
showing secretary ducts (SD); C: longitudinal section
of bark showing crystal (CR), medullar ray (MR), and
fivers (F); D: transversal section of bark showing stone
cells (ST); E: transversal section of root showing
vessels (V) and rays (R); F: vessel.
 NAVARRETE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 1, 2006 7
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