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Journal of Ethnopharmacology 114 (2007) 72–77

Anti-Helicobacter pylori activity of anacardic acids


from Amphipterygium adstringens
Israel Castillo-Juárez a , Fausto Rivero-Cruz b , Heliodoro Celis c , Irma Romero a,∗
aDepartamento de Bioquı́mica, Facultad de Medicina, Universidad Nacional Autónoma de México, México D.F., C.P. 04510, Mexico
b
Departamento de Farmacia, Facultad de Quı́mica, Universidad Nacional Autónoma de México, México D.F., C.P. 04510, Mexico
c Departamento de Bioquı́mica, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, México D.F., C.P. 04510, Mexico

Received 14 March 2007; received in revised form 22 June 2007; accepted 23 July 2007
Available online 27 July 2007

Abstract
Amphipterygium adstringens (Schltdl.) Standl. (Anacardiaceae) is widely used in traditional Mexican medicine for the treatment of gastritis and
ulcers. In this work, we studied the anti-Helicobacter pylori activity of its bark, this Gram-negative bacterium is considered the major etiological
agent of chronic active gastritis and peptic ulcer disease, and it is linked to gastric carcinoma. From a bio-guided assay of the fractions obtained form
a continuous Soxhlet extraction of the bark, we identified that petroleum ether fraction had significant antimicrobial activity against Helicobacter
pylori. From this fraction, we isolated an anacardic acids mixture and three known triterpenes: masticadienonic acid; 3␣-hydroxymasticadienonic
acid; 3-epi-oleanolic; as well as the sterol ␤-sitosterol. Only the anacardic acids mixture exhibits a potent dose-dependent antibacterial activity
(MIC = 10 ␮g/ml in broth cultures). It is enriched in saturated alkyl phenolic acids (C15:0 , C16:0 , C17:0 C19:0 ) which represents a novel source of
these compounds with potent anti-Helicobacter pylori activity. The promising use of anacardic acids and Amphipterygium adstringens bark in the
development of an integral treatment of Helicobacter pylori diseases is discussed.
© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Helicobacter pylori; Anacardic acids; Antibacterial activity; Amphipterygium adstringens; Long-chain phenols; Anacardiaceae

1. Introduction zole given for 7–14 days. However, approximately 20% of


patients fail to obtain eradication; thus a second-line therapy,
Helicobacter pylori is a spiral-shaped Gram-negative bac- a quadruple regimen composed by tetracycline, metronida-
terium that colonizes the stomach in more than 80% of zole (or others depending of the initial treatment), bismuth
the population in developing countries, and under 40% in salts and a proton pump inhibitor is recommended in these
industrialized countries. Helicobacter pylori infection is now cases. An indiscriminate use of antibiotics to eradicate Heli-
recognized as the major etiological agent of chronic active cobacter pylori also from healthy carriers, sometimes leads
gastritis and peptic ulcer disease, and it is linked to gastric to severe problems with bacterial resistance against these
carcinoma. It is estimated that Helicobacter pylori-positive important drugs. Moreover, the emergence of any significant
patients have a 10–20% risk of developing ulcer disease and side adverse effect caused by antibacterial drugs may result
1–2% risk of developing distal gastric cancer (Kusters et al., in need to stop eradication therapy even if the aim is not
2006). achieved. Therefore, there is an intense demand for a thera-
According to international guidelines, first-line therapy peutic regimen having the same or higher beneficial properties
for treating Helicobacter pylori infection consists of pro- of antibiotics but with reduced side effects (Di Mario et al.,
ton pumping inhibitor or ranitidine bismuth citrate with any 2006).
two antibiotics of amoxicillin, clarithromycin or metronida- The ethnopharmacological approach has been an important
source to find new anti-Helicobacter pylori agents. Crude drugs
and isolated compounds from plants used in herbal traditional
∗ Corresponding author. Tel.: +52 55 5623 2511; fax: +52 55 5616 2419. medicines of different parts of the world have been tested for
E-mail address: iromero@ifc.unam.mx (I. Romero). anti-Helicobacter pylori activity in vitro, in animal models,

0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2007.07.022
I. Castillo-Juárez et al. / Journal of Ethnopharmacology 114 (2007) 72–77 73

as well as in clinical studies, as possible sources for alter- Petroleum ether extract: 1.0 kg of powdered bark was exhaus-
native eradication therapies (Borrelli and Izzo, 2000; Kawase tively extracted by maceration at room temperature with 2.5 l
and Motohashi, 2004; Mahady, 2005). With this concern in petroleum ether. After filtration, the extract was concentrated
mind, we have searched for new antibacterial agents from under vacuum to yield 27 g of a dark green oily residue. The
the Anacardiaceae plant Amphipterygium adstringens (Schltdl.) extract was subjected to a silica gel (0.5 kg) vacuum col-
Standl. This traditional medicinal tree is known in Mexico umn chromatography and eluted with a gradient mixture of
as “cuachalalate”, “cuachalala”, “matixeran”, “volador” or in hexane–ethyl acetate (1:0 → 0:1, 1 l per fraction) to give seven
Nahuatl language “cuachalalatl”, and its bark decoction is com- pooled fractions (F1–F7). A crystalline solid precipitated from
monly used for the treatment of numerous conditions such as F4 and was identified as 3␣-hydroxymasticadienonic acid. Frac-
cholelithiasis, fresh wounds, fevers, gastritis, gastric ulcers, gas- tions F2 and F3 were separately rechromatographed in a silica
trointestinal cancer, gum diseases and various inflammatory gel column using a gradient mixture of hexane–ethyl acetate.
conditions (Olivera Ortega et al., 1999). These processes yielded an anacardic acids mixture from F2;
Previous phytochemical investigation on Amphipterygium and, masticadienonic acid, 3-epi-oleanoic acid and ␤-sitosterol
adstringens bark resulted in the isolation and identification of from F3. All the structures were established by comparing spec-
several triterpenoids (Soriano-Garcı́a et al., 1987; Watson et tral and physical data with those reported previously in the
al., 1987; Navarrete et al., 1989; Makino et al., 2004); long- literature (Mata et al., 1991), and by direct comparison with
chain phenols (Navarrete et al., 1989; Mata et al., 1991); and, authentic samples.
the 3-dodecyl-1,8-dihydroxy-2-naphtoic acid (Rivero-Cruz et
al., 2005). Pharmacological studies have revealed an hypoc- 2.3. Preparation of derivatives of the alkyl phenolic acids
holesterolaemic effect of an hexane extract of the bark (Mata for GC/MS
et al., 1991). A methanolic extract was found to have a gas-
troprotective effect (Navarrete et al., 1998), and the isolated A sample of anacardic acids mixture (10 mg) was reacted
triterpenes: 3␣-hydroxymasticadienonic acid, 3-epi-oleanolic with a freshly distilled diazomethane ether solution for 24 h at
acid, and the sterol, ␤-sitosterol were identified as the active room temperature to yield 11.1 mg of the methylated mixture.
principles (Arrieta et al., 2003). The aqueous and hexane extracts
of the stem-bark showed anti-inflammatory activity with mas-
2.4. Gas chromatography/mass spectrometry
ticadienonic and 3␣-hydroxymasticadienonic acids as active
compounds (Oviedo-Chavez et al., 2004). A methanol extract
Samples were injected to an Agilent 6890N gas chro-
showed moderate trypanocidal activity against Trypanosoma
matograph with an automatic liquid sampler Agilent 7683B
cruzi (Abe et al., 2005). Finally, several tirucallane-type triter-
coupled to a LECO Pegasus 4D mass spectrometer. The col-
penes were found to exhibit moderate growth inhibitory activity
umn was an HP-5MS 10 m × 180 ␮m, film thickness 0.18 ␮m.
against leukemia cells (L-1210) (Makino et al., 2004).
Helium at 1 ml/min was used as the carrier gas. The col-
In spite of the large quantity of studies about the compounds
umn oven was temperature-programmed from 180 to 280 ◦ C
of Amphipterygium adstringens, there are no reports concerning
at 8 ◦ C/min. The injector and detector temperatures were both
their anti-Helicobacter pylori activity. In this work we evaluate
280 ◦ C; electron energy 70 eV. Masses scanned 33–600 a.u.
the in vitro anti-Helicobacter pylori activity of Amphipterygium
The constituents of the anacardic acids mixture were iden-
adstringens bark.
tified by matching their 70 eV mass spectra with compound
libraries.
2. Materials and methods

2.1. Plant material 2.5. Bacterial strains and culture conditions

The bark of Amphipterygium adstringens was obtained from Helicobacter pylori standard strain ATCC 43504 was used
a Mexican gathering center for medicinal plants located in Jolal- in this study. Stock cultures were stored at −70 ◦ C in Brucella
pan (Puebla). A voucher specimen was deposited in the National broth with 10% fetal bovine serum (GIBCO BRL) and 10% glyc-
Herbarium (MEXU) Institute of Biology at the Universidad erol. Helicobacter pylori was grown on Casman agar (DIFCO)
Nacional Autónoma de México, in Mexico City, Mexico. plates, 0.2% ␤-cyclodextrin, and 10 ␮g/ml vancomycin for 3–5
days at 37 ◦ C under microaerophilic conditions (10% CO2 ). Liq-
2.2. Extraction and fractionation uid cultures were performed in 3 ml of Brucella broth (DIFCO)
plus 0.2% ␤-cyclodextrin and 10 ␮g/ml vancomycin, maintained
Thirty gram of powdered bark was successively extracted under gentle shaking (150 rpm) for the time of the experiment
with increased polarity solvents in a Soxhlet extraction appa- in the aforedescribed CO2 enriched atmosphere. The strains
ratus. The obtained yields were: petroleum ether 3.05%; were identified by Gram staining morphology and biochemi-
anhydrous ether 0.79%; chloroform 0.37%; anhydrous ethanol cally tested (catalase, urease, and oxidase positive). Escherichia
7.50%; 70% ethanol 13.57%; and, water 7.13%. Each sample coli K12 was also used in this study; Luria–Bertani broth (LB)
was dissolved in water or DMSO according to its polarity and and LB agar plates were used for its growth and for the inhibition
tested for antimicrobial activity. experiments.
74 I. Castillo-Juárez et al. / Journal of Ethnopharmacology 114 (2007) 72–77

2.6. Minimal inhibition concentration and bactericidal Table 1


activity Effect of anacardic acids mixture isolated from Amphipterygium adstringens
against Helicobacter pylori

MIC was determined in broth cultures by using an in vitro Tested compounds ␮g/ml Percentage growth inhibition
killing assay. Different concentrations of the compounds to be Anacardic 2 48
tested (in a maximal volume of 20 ␮l DMSO), were added to acids 10 109
3 ml of Helicobacter pylori broth culture at the beginning of mix- 16 112
ture
the exponential growth phase (∼107 CFU/ml). A660 was cal- Metronidazolea 300 90
culated after maintaining the tubes for around 14 h at 37 ◦ C in Amoxicillina 0.1 90
a microaerophilic atmosphere with gentle shaking. A660 was Percentage of inhibition was estimated with respect to a control that was incu-
used to calculate the growth percentage of inhibition with respect bated only with the solvent (DMSO).
a Reference antibiotics.
to a control that was grown only with DMSO. In order to deter-
mine the bactericidal action, samples (100 ␮l) were taken at
various growing times for viable-cell counting. Serial 10-fold 16 ␮g/ml, respectively). Column chromatography separation of
dilutions were made with Brucella broth and plated onto Cas- this active fraction resulted in the isolation and identification
man agar prepared as described before. Viability was measured of 3␣-hydroxymasticadienonic acid, masticadienonic acid, 3-
by the plate colony count method after 5–10 days of incubation. epi-oleanoic acid, ␤-sitosterol, and a long-chain phenolic acids
Also, samples for morphological study were taken at different (anacardic acids) mixture. The GC/MS analysis of the methy-
growing times. lated anacardic acids mixture revealed that the phenolic acids
The final DMSO concentration in the assay never exceeded mixture was composed of four long-chain phenolic acids pos-
0.66% (v/v) and did not have any effect in the growth at this sessing C15 (46.8%), C16 (7.2%), C17 (29.9%), and C19 (7.5%)
concentration. side saturated chains as well as one C19 monounsaturated side
chain phenol (8.6%). In all cases, the mass spectra of the methyl
2.7. Cytotoxic test derivatives were consistent with those previously reported in the
literature (Mata et al., 1991).
Cellular toxicity was assessed by MTT method in peripheral
blood mononuclear cells (PBMC) as described before (Vega et 3.1. Antibacterial activity of anacardic acids and other
al., 1999). Briefly, PBMC were harvested and inoculated into isolated compounds from Amphipterygium adstringens
96-well microtitler plates at 105 cells/well, with various concen-
trations of anacardic acids mixture dissolved in DMSO (0.2% The effect of anacardic acids mixture, masticadienonic acid,
total volume). After incubation for 3, 24 and 48 h, 20 ␮l of 3␣-hydroxymasticadienonic acid, and 3-epi-oleanoic acid (200,
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide 100, 50, 12.5 and 6.25 ␮g/ml) on the growth of Helicobacter
(MTT, 5 mg/ml in PBS) was added for 3 h at 37 ◦ C. The for- pylori ATCC 43504 was determined using broth cultures. The
mazan dye was solubilized adding 100 ␮l DMSO to each well effect of ␤-sitosterol was not tested because of its insolubility
followed by gentle shaking and, the absorbance was determined in culture media. Only anacardic acids completely inhibited the
on an ELISA-plate reader at 545 nm. The absorption obtained growth of the bacteria; 3-epi-oleanoic acid inhibits 70% with
in DMSO treated cultures was considered as 100% viability. 50 ␮g/ml, and the other terpenoids did not inhibit its growth
(data not shown). It should be noted that all the terpenoids at the
2.8. Transmission electron microscopy

Cells were fixed at 4 ◦ C with 3% glutaraldehyde for 2 h. The


samples were rinsed with PBS buffer; post-fixed for 2 h at 4 ◦ C
using a 1% OsO4 solution in PBS; then gradually dehydrated
with ethanol series; and finally embedded in Epon resin. Ultra-
thin sections (90 nm) were mounted on copper grids and stained
with 2% uranyl acetate followed by 2% lead citrate and observed
under a JEOL 1200CXII electron microscope at an accelerating
voltage of 60 kV.

3. Results

Amphipterygium adstringens is a highly valued plant used


in traditional Mexican medicine for the treatment of gastritis Fig. 1. Bactericidal activity of anacardic acids mixture against Helicobacter
pylori. Broth cultures of Helicobacter pylori ATCC 43504 were exposed to
and ulcer. From a bio-guided assay of the fractions obtained
anacardic acids mixture at concentrations of 0 (), 12.5 (), 25 (), and 50
from a continuous Soxhlet extraction of the bark, it was found (䊉) ␮g/ml. Culture samples were taken at the indicated times, and viability was
that petroleum ether fraction exhibited antimicrobial activity measured by plate colony count technique. Reference antibiotics: metronidazole
against Helicobacter pylori (100 and 90% inhibition at 160 and (), 250 ␮g/ml and amoxicillin (), 0.1 ␮g/ml.
I. Castillo-Juárez et al. / Journal of Ethnopharmacology 114 (2007) 72–77 75

Fig. 2. Transmission electron micrograph of Helicobacter pylori culture exposed to anacardic acids mixture. (a) Typically curved bacteria in untreated controls are
seen. (b) Destroyed and empty envelops predominated after treatment for 8 h with 30 ␮g/ml anacardic acids mixture. (c) Magnification of the last condition.

highest tested concentration have solubility problems evidenced atively homogeneous (Fig. 2a). Cultures exposed to anacardic
as culture turbidity. The MIC100 determined for anacardic acids acids mixture at 30 ␮g/ml for 8 h revealed smaller degenerative
mixture by this method is 10 ␮g/ml; this antibacterial activity cells surrounded by discontinuous or broken membranes; and, in
is comparable with the one observed in the assay for reference some of them, the cytoplasm appeared to be separated from the
antibiotics metronidazole and amoxicillin (Table 1). cell wall. A considerable number of empty round membranes is
The killing kinetic of anacardic acids mixture is summarized also observed (Fig. 2b and c). These observations are consistent
in Fig. 1. The bactericidal effect is time and dose-dependent; with viability data and with the decrease observed in the culture
the number of viable Helicobacter pylori cells decreased by absorption.
exposure to anacardic acids mixture reaching total inhibition In order to determine if anacardic acids mixture could exert
in 15 h with 25 ␮g/ml, and in only 2 h with 50 ␮g/ml. Fig. 1 a toxic effect over human peripheral blood mononuclear cells
also shows the kinetic for metronidazole and amoxicillin; it is (PBMC), we treated resting human PBMC for 3, 24, and 48 h
interesting that with the first reference antibiotic, the maximal with eight concentrations of the mixture (0.01–50 ␮g/ml). The
bactericidal effect was reached in 8 h with 250 ␮g/ml, and in assay revealed that anacardic acids mixture did not affect the
23 h with 0.1 ␮g/ml of amoxicillin. viability of PBMC at 3 and 24 h of incubation; however, a slightly
It is remarkable that none of the tested compounds in (6.3–12.8%) not dose-dependent effect was observed at 48 h
this study revealed significant antibacterial activity against (Table 2).
Escherichia coli (data not shown).
The results of morphological analysis by transmission elec-
4. Discussion
tron microscopy showed that most of the untreated control cells
appeared as slightly curved bacilli, and the cytoplasm was rel-
In this study, we investigated the in vitro antibacterial activity
of various compounds isolated from Amphipterygium adstrin-
Table 2 gens bark against Helicobacter pylori. Among them, anacardic
Viability of PBMC treated for 48 h with anacardic acids mixture
acids mixture was the most effective in inhibiting bacterial
Anacardic acids mixture (␮g/ml) A545 %Viability growth, having a MIC of 10 ␮g/ml in broth cultures. This value is
0 1.27 ± 0.08 100.00 in the inhibitory range of reference antibiotics used to test antimi-
0.01 1.18 ± 0.07 92.96 crobial susceptibility and for the eradication of Helicobacter
0.05 1.18 ± 0.01 92.59 pylori.
0.1 1.11 ± 0.06* 87.49 Anacardic acids have been previously isolated from var-
0.5 1.15 ± 0.03* 90.24
1.0 1.19 ± 0.07 93.74
ious parts of Anacardium occidentale (Tyman, 1979), and
5.0 1.11 ± 0.04* 87.02 subsequently their diverse biological activities have also been
10.0 1.17 ± 0.02* 91.73 reported. For example, their molluscicidal (Sullivan et al., 1982),
50.0 1.17 ± 0.01* 92.28 antibacterial (Gellerman et al., 1969; Kubo et al., 1993a; Kubo
Absorbance results are expressed as the mean ± S.E., n = 3. *P < 0.05 compared et al., 1999), and cytotoxic (Kubo et al., 1993b) activities have
with untreated cells (Student’s t-test). been described. Anacardic acids are also known to inhibit vari-
76 I. Castillo-Juárez et al. / Journal of Ethnopharmacology 114 (2007) 72–77

ous enzymes such as prostaglandin endoperoxide synthase and siderably affected by them (Kubo et al., 1993a). However, the
lipoxygenase (Grazzini et al., 1991), ␣-glucosidase, invertase lipid composition of Helicobacter pylori has special features
and aldose reductase (Toyomizu et al., 1993), urease (Kubo et (Hirai et al., 1995) and may be specifically susceptible to this
al., 1999), and xanthine oxidase (Trevisan et al., 2006). kind of compounds.
It has been particularly demonstrated that some anac- The cytotoxic assay reveals that Amphipterygium adstringens
ardic acids exhibited a potent antibacterial activity against anacardic acids mixture did not affect significantly the viability
Gram-positive bacteria (MIC 0.78–100 ␮g/ml and in general, of PBMC. It was reported that the mixture of anacardic acid
increasing the MIC as decreased the double bonds in the methyl esters was not toxic to brine shrimp Artemia salina
chain) (Kubo et al., 1993a). Furthermore, the bactericidal activ- (LC > 1000 ppm) (Navarrete et al., 1989). The cytotoxic and
ity of cashew apple (C15:3 ), (C15:2 ), (C15:1 ), anacardic acids, genotoxic effects of Amphipterygium adstringens C19:0 anac-
and the synthetic analogues (C15:0 ) and (C12:0 ) against the ardic acid and its methyl ester have been recently studied. The
Gram-negative Helicobacter pylori have also been studied. The first, at 10 mg/kg for 24 h, lowers the ratios of polychromatic ery-
long-chain phenols with C15:3 , C15:2 and C12:0 were the most throcytes to normochromatic erythrocytes in mice; the methyl
potent with MICs of 200 ␮g/ml when tested through the agar ester showed no cytotoxic activity. The administration of either
dilution method, and with MIBs of 800 ␮g/ml for C15:3 and of the compounds did not lead to chromosome damage at the
C12:0 . While the salicylic acid was inactive (Kubo et al., 1999). evaluated doses (Acevedo et al., 2006). It is interesting to note
Anacardic acids with a saturated alkyl side chain seem to that in the case of Anacardium occidentale, the apple anacardic
be relatively rare and to occur only in traces and mixed with acids content is 1.1 g/kg (Trevisan et al., 2006); people regu-
unsaturated analogues (Spencer et al., 1980). Nevertheless, it larly consume directly the fruit, as well as, different processed
has been reported the presence of anacardic acids with C15:0 , products derived from this plant, thus, it would seem that their
C16:0 , C17:0 C19:0 and one unsaturated, 6[15 (Z)-nonadecenyl]- toxicity may not be serious or that it has been overlooked. Nev-
salicylic acid, (C19:1 ) in Amphipterygium adstringens (Mata et ertheless, the type of anacardic acids of Anacardium occidentale
al., 1991). is different to that of Amphipterygium adstringens.
The anacardic acid mixture utilized in this work, enriched in Conventional therapies suppress not only Helicobacter pylori
saturated alkyl phenolic acids, represents a novel source with but also the intestinal bacterial flora which produces side-effects
potent anti-Helicobacter pylori activity. The length of the side as abdominal pain and diarrhoea. In regards to this, since anac-
chains of three of these compounds (C16:0 , C17:0 C19:0 ) present in ardic acids seem to have a restricted antibacterial spectrum
the mixture isolated from Amphipterygium adstringens is larger (Gellerman et al., 1969; Kubo et al., 1993a), these compounds
and different than the ones of anacardic acids previously tested arise as an option in the management of the bacterial infection.
for antimicrobial activity (Gellerman et al., 1969; Kubo et al., On the other hand, Amphipterygium adstringens bark appears
1993a; Kubo et al., 1999); therefore, the specific effect of the as a candidate for the development of an integral treatment of
length chain should be further explored. Helicobacter pylori diseases because, as it has been demon-
Kubo et al. (1999) observed that when the MIC was deter- strated in this work, it possesses an antibacterial capacity due
mined through the agar dilution method, the two saturated side to anacardic acids in addition to a gastroprotective and anti-
chain anacardic acids, C15:0 and C12:0 , were hardly soluble in inflammatory activity due to its terpenoids as has also been
the water-based medium; consequently, an exact value could not previously proved (Olivera Ortega et al., 1999; Arrieta et al.,
be established unequivocally. As these compounds are organic 2003; Oviedo-Chavez et al., 2004).
solvent soluble, we think that the best way to test their antibac- In conclusion, anacardic acids derived from Amphipterygium
terial activity is through the broth method as it is easier for adstringens have an antibacterial activity against Helicobacter
them to reach the membranes rather than with the agar dilution pylori. These compounds together with proton pump inhibitors
one, where they are dissolved in the agar matrix first, making could be potentially used as a therapy against Helicobacter
their diffusion to the membranes less effective. In fact, when we pylori. Nevertheless, it is necessary to carry out further in vivo
tested the Amphipterygium adstringens anacardic acids inhibi- experiments to determine the therapeutic value of these com-
tion through the agar dilution method recommended by NCCLS pounds. The precise antibacterial action of these compounds
the compound made aggregates in the plate. Hence, the broth remains to be elucidated.
method could be useful to test this and other hydrophobic com-
pounds. Acknowledgements
The lipophilic nature of these compounds suggests an inter-
action with cell membranes. Anacardic acids are salicylic acid The authors are grateful to Dr. Rachel Mata for her critical
derivatives with a linear long alkyl chain and the salicylic acid feedback and generous support. The authors also thank Dr. Libia
moiety gives them some degree of hydrophilicity. It has been Vega Loyo, Sección Externa de Toxicologı́a, CINVESTAV-IPN,
demonstrated that four anacardic acids (C15:0 , C15:1 , C15:2 and for her help in the cytotoxicity assay; as well as M.S. Edelmira
C15:3 ) may act as protonophores, and when the carboxyl group Linares, and Ph.D. Robert Bye, Jardı́n Botánico, Instituto de
in anacardic acid was removed, the uncoupling activity dra- Biologı́a, Universidad Nacional Autónoma de México, for the
matically decreased (Toyomizu et al., 2000). Nevertheless, this taxonomical identification of the plant; M.S. Silvia Escobedo
uncoupler action may not be the only mechanism of action since for technical assistance; and, Rodolfo Paredes for assistance in
Escherichia coli and other Gram-negative bacteria are not con- the electron microscope studies.
I. Castillo-Juárez et al. / Journal of Ethnopharmacology 114 (2007) 72–77 77

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