articulo de propóleos y trofozoitoz de guardia lamblia

1. Complimentary and personal copy for
Samuel Alday-Provencio, Gabriela Diaz, Lucila Rascon,
Jael Quintero, Efrain Alday, Ramón Robles-Zepeda,
Adriana Garibay-Escobar, Humberto Astiazaran,
Javier Hernandez, Carlos Velazquez
www.thieme.com
Sonoran Propolis and Some of
its Chemical Constituents Inhibit
in vitro Growth of Giardia lamblia
Trophozoites
DOI 10.1055/s-0035-1545982
Planta Med
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2. Introduction
!
Giardia lamblia (Lambl, 1859) (Kofoid and Christi-
ansen, 1915) (syn. Giardia intestinalis, Giardia du-
odenalis) is a flagellated protozoan that inhabits
the upper small intestine of humans and other
mammals and is the causal agent of giardiasis [1].
It is one of the most frequent causes of intestinal
disease worldwide [2]. This parasitosis has a clin-
ical manifestation that varies from asymptomatic
infection to acute or chronic infection, and is asso-
ciated with diarrhea, flatulence, and abdominal
pain, among other symptoms. G. lamblia infection
is transmitted by contaminated water or food in-
gestion with G. lamblia cysts (infective form).
Among drugs used to control this parasitic infec-
tion are the members of the imidazole family such
as albendazole, metronidazole, and imidazole; 5-
nitrofuranes, acrydines, quinolones, and amino-
glycosides are also used [3]. The high prevalence
of giardia infection as well as the inadequate drug
administration could favor the emergence of re-
Abstract
!
Propolis is a cereus resin with a complex chemical
composition that possesses a wide range of bio-
logical activities. The aim of this study was to
evaluate the in vitro anti-Giardia lamblia activity
of Sonoran propolis collected from three different
areas of Sonoran Desert in northwestern Mexico
(Caborca, Pueblo de Alamos, and Ures) and some
of its chemical constituents. Additionally, we also
analyzed the seasonal effect on the anti-G. lamblia
activity of propolis. G. lamblia trophozoite cul-
tures were treated with different concentrations
of Sonoran propolis or chemical compounds dur-
ing 48 h cell proliferation and cell viability were
determined. Ures propolis showed the highest in-
hibitory activity against G. lamblia (IC50 63.8 ±
7.1 µg/mL) in a dose-dependent manner (Ures >
Pueblo de Alamos > Caborca). Season had a signif-
icant effect on the in vitro anti-G. lamblia activity
of Ures propolis. Summer propolis showed the
highest inhibitory effect on the G. lamblia tropho-
zoite growth (IC50 23.8 ± 2.3 µg/mL), followed by
propolis collected during winter (IC50 59.2 ±
34.7 µg/mL), spring (IC50 102.5 ± 15.3 µg/mL), and
autumn (IC50 125.0 ± 3.1 µg/mL). Caffeic acid phe-
nethyl ester, an Ures propolis exclusive constitu-
ent, had the highest growth-inhibitory activity
towards G. lamblia [IC50 63.1 ± 0.9 µg/mL (222.1 ±
3.2 µM)]. To our knowledge, this is the first study
showing that caffeic acid phenethyl ester pos-
sesses antiparasitic activity against G. lamblia.
Naringenin [IC50 125.7 ± 20.7 µg/mL (461.8 ±
76.3 µM)], hesperetin [IC50 149.6 ± 24.8 µg/mL
(494.9 ± 82.2 µM)], and pinocembrin [IC50 174.4 ±
26.0 µg/mL (680.6 ± 101.7 µM)] showed weak
anti-G. lamblia activity. On the other hand, chrys-
in and rutin did not show significant antiparasitic
activity. In conclusion, our results suggest that So-
noran propolis and some of its chemical constitu-
ents had inhibitory effects on the in vitro growth
of G. lamblia trophozoites.
Abbreviations
!
AP: autumn propolis
CAPE: caffeic acid phenethyl ester
CP: Caborca propolis
PAP: Pueblo de Alamos propolis
SP: spring propolis
SuP: summer propolis
UP: Ures propolis
WP: winter propolis
Supporting information available online at
http://www.thieme-connect.de/products
Sonoran Propolis and Some of its
Chemical Constituents Inhibit in vitro Growth
of Giardia lamblia Trophozoites
Authors Samuel Alday-Provencio1
, Gabriela Diaz2
, Lucila Rascon1
, Jael Quintero1
, Efrain Alday1
, Ramón Robles-Zepeda1
,
Adriana Garibay-Escobar1
, Humberto Astiazaran3
, Javier Hernandez4
, Carlos Velazquez1
Affiliations The affiliations are listed at the end of the article
Key words
l" Giardia lamblia
l" propolis
l" seasonal effect
l" chemical compounds
received Dec. 13, 2014
revised March 28, 2015
accepted April 1, 2015
Bibliography
DOI http://dx.doi.org/
10.1055/s-0035-1545982
Published online
Planta Med © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Dr. Carlos Velazquez
University of Sonora
Department of
Chemistry-Biology
Blvd. Luis Encinas y Rosales s/n.
Hermosillo
Sonora 83000
Mexico
Phone: + 526622592163
Fax: + 526622592163
velaz@guayacan.uson.mx
Alday-Provencio S et al. Sonoran Propolis and… Planta Med
Original Papers
Electronicreprintforpersonaluse

3. sistant Giardia strains [4,5]. For this reason, the identification of
potential new drug candidates for anti-G. lamblia is of paramount
importance [3,6].
Propolis is a resinous material collected by bees (Apis mellifera)
from different plant exudates [7]. Over 300 chemical compounds
have been identified in propolis and among them are aglycans
flavonoids, phenolic acids and esters, p-coumaric prenylated ac-
ids, and ketophenones. Other identified compounds in propolis
are lignans, di- and triterpenic acids, caffeoylquinic acids, mono-
terpens, sesquiterpens, isoflavonoids, chalcones, and pterocar-
pans [8–10]. Propolis has a wide spectrum of biological activities
such as anti-inflammatory, antiproliferative, antimicrobial, anti-
oxidant, and antiparasitic, among others [9–16]. Previously, we
have shown that Sonoran propolis possesses a strong antiprolif-
erative activity against different cancer cell lines [12,17]. It has
been demonstrated that the chemical composition of propolis
can vary during the different seasons of the year, which could
modify its biological activities [17–19].
There are limited data available about the antiparasitic activities
of propolis. It has been reported that Brazilian propolis inhibited
the growth of Trypanosoma cruzi [7,13], Trichomonas vaginalis
[20], and Leishmania amazonensis [20–22]. Furthermore, propo-
lis has immunoregulatory properties on peripheral blood mono-
nuclear cells from leishmaniasis patients [23]. The enhancing ef-
fect of propolis on immune response in an experimental mouse
model of giardiasis, also resulting in the reduction of the parasite
load, has been demonstrated [24]. Additionally, it has been re-
ported that Brazilian propolis inhibited the in vitro proliferation
of G. lamblia trophozoites [14].
The aim of the present study was to evaluate the antiparasitic ac-
tivity of Sonoran propolis collected in three different geographic
areas of Sonora, Mexico, and commercial samples of some of its
chemical constituents as well as to analyze the seasonal effect
on the anti-G. lamblia activity of Sonoran propolis collected at
Ures.
Results
!
In order to evaluate the antiparasitic activity of Sonoran propolis
on G. lamblia trophozoites, we used several concentrations of
propolis extracts (0, 25, 50, 100, and 200 µg/mL). UP showed the
highest inhibition of G. lamblia trophozoite growth (IC50
63.8 ± 7.1 µg/mL), followed by PAP (IC50 222 ± 68.4 µg/mL). The
CP extract did not show any significant effect on G. lamblia prolif-
eration. The antiparasitic drug albendazole (10 µM) was used as a
growth inhibition control, which inhibited more than 90% of the
trophozoite proliferation. DMSO did not show any significant ef-
fect on proliferation or viability of G. lamblia trophozoites
(l" Fig. 1).
Microscopic analysis showed that UP induced evident morpho-
logical changes on G. lamblia trophozoites. Control G. lamblia cul-
tures showed trophozoite monolayers attached to the surface
tubes, showing slightly elongated trophozoites (l" Fig. 2A,B).
After incubation (48 h) with albendazole (l" Fig. 2C) and UP
(l" Fig. 2D to G), the trophozoites were detached from the surface
of the culture tubes, and some of them acquired a round and
elongated form. Additionally, in cultures with detached tropho-
zoites (60–80%), we observed abundant cellular debris as well as
swollen trophozoites with slow movements, mainly at the high-
est propolis concentration evaluated (200 µg/mL). PAP and CP ex-
tracts did not show evident morphological changes on tropho-
zoites at the propolis concentrations tested (data not shown).
Since UP showed the highest growth-inhibitory activity on G.
lamblia trophozoites, we decided to investigate the seasonal ef-
fect on the antiparasitic activity of UP. We collected propolis dur-
ing the different seasons of the year in the region of Ures, Sonora
(see Materials and Methods). The collected seasonal samples of
propolis (SP, SuP, AP, and WP) were evaluated at different concen-
trations (0, 25, 50, 100, and 200 µg/mL). All propolis extracts
showed antiparasitic activity. The SuP extract showed the highest
anti-G. lamblia activity (IC50 23.8 ± 2.3 µg/mL), followed by the
WP extract (IC50 59.2 ± 34.7 µg/mL), SP (IC50 102.5 ± 15.3 µg/mL),
and AP (IC50 125.0 ± 3.1 µg/mL). DMSO did not affect G. lamblia
trophozoite growth (l" Fig. 3).
In order to understand more about the antiparasitic activity of
UP, we evaluated the growth-inhibitory effect of some of the
main chemical constituents of Sonoran propolis on G. lamblia
trophozoites. Previously, we identified phenolic compounds,
mostly flavonoids, as the main constituents of Sonoran propolis
(UP, PAP, and CP) (l" Table 1) [12]. Therefore, the antiparasitic
effect of six chemical constituents of Sonoran propolis (CAPE,
naringenin, hesperetin, pinocembrin, chrysin, and rutin) was
evaluated on G. lamblia trophozoites. CAPE was the chemical
constituent that showed the highest anti-G. lamblia activity
[IC50 63.1 ± 0.9 µg/mL (222.1 ± 3.2 µM)], followed by naringenin
[IC50 125.7 ± 20.7 µg/mL (461.8 ± 76.3 µM)]. Hesperetin [IC50
149.6 ± 24.8 µg/mL (494.9 ± 82.2 µM)] and pinocembrin [IC50
174.4 ± 26.0 µg/mL (680.6 ± 101.7 µM)] had weak inhibitory ac-
tivity. In contrast, chrysin and rutin did not show antiparasitic ac-
tivity at the evaluated concentrations (l" Fig. 4).
Fig. 1 Antiparasitic
activity of Sonoran
propolis. The antipara-
sitic activity of the So-
noran propolis was
evaluated on the in vitro
growth of G. lamblia
trophozoites at differ-
ent concentrations (0,
25, 50, 100, and
200 µg/mL). Propolis
tested: A UP, B PAP,
C CP. Albendazole was
used as a growth inhibi-
tion control, which in-
hibited more than 90%
(IC90 10 µM) of the
trophozoite prolifera-
tion. The tests were
performed in triplicate
(± SD) and at least two
experiments in each
condition were done.
The antiparasitic activ-
ity was evaluated at
48 h after the addition
of propolis extract. The
asterisks on the bars
show significant differ-
ences (p < 0.05) with
respect to the control
culture.
Alday-Provencio S et al. Sonoran Propolis and… Planta Med
Original PapersElectronicreprintforpersonaluse

4. Discussion
!
In this study, we evaluated the antiparasitic activity of Sonoran
propolis collected from three different areas of Sonora, Mexico,
and some of its chemical constituents. Additionally, we analyzed
the seasonal effect on the growth-inhibitory activity of Sonoran
propolis on G. lamblia trophozoites. Our data showed that UP
had the highest growth-inhibitory activity on G. lamblia tropho-
zoites, followed by PAP with moderate activity, and finally CP,
which did not show any anti-G. lamblia activity at the tested con-
centrations. The difference in the antiparasitic activity of Sonoran
propolis with a dissimilar geographical origin can be explained
by qualitative and quantitative differences in the chemical com-
position of propolis [25–30].
The antiparasitic activity of propolis has been determined in dif-
ferent parasites, including T. cruzi, L. amazonensis, T. vaginalis,
and G. lamblia [7,13,14,20–22]. Specifically on G. lamblia (BTU-
10 strain recovered from an asymptomatic host), Brazilian prop-
olis showed antiparasitic activity at 125 µg/mL (IC50) [14]. Inter-
estingly, our data showed that UP had a higher anti-G. lamblia ac-
tivity (GS/M‑83-H7 strain, ATCC 50581) than Brazilian propolis
[twice effective (IC50 63.8 ± 7.1 µg/mL)]. These differences could
be due to the propolis chemical composition. Brazilian propolis
is mainly constituted by phenolic compounds (flavonoids, aro-
matic acids, benzopyranes), di- and triterpenes, and essential
oils, among others, where artepillin C is its major constituent
[14,31], while UP constitution is mainly comprised by flavonoids,
phenolic acids and its esters, with pinocembrin being its major
constituent [12,17]. In addition, it is important to notice that dif-
ferent G. lamblia strains were evaluated against Brazilian and UP
propolis; thus, the differences found in the anti-Giardia activity
of propolis could be due to the susceptibility of the G. lamblia
strains used in both studies.
We observed that season had an important effect on the anti-G.
lamblia activity of UP. The propolis collected in summer (SuP)
showed the highest parasite growth-inhibitory activity, followed
by WP > SP > AP. Despite the observed differences on the biolog-
ical activity, season did not have a significant effect on the chem-
ical profile of UP (Table 1S, Supporting Information) [17]. The dif-
Fig. 3 Seasonal effect on the growth-inhibitory
activity of propolis on G. lamblia trophozoites. The
seasonal effect was evaluated by using extracts of
propolis collected during different seasons of the
year. Propolis was evaluated at different concentra-
tions (0, 25, 50, 100, and 200 µg/mL). The propolis
collected in A SuP, B WP, C SP, and D AP inhibited
the growth of G. lamblia trophozoites. Albendazole
was used as a growth inhibition control, which in-
hibited more than 90% (IC90 10 µM) of the tropho-
zoite proliferation. The tests were performed in
triplicate (± SD) and at least two experiments in
each condition were done. The antiparasitic activity
was evaluated at 48 h after the addition of propolis
extract. The asterisks on the bars show significant
differences (p < 0.05) with respect to the control
culture.
Fig. 2 Morphological changes induced by UP on G. lamblia trophozoites.
In order to observe the morphological changes induced by UP on G. lamblia
trophozoites, photographs were taken in an inverted optical microscope
(100X, total magnification). A Cells in monolayer, B control DMSO
(0.06%), C albendazole (IC90 10 µM), D UP (200 µg/mL), E UP (100 µg/
mL), F UP (50 µg/mL), and G UP (25 µg/mL).
Alday-Provencio S et al. Sonoran Propolis and… Planta Med
Original Papers
Electronicreprintforpersonaluse

5. ferences found on anti-G. lamblia activity of seasonal UP could be
due to small quantitative variations in its constituents, which are
secondary metabolites generated by the botanical source under
the influence of climatic conditions of the Ures region. Another
possible explanation for those observations could be quantitative
variations of unidentified compounds with potent anti-G. lamblia
activity in the seasonal propolis tested. Further experiments are
needed to know the chemical basis of the seasonal effect on anti-
G. lamblia activity of the Sonoran propolis.
In this study, we evaluated some chemical constituents present
in the Sonoran propolis. Pinocembrin and chrysin are com-
pounds present in the three propolis samples evaluated. CAPE is
a UP exclusive constituent, while rutin, naringenin, and hespere-
tin are exclusive constituents of PAP (l" Table 1). CAPE was the
most effective chemical constituent tested of Sonoran propolis
and showed the highest growth-inhibitory activity on G. lamblia
trophozoites [IC50 63.1 µg/mL (222.1 µM)]. These results could
suggest that CAPE is one of the chemical constituents involved
in the anti-G. lamblia activity of UP. However, the antiparasitic ac-
tivity of the control drug albendazole (IC90 10 µM) was more ef-
fective than CAPE. It is important to consider that propolis has a
complex chemical composition, where CAPE represents only
1.14% of the UP total weight (Table 1S, Supporting Information).
When we compared the antiparasitic activity (IC50) of CAPE
(63.1 ± 0.9 µg/mL) and UP (63.8 ± 7.1 µg/mL), it was evident that
UP is more efficient than CAPE, suggesting that there are other
compounds in UP that could be involved in anti-G. lamblia activ-
ity. Further experiments need to be conducted in order to estab-
lish the chemical basis of anti-G. lamblia activity of this natural
product and CAPE. To our knowledge, this is the first work that
reports the antiparasitic activity of CAPE.
Pinocembrin is widely distributed in nature, and it is one of the
main components of the propolis of various geographic areas,
which presents a broad spectrum of biological activities [9,32,
Fig. 4 Antiparasitic activity of Sonoran propolis chemical constituents. The
anti-G. lamblia activity of several chemical constituents of Sonoran propolis
were evaluated at different concentrations (0, 25, 50, 100, and 200 µg/mL or
its equivalent in µM). A CAPE (0, 88, 176, 352, and 703 µM), B naringenin
(0, 92, 184, 367, and 734 µM), C hesperetin (0, 83, 165, 331, and 662 µM),
D pinocembrin (0, 97, 195, 390, and 780 µM), E chrysin (0, 89, 197, 393,
and 787 µM), and F rutin (0, 41, 82, 164, and 328 µM). Albendazole was used
as a growth inhibition control, which inhibited more than 90% (IC90 10 µM) of
the trophozoite proliferation. The tests were performed in triplicate (± SD)
and at least two experiments in each condition were done. The antiparasitic
activity was evaluated at 48 h after the addition of propolis extract. The as-
terisks on the bars show significant differences (p < 0.05) with respect to the
control culture.
Table 1 Identification and quantification of the major constituents of Sonoran propolis by HPLC‑DADa
.
Compound R.T. (min) λmax Propolisb
UP PAP CP
Rutin 22.1 256, 355 N.d. 4.1 ± 0.0 N.d.
Naringenin 30.9 292 N.d. 21.9 ± 0.1 N.d.
Hesperetin 33.1 243, 289 N.d. 3.7 ± 0.0 N.d.
Pinocembrin 46.7 243, 294 218.4 ± 1.5 62.7 ± 1.3 60.6 ± 1.2
CAPE 52.3 242, 328 11.4 ± 0.5 N.d. N.d.
Chrysin 54.0 270, 313 58.9 ± 0.6 6.3 ± 0.1 11.4 ± 0.8
All values represent the mean of triplicate determinations ± standard deviation; a
Data obtained from Hernandez et al. [16]; b
Quantification (mg/g) propolis; N.d.: Not detectable
Alday-Provencio S et al. Sonoran Propolis and… Planta Med
Original PapersElectronicreprintforpersonaluse

6. 33]. Pinocembrin is the most abundant constituent in UP
(218.4 mg/g) and has weak antiparasitic activity (IC50 174.4 ±
26.0 µg/mL or 680.6 ± 101.7 µM), suggesting that pinocembrin is
not properly related to the antiparasitic activity of UP. The exclu-
sive constituents of PAP, naringenin (IC50 125.7 ± 20.7 µg/mL or
461.8 ± 76.3 µM), and hesperetin (IC50 149.6 ± 24.8 µg/mL or
494.9 ± 82.2 µM) showed significant anti-G. lamblia activity. Our
data suggested that naringenin and hesperetin are involved in
the anti-G. lamblia activity of PAP. Additional studies will be
needed to determine the structural-activity relationship of these
chemical compounds of Sonoran propolis.
In conclusion, our results suggest that Sonoran propolis and some
of its chemical constituents had an effect on the in vitro growth of
G. lamblia trophozoites. Additionally, season had a significant ef-
fect on the anti-G. lamblia activity of Sonoran propolis. Although
the antiparasitic activity of propolis from other sources is known,
to our knowledge, this is the first study reporting the antipara-
sitic activity of propolis collected from arid and semidesert areas
of Sonora, Mexico.
Materials and Methods
!
Giardia lamblia culture
G. lamblia trophozoites were obtained from the American Type
Culture Collection (GS/M‑83-H7, ATCC 50581), and were axeni-
cally maintained in TYI‑S‑33 culture medium supplemented with
10% newborn calf serum (NBCS; Gibco) with antibiotics (ceftriax-
one 100 µg/mL) at 37°C [34].
Propolis and methanolic extracts from propolis
Sonoran propolis was collected in different arid and semiarid re-
gions in northwest Mexico (Sonora), Pueblo de Alamos, Caborca,
and Ures regions [11,12]. PAP was collected between June 2001
and February 2003 (N29°15′, W 109°59′), CP (N30°46′, W 112°
05′) was collected between March and June 2006, and UP (N29°
27′, W 110°22′398) was collected between March 2008 and
March 2009. Previously, we observed that there were not qualita-
tive variations in the chemical composition of UP during the year
(Fig. 1S, Supporting Information) [17]. Therefore, to perform the
antiparasitic activity of Sonoran propolis on G. lamblia, we de-
cided to use UP collected in summer (2008) to compare with
PAP and CP. To evaluate the seasonal effect on the antiparasitic ac-
tivity of Sonoran propolis, UP was collected in different seasons
of the year (SuP: June 22, 2008 to September 22, 2008; SP: March
21, 2008 to June 21, 2008; AP: September 23, 2008 to December
21, 2008; and WP: December 22, 2008 to March 20, 2009). Sea-
sonal propolis was collected from several hives in the Ures area,
as reported by Valencia et al. 2012. The propolis extractions were
performed according to Hernandez et al. 2007 and Valencia et al.
2012 and, subsequently, the propolis methanolic extracts were
stored at − 80°C [12,17].
Sonoran propolis chemical compounds
The following authentic flavonoids standards were commercially
acquired from INDOFINE: naringenin, hesperetin, chrysin (≥ 98%
purity), and rutin (≥ 95% purity). Albendazole (≥ 98% purity) was
acquired from Sigma-Aldrich. Pinocembrin (≥ 95% purity) was
purified from UP in our lab (HPLC‑DAD‑MS). CAPE (≥ 97% purity)
was synthetized by our group [35].
Antiparasitic activity of Sonoran propolis
and chemical constituents
In order to evaluate the growth-inhibitory capacity of propolis or
chemical compounds against G. lamblia trophozoites, we used
propolis obtained from three different Sonoran regions (PAP, CP,
and UP) and some of its main chemical constituents (CAPE, nar-
ingenin, hesperetin, pinocembrin, chrysin, and rutin). G. lamblia
trophozoites cultures (1 × 105
/tube) were incubated at 37°C for a
period of 12 h in 13 × 100 glass tubes with a screw cap. Then,
trophozoite cultures were treated with different concentrations
of propolis or chemical compounds (0, 25, 50, 100, and 200 µg/
mL) during 48 h. Each experiment was performed in triplicate,
and at least two experiments were done in each condition eval-
uated. Dimethyl sulfoxide (DMSO; Sigma-Aldrich) was used as a
solvent control, and albendazole (10 µM) as a positive control
for parasite inhibition. Cell proliferation and cell viability were
determined by using a Neubauer chamber and by trypan blue
(Sigma-Aldrich) dye exclusion assay, respectively. The results
were analyzed using GraphPad Prism v5.01 software.
Microscopic analysis
Morphological changes induced by Sonoran propolis on G. lam-
blia trophozoites were observed and photographed at 100× on a
Nikon inverted optical microscope (Inverses Mikroskop Eclipse
TS 100) and analyzed with Lumera Corporation Infinity Analyze
software (Current revision 6.0.0).
Statistical analysis
Data were processed and analyzed by the nonparametric method
of Kruskal-Wallis. The statistical analyses were made in SPSS
software. Statistical significance was accepted when p < 0.05.
Supporting information
HPLC‑DAD/UV chromatograms of UP, and the chemical structures
and relative abundance of its constituents are available as Sup-
porting Information.
Acknowledgements
!
We are grateful to the professional beekeeper Gilberto Valenzuela
for all the facilities provided during the collection of propolis. We
appreciate the support provided for the partial realization of this
work by the project CONACYT (CB-2010-01/155224).
Conflict of Interest
!
The authors declare that they have no financial or nonfinancial
competing interests.
Affiliations
1
Department of Chemistry-Biology, University of Sonora, Hermosillo, Sonora,
Mexico
2
Department of Chemistry-Biology and Agropecuary Sciences, University of
Sonora, Navojoa, Sonora, Mexico
3
Department of Nutrition and Metabolism, Centro de Investigación en
Alimentación y Desarrollo A.C. Hermosillo, Sonora, México
4
Unidad de Servicios de Apoyo en Resolución Analítica, Universidad
Veracruzana, Xalapa, Veracruz, México
Alday-Provencio S et al. Sonoran Propolis and… Planta Med
Original Papers
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