JOURNAL OF MEDICINAL FOOD

J Med Food 00 (0) 2018, 1–8

# Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2017.0137

Anti-Helicobacter pylori Potential of Three Edible Plants

Known as Quelites in Mexico
Erika Gomez-Chang,1 Guadalupe Vanessa Uribe-Estanislao,1
Maricruz Martinez-Martinez,1 Amanda Gálvez-Mariscal,2 and Irma Romero1
1
Department of Biochemistry, Faculty of Medicine, Universidad Nacional Autónoma de México,
Ciudad Universitaria, Cd. Mx., México.
2
Department of Food and Biotechnology, Faculty of Chemistry,
Universidad Nacional Autónoma de México, Ciudad Universitaria, Cd. Mx., México.
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ABSTRACT ‘‘Quelites’’ are edible plants that are part of the traditional agro-ecosystems in Mexico. These plants, despite
their already known nutritional properties, are now considered neglected and underutilized species. With the objective of
promoting their reinsertion in the markets and mainly, in daily diets, efforts have been made to study them from multidis-
ciplinary approaches to demonstrate their beneficial properties. To generate evidence of an added health-promoting value that
would encourage quelites consumption, in the present work, the anti-Helicobacter pylori activity of three representative
quelite species, Anoda cristata (Alache), Cnidoscolus aconitifolius (Chaya), and Crotalaria pumila (Chepil), was tested. H.
pylori is considered the etiological agent of gastritis, ulcer, and gastric cancer, and represents a public health problem in
Mexico and worldwide. Aqueous (AQ) and dichloromethane–methanol (DM) extracts were obtained from the three species of
quelites to investigate their effect on H. pylori growth and on two of its colonization factors (adherence and urease activity).
DM extracts from Chaya, Chepil, and Alache exert the best inhibitory effect on bacterial growth, with minimum inhibitory
concentrations of 62.5, 125, and 250 lg/mL, respectively. AQ and DM extracts inhibit bacterial adhesion by 30% to 50%.
None of them has an effect on urease activity. The two flavonoids present in A. cristata, acacetin and diosmetin, inhibit H.
pylori growth by *90% with 3.9 lg/mL. These results provide new information about the anti-H. pylori potential of three
edible quelites, and give an added value, since their routine consumption may impact on the prevention and/or control of H.
pylori-associated diseases.

KEYWORDS:  acacetin  Anoda cristata  antibacterial  antiadherent  Cnidoscolus aconitifolius  Crotalaria

pumila  diosmetin  Helicobacter pylori  urease

INTRODUCTION The traditional agro-ecosystem in Mexico, known as

‘‘milpa,’’ houses besides the Mesoamerican triad (beans,
T he ‘‘quelites’’ (from the Nahuatl language quilitl,
comestible herb) are defined as edible green plants
usually derived from young and tender annual herbs; nev-
corn, and squash) several edible plants considered Ne-
glected and Underutilized Species (NUS),2 among which
quelites are included.3
ertheless, flowers, inflorescences, and stem tips of perennials
Currently in Mexico, quelites are used by different ethnic
may also be included. These plants are usually consumed
groups in all regions of the country. The form of con-
when they are immature and are often eaten raw or lightly
sumption is varied and, due to their importance in local
cooked in boiling water.1 The quelites have been consumed
traditions and to their nutritional value, these plants are
in Mexico from pre-Hispanic times to nowadays; however,
promising species with the potential to enrich nutrition in
in current diets, the populations tend to include only a few
areas of the country with greater food vulnerability and to
species of ‘‘major’’ crops (i.e., rice, maize, and wheat),
empower marginalized communities by strengthening their
which has led to the displacement of quelites and other
local identity.4 Unfortunately, in terms of production and
species of the so-called ‘‘minor’’ crops.
market value, the quelites, like other NUS, have a minor
significance and have been ignored by agricultural re-
searchers, plant breeders, and policymakers.
Manuscript received 29 October 2017. Revision accepted 10 June 2018. With the objective of rescuing the consumption of que-
lites and their reintegration in the Mexican markets and in
Address correspondence to: Irma Romero, PhD, Department of Biochemistry, Faculty of
Medicine, Universidad Nacional Autónoma de México, Ciudad Universitaria, Cd. Mx.,
the daily diet, a multidisciplinary project was initiated with
04510. México, E-mail: irma@bq.unam.mx the objective of studying the following three representative

1
 2 GOMEZ-CHANG ET AL.
native species of quelites: Anoda cristata (L.) Schltdl.,
Cnidoscolus aconitifolius (Mill.) I.M. Johnst., and Crota-
laria pumila Ortega, commonly known as Alache, Chaya,
and Chepil, respectively. In addition to their well-known use
as food, two of these species have been traditionally rec-
ognized for having beneficial health properties. In some
areas, A. cristata is used to treat fever, cough, skin wounds,
alopecia, renal disorders, and digestive ailments such as
stomachache.5–8 Moreover, some pharmacological studies
using different animal models have reported the hypogly-
cemic and antihyperglycemic effects of this plant.9 In the
case of C. aconitifolius (Chaya), its traditional usage in
muscular discomforts, rheumatism, and arthritis has been
documented. The consumption of Chaya has also been re-
ported for the treatment of other diseases such as diabetes,
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hypercholesterolemia, hypertension, renal and urinary ma-
ladies, and digestive disorders.5,6,8,10 Some pharmacological
approaches have assessed Chaya’s anti-inflammatory and
cardioprotective activities.11 In this context, in vivo experi-
ments in diabetic rats have demonstrated that a 4-week Chaya
aqueous (AQ) extract administration produced hypoglycemic
effects and was also helpful in reducing hyperlipidemia.12 More
recently, antiprotozoal, antibacterial, and anti-inflammatory
activities of a Chaya CHCl3-MeOH extract have been re-
ported.13 Regarding C. pumila (Chepil), in the XVI century,
this plant was traditionally used for skin conditions and cough;
however, nowadays these uses seem to be discontinued.5
In the present work, the anti-Helicobacter pylori potential
of the three mentioned quelites was explored.
H. pylori is a Gram-negative bacteria that colonizes the
stomach of more than half of the world’s population and is
the main etiological factor of gastritis, peptic ulcer, and
gastric cancer.14–16 H. pylori uses unique strategies (colo-
nization, virulence, and survival factors) to overcome the
protective elements of the gastric mucosa and to evade the
host immune response.17
For the present study, two extracts were obtained from
each one of the three species of quelites to test, in vitro, their
anti-H. pylori activity and the effect over two colonization
factors (urease and adhesion to gastric epithelial cells). In
addition, the anti-H. pylori activity of two flavonoids present
in A. cristata was investigated. The purpose of this work is
to generate evidence to give an added health-promoting
value to these plants, besides their already known nutritional
properties, which would encourage their consumption.
MATERIALS AND METHODS
Chemicals
Gallic acid, quercetin, and acacetin were purchased from
Sigma-Aldrich. Diosmetin was from Santa Cruz Biotechnol-
ogy. Analytical-grade solvents were from Merck.
Plant material
A. cristata (L.) Schtdl (Alache) was collected and iden-
tified by MS Edelmira Linares and Dr. Robert Bye (Instituto
de Biologı́a, UNAM) at Tepetlixpa, Estado de México
(18 580 57.500 N, 98 490 40.100 W), on June 2, 2015. A
voucher specimen is deposited in the Herbario Nacional
(MEXU 1445645) located at the Universidad Nacional
Autónoma de México. C. aconitifolius (Mill.) I.M. Johnst
(Chaya) was collected at Timucuy, Mérida, Yucatán (20
480 43.500 N, 89 300 56.800 W) by Dr. Amanda Gálvez and
identified by MS Clarisa Jiménez (Unidad de Recursos
Naturales del Centro de Investigación Cientı́fica de Yuca-
tán) on August 28, 2015. A voucher specimen is deposited in
the Herbario CICY (068784). C. pumila Ortega (Chepil) was
collected at Ocotlán de Morelos, Oaxaca (16 480 30.700 N,
96 400 21.300 W), on September 2, 2015, and identified by
MS Magali Martı́nez. A voucher specimen is deposited in
the Herbario Nacional (MEXU 1457315).
Extract preparation
The leaves were separated and cleaned. After air drying,
the material was pulverized. The dichloromethane–methanol
(DM) (1:1) extracts were prepared by maceration of 50 g of
plant material in 500 mL of the solvent mixture for 72 h (this
procedure was repeated three more times). The solvents were
filtered and evaporated to dryness under reduced pressure to
obtain the DM extracts. The AQ extracts were obtained by
preparing an infusion with 10 g of dried plant material in
500 mL of boiling distilled water and left to stand for 30 min.
After filtration the extract was lyophilized. A. cristata and
C. pumila contain mucilage, which was removed from the
filtered infusion by precipitation with ethanol (1:1, v/v). The
precipitated mucilage was collected by centrifugation and
then oven dried (70C, 5 h).
Quantification of total phenolic and flavonoid contents
For each extract, a 1 mg/mL stock solution was prepared
(methanol or bidistilled water was used as solvent for DM or
AQ extracts, respectively).
Total phenolic concentration was determined according to
Garcı́a-Rodrı́guez et al.11 with modifications. Briefly,
0.05 mL of each extract solution was mixed with 2.5 mL of
10% (v/v) Folin–Ciocalteu reagent and 2 mL of 7.5% (w/v)
Na2CO3. The absorbance was measured at 765 nm after a 15-
min incubation at 45C in a water bath. Gallic acid was used
for the standard calibration curve. Results were expressed as
lg of gallic acid equivalent (GAE) per mg of extract.
Total flavonoid quantification was performed according to
Formagio et al.18 with modifications. To 0.5 mL of each ex-
tract solution, 1.5 mL of methanol was added and then mixed
with 0.01 mL of 10% AlCl3 (w/v) and 0.1 mL of 1 M
CH3COONa $3H2O. The final volume was adjusted with
water to 5 mL. The absorbance was measured at 415 nm after
a 30-min incubation in the dark. Quercetin was used for the
standard calibration curve. Results were expressed as lg of
quercetin equivalent per mg of extract.
Anti-H. pylori activity
H. pylori strain ATCC 43504 was cultured on Casman
agar base as previously reported.19
 ANTI-H. PYLORI ACTIVITY OF EDIBLE QUELITES
The antibacterial activity of the extracts, acacetin, dios-
metin, and the reference antibiotics (amoxicillin and metro-
nidazole), was assessed by the broth dilution method using
Mueller-Hinton broth (DIFCO), supplemented with 0.2% b-
-cyclodextrin, 10 mg/L vancomycin, 5 mg/L trimethoprim,
2 mg/L amphotericin B, and 2.5 mg/L polymyxin B.19 A
volume of 0.01 mL of different concentrations of the extracts,
dissolved in bidistilled water or DMSO, was added to 1.5 mL
of H. pylori broth culture at the beginning of the exponential
growth phase (*108 CFU/mL). DA600 was measured after
24 h of incubation at 37C under microaerophilic conditions
and with shaking (150 rpm). The value obtained was used to
calculate the percentage of growth inhibition with respect to
a control that grew only in the presence of water or DMSO
(0.66% final concentration, without any effect on bacterial
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growth). The minimum inhibitory concentration (MIC) was
defined as the lowest concentration of the extract that inhibited
100% of bacterial growth.
Inhibition of bacterial adhesion to AGS cells
Human gastric adenocarcinoma cells (AGS) ATTCC
1739 were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 5% (v/v) heat-inactivated
FBS, 2 mM l-glutamine, 10 mg/mL insulin, 2 mM sodium
pyruvate, and 100 U/mL/100 mg/mL penicillin/streptomy-
cin in a humidified atmosphere with 5% CO2 at 37C.
H. pylori was collected from a liquid culture at the be-
ginning of the exponential growth phase, suspended in PBS
(1 · 109 UFC/mL), and incubated with 10 lL of 0.1% fluo-
rescein isothiocyanate (FITC) diluted in DMSO for 1 h at
37C in the dark. The excess FITC was removed with two
washing steps in PBS. Formerly, 1 · 105 AGS cells/well
were plated in 96-well plates with supplemented DMEM
and incubated for 24 h in a 5% CO2 atmosphere at 37C.
Subsequently, AGS cells were washed and infected with
1 · 108 FITC-labeled H. pylori in a final volume of 110 lL
of DMEM. After 1 h of incubation under 5% CO2 and 37C
in the presence or absence of the dissolved extracts (0.025–
1 mg/mL), AGS cells were washed with PBS for eliminating
nonadherent bacteria and the extract remnants. The adher-
ence was quantified fluorometrically by measuring the
FITC-labeled H. pylori bound to AGS cells (FITC excited at
460 nm and detected at 544 nm).
Obtention of urease
H. pylori cells were collected from a liquid culture at the
exponential growth phase and washed twice with PBS pH
7.2. Bacteria suspended in PBS-protease inhibitor cocktail
(cOmplete Roche) were lysed by sonication (Branson
Sonifier 250 at 40 W for 90 sec at 4C). The lysate was
centrifuged at 17,000 g at 4C and the supernatant was
collected and ultracentrifuged at 130,000 g at 4C. The su-
pernatant was used as the urease source and stored at 4C
until its utilization. Protein content was determined by
Lowry’s method.20
3
Urease inhibition assay
The urease activity was determined by measuring the
amount of ammonia released in the reaction, which was
detected by the Berthelot’s method with some modifica-
tions.21 The reaction mixture consisted of 2 lg protein of H.
pylori urease, 10 lL of the DM or AQ extracts dissolved in
DMSO or in bidistilled water, respectively (1.95–250.0 lg/
mL), and PBS in a final volume of 130 lL. The reaction was
initiated with the addition of 20 lL 37.5 mM urea (5 mM
final concentration). After a 10-min incubation at 37C, the
reaction was stopped by adding 50 lL of solution A
(0.714 M phenol). To start the colorimetric reaction, 100 lL
of solution B (0.714 M NaOH/0.357 M NaOCl) was added.
After a 5-min incubation at room temperature, the absor-
bance was read at 600 nm in a Bio-Rad 2550 EIA Reader.
(NH4)2SO4 was used as standard for the calibration curve.
The activity of noninhibited urease was considered 100%
enzyme activity (negative control). Acetohydroxamic acid
(AHA) was used as positive control. The percentage of ac-
tivity inhibition was calculated with respect to the negative
control.
Statistical analysis
All experiments were performed in triplicate and at least
three independent experiments were recorded. Data are
presented as mean – SEM. A one-way analysis of variance
followed by the Bonferroni’s multiple comparison test was
performed. A P value <.05 was considered to be statistically
significant.
RESULTS
Yields and total phenolic and flavonoid contents
of DM and AQ extracts
Table 1 shows the extraction yields obtained from A.
cristata, C. aconitifolius, and C. pumila, and the abbrevia-
tions assigned to each one of the extracts. The total phenolic
(TPC) and flavonoid contents (TFC) of each extract are also
presented. The TPC of the three analyzed quelites ranges
from 0.4% to 1.2% (per 100 g of dried plant), independently
of the solvent used for extraction. It can be observed that in
the AQ extracts, the TPC is higher in A. cristata and in C.
pumila, compared with the corresponding DM extracts,
whereas in the case of C. aconitifolius, the TPC is practically
the same with either solvent used. Regarding the TFC, the
range varies from 0.1% to 1%. However, a higher content of
flavonoids was quantified in the three species of quelites
when DM was used for extraction.
Anti-H. pylori activity
The effect of DM extracts against H. pylori growth is
shown in Figure 1. The results demonstrate that DM ex-
tracts obtained from the three species of quelites inhibited
the growth of H. pylori in a concentration-dependent man-
ner but with different effectiveness. The Cha-DM extract
exerted the highest inhibitory effect on H. pylori with an
 4 GOMEZ-CHANG ET AL.
Table 1. Abbreviation, Yield, and Total Phenolic and Flavonoid Contents of the Quelite Extracts
Extract
Species (common name) abbreviation Yielda (%)
A. cristata (Alache) Ala-DM 13.71
Ala-AQ 19.20b
C. aconitifolius (Chaya) Cha-DM 14.16
Cha-AQ 19.38
C. pumila (Chepil) Che-DM 18.55
Che-AQ 16.50b
Data are presented as mean – SEM of at least three independent experiments in triplicate.
a
Yields are expressed with respect to 100 g of dry plant material.
b
Mucilage-free yield.
AQ, aqueous extract; DM, dichloromethane–methanol extract; GAE, gallic acid equivalent; QE, quercetin equivalent.
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MIC = 62.5 lg/mL, followed by the DM-Che and DM-Ala
extracts, with MIC values of 125 and 250 lg/mL, respec-
tively (Table 2). The anti-H. pylori activity of DM extracts
obtained from C. aconitifolius and C. pumila was even more
effective than the reference antibiotic metronidazole (Fig. 1
and Table 2), but less effective than amoxicillin (Table 2).
With respect to AQ extracts, none of them had activity
against H. pylori. The same lack of antibacterial activity was
observed in the case of mucilages obtained from the AQ
extracts of Alache (Ala-MU) and Chepil (Che-MU). The
MIC values obtained for all the quelite extracts are sum-
marized in Table 2.
Antiadherent activity of H. pylori to AGS cells
All quelite extracts exhibited an incomplete inhibition. At
the highest tested concentration (1000 lg/mL), the three DM
extracts inhibited the adhesion around 50%, (49.8%, 50.2%,
and 47.6% for Alache, Chaya, and Chepil, respectively)
(data not shown). This percentage was almost achieved with
100 lg/mL of DM-Ala and DM-Che extracts (Table 2).
FIG. 1. Effect of DM extracts on H. pylori growth. The antibacterial
activity was assessed by broth dilution method. The percentage of growth
inhibition was calculated with respect to a control that grew only in the
presence of DMSO. Data are presented as mean – SEM of at least three
independent experiments in triplicate. Ala, A. cristata (Alache); Cha, C.
aconitifolius (Chaya); Che, C. pumila (Chepil); DM, dichloromethane–
methanol extract; Met, metronidazole (positive control).
Total phenolic Flavonoids
(lg GAE/mg dried extract) (lg QE/mg dried extract)
31.0 – 1.9 66.1 – 7.1
54.2 – 5.5 17.4 – 0.5
40.0 – 4.3 69.3 – 3.5
39.4 – 5.6 6.1 – 1.1
54.5 – 3.2 38.9 – 2.1
75.3 – 7.8 10.3 – 0.9
The AQ extracts, in general, were less efficient than DM
extracts at all the tested concentrations. Nevertheless, with
100 lg/mL, the Cha-AQ extract was more effective (33% of
adherence inhibition) than the corresponding DM extract
(Table 2). The mucilages studied also presented a low but
significant antiadherent activity.
Urease inhibition
In the last column of Table 2, it is shown that, compared
to the positive control, none of the quelite extracts provoked
a significant inhibitory effect on H. pylori urease activity,
even at the highest concentration tested (250 lg/mL, data
not shown).
Anti-H. pylori activity of acacetin and diosmetin
Figure 2 shows the effect of diosmetin and acacetin on H.
pylori growth. Acacetin shows an MIC value of 62.5 lg/mL,
meanwhile diosmetin inhibited a maximum of about 90% at
this concentration. Interestingly, the inhibitory effect is
practically achieved with a very low concentration. In the
case of acacetin, it inhibited the bacterial growth by 86.70%
with only 3.9 lg/mL, while diosmetin by 91.94%.
DISCUSSION
Antibiotic phytocomponents can offer an alternative for
the treatment of infectious diseases.22 In the present work, the
effect on H. pylori growth of some edible Mexican quelites
was investigated for determining whether they could have,
besides their nutritional value, some other health benefits.
Several previous reports, working with different plant ex-
tracts, have classified their anti-H. pylori activity from strong
to null according to their MIC values.23,24 Taking into account
these classifications, we considered five categories of anti-
bacterial activity depending on the MIC: (1) strong (MIC
£15.6 lg/mL); (2) good (MIC 15.6–125 lg/mL); (3) moderate
(MIC 125–300 lg/mL); (4) low (MIC >300–500 lg/mL); and
(5) null (MIC >500 lg/mL). From this perspective, all quelite
DM extracts exerted inhibition on H. pylori growth. The anti-
H. pylori activity produced by the Ala-DM extract can be
considered moderate, while both Cha-DM and Che-DM
 ANTI-H. PYLORI ACTIVITY OF EDIBLE QUELITES 5

Table 2. Summary of the Effect of Three Species of Quelites (Extracts and Mucilages) Against H. pylori
and Two Colonization Factors (Adhesion to Cells and Urease Activity)

Adhesion inhibitiona (%) Urease activity inhibitiona (%)

Species (common name) Extract or compound MIC (lg/mL) 100 lg/mL 125 lg/mL
A. cristata (Alache) Ala-DM 250.00 46.8 – 2.6 -7.2 – 0.9
Ala-AQ >500.00 25 – 2.0 1.2 – 0.9
Ala-MU >500.00 24.3 – 2.7 9.2 – 1.9
C. aconitifolius (Chaya) Cha-DM 62.50 23.5 – 3.4 9.6 – 3.3
Cha-AQ >500.00 33.8 – 2.5 -7.1 – 4.1
C. pumila (Chepil) Che-DM 125.00 41.4 – 6.3 9.8 – 1.5
Che-AQ >500.00 17.8 – 3.0 4.5 – 3.5
Che-MU >500.00 21 – 2.6 6.3 – 1.5
Positive control Amoxicillin 0.005b — —
Metronidazole 250.00b — —
Fucose 100 lg/mL — 21.7 – 6.5 —
Sialic acid 100 lg/mL — 20.2 – 5.8 —
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AHA 200 lg/mL — — 99.7 – 0.3

a
Data are presented as mean – SEM of at least three independent experiments in triplicate.
b
The MIC was defined as the lowest concentration of the extract that inhibited 100% of bacterial growth. MIC values are within the range reported by the Clinical
and Laboratory Standards Institute (CLSI) and were determined by broth dilution method.
AHA, acetohydroxamic acid; MIC, minimum inhibitory concentration; MU, mucilage obtained from the AQ extract.

extracts had a good activity. On the contrary, AQ extracts did
not have antibacterial activity (Table 2).
Some concentrations of DM extracts produced more than
100% of bacterial growth inhibition (Fig. 1), an effect that is
due to a decrease in the culture absorbance at the end of the
experiment with respect to the initial one. This absorption
decrement indicates that the extracts are not only promoting
inhibition of bacterial growth but also bacterial lysis.
When comparing the obtained MICs of DM extracts with
the MIC of amoxicillin (0.005 lg/mL), it is evident that
much higher concentrations of DM extracts are required to
inhibit bacterial growth; however, the antibacterial activity
displayed by each one of the DM extracts was equal or even
FIG. 2. Effect of the flavonoids acacetin and diosmetin on H. pylori
growth. The antibacterial activity was assessed by broth dilution
method. The percentage of growth inhibition was calculated with
respect to a control that grew only in the presence of DMSO. Data are
presented as mean – SEM of at least three independent experiments in
triplicate.
better than the other positive control, metronidazole
(250 lg/mL) (Table 2). The latter antibiotic is widely used in
the H. pylori eradication therapy, despite its high resistance
rates.25
Adherence of H. pylori to the gastric epithelium is a
primary step for a successful infection. Many studies have
shown that the binding of bacteria is mediated by multiple
adhesins; among the most studied are BabA and SabA,
which are known to interact through fucosylated or sialy-
lated blood group antigens,26 respectively. It is also known
that there are other bacterial proteins involved in H. pylori
adhesion, however, for most of them, their corresponding
host receptors remain unknown. Thus far, the adherence
receptor network related to H. pylori infection is not com-
pletely elucidated.27,28
In this context, antiadhesive compounds capable of in-
hibiting the docking process of H. pylori to gastric cells have
drawn great attention in recent years as a new strategy to
prevent and combat bacterial infection.
In the present work, the evaluation of antiadhesive po-
tential of quelite extracts showed that, at the highest con-
centration tested (1000 lg/mL), DM extracts inhibited
around 50% the adhesion of bacteria to AGS cells, while the
AQ extracts and mucilages blocked the interaction between
bacteria and cells in a lesser (30–40%), but statistically
significant, extent. It is possible that the compounds re-
sponsible for the antiadherent activity are different in each
one of the extracts (DM, AQ, or mucilage); hence, their
combination could enhance the antiadherent activity over
the individual value obtained for each extract.
Other research works have demonstrated a partial inhi-
bition of H. pylori adherence to cells with different kinds of
extracts29–32; and only a few works have reported a total
inhibition, just as in the case of a chloroform extract ob-
tained from Phyllanthus urinaria, which inhibited the ad-
hesion to AGS cells by 100% with 0.5 mg/mL.33
 6 GOMEZ-CHANG ET AL.
As stated before, bacterial binding to epithelial cells is
mediated by multiple adhesins, which explains the difficulty
to reach a complete inhibition of H. pylori attachment to
cells with only one compound. Considering this, the anti-
adherent activity exhibited by the quelite extracts could be
part of a prophylactic or eradication therapy by participating
in the inhibition of the bacterial binding to host cells.
For a successful infection, H. pylori also requires the
participation of urease.34 The importance of this enzyme can
be appreciated considering that urease-negative mutant
strains are unable to colonize animal models.35 Regarding
our results, we can conclude that the anti-H. pylori effect
observed with the quelite extracts does not rely on the
abolishment of the urease activity.
Collectively, the results obtained for DM extracts indicate
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that they could be a good source for the isolation of com-
pounds with antibiotic or antiadhesive activity.
Dietary plant phenolic compounds exert a variety of bi-
ological functions (including the antimicrobial activity)36–38
and have received particular attention because of their
beneficial health properties. In this work, we determined that
all the assayed extracts contain both kinds of compounds
(Table 1).
Only a few studies have assessed the phytochemical com-
position of the quelite species utilized in this work. With respect
to phenolic compounds, in C. aconitifolius has been reported the
presence of some flavonoids, including amentoflavona, quer-
cetin, kaempferol, catechin, hesperidin, kaempferol-3,7-
dimethyl ether, epigallocatechin gallate, naringenin, and some
flavonoid glycosides of quercetin and kaempferol. In addition,
phenolic acids such as protocatechuic, chlorogenic, 4 hydro-
xybenzoic, caffeic, p-coumaric, ferulic, and ellagic acids,
among others, have been detected in the species.12,13,39–41 In the
case of A. cristata, only two flavones have been reported: aca-
cetin and diosmetin.9 For C. pumila, none compound of this
group has been identified.
Several studies have determined the anti-H. pylori ac-
tivity of phenolic acids and flavonoids,23 including the
majority of those reported for C. aconitifolius42–47; never-
theless, concerning the two flavonoids of A. cristata, only
one work describes some effect of diosmetin on H. pylori,48
but there is no report regarding acacetin. With this back-
ground, we decided to study the effect of both flavonoids on
bacterial growth.
In our experiments, the MIC obtained for diosmetin
(Fig. 2) was 62.5 lg/mL. With the same H. pylori strain
that we used in this work, Bae et al.48 reported an MIC
>100 lg/mL. The discrepancy could be due to the source of
the compound or to the method they used in the inhibition
assay (agar dilution method). However, for both flavones,
we obtained a very good inhibitory effect (*90%) with only
3.9 lg/mL. The results indicate that it is possible that these
flavonoids may be, in part, responsible for the anti-H. pylori
activity achieved with the Ala-DM extract.
The anti-H. pylori activity and the inhibitory effect of the
bacterial adhesion to AGS cells obtained with the quelites
are probably due not only to a particular compound but to
the combined effect of several molecules as well. Further-
more, the two main flavones of A. cristata, acacetin and
diosmetin, wield a strong activity against the bacteria, and
could represent new anti-H. pylori agents of natural origin.
H. pylori has lived with us for thousands of years and it is
estimated that around 50% of the world’s population is in-
fected. It is associated with a wide group of human gastro-
intestinal diseases, but only some of the infected people
develop any pathology. The main factors that influence the
transition from H. pylori infection to an overt clinical disease
are bacterial polymorphism, host susceptibility, and diet.
Since the beginning of mankind, plants have fulfilled
basic functions for humans, including food or/and medicine.
The proposed mechanism by which plants and plant-derived
drugs exert their action against H. pylori is, on the one hand,
by preventing, mitigating, or eradicating the infection; and
on the other hand, by protecting against the damage pro-
voked by the bacteria to the host. Even though plant alter-
native therapies do not achieve permanent eradication of
H. pylori, the routine consumption of some edible and/or
medicinal plants may help to maintain a delicate balance
between health and illness, where bacteria remain in the
gastric mucosa without causing the most severe clinical
outcomes such as peptic ulcer or gastric cancer.
In the case of the three species of quelites studied in this
work, the results suggest that, considering their antibacterial
and antiadhesive properties, these plants not only represent a
promising source for a complementary therapy but also their
regular consumption may play an important key role in re-
ducing bacterial colonization.
This work provides new information about the anti-H.
pylori potential of three edible quelites and provides the
basis for further studies to establish whether their con-
sumption in a daily diet could have an impact on the pre-
vention and/or control of H. pylori-associated diseases.
ACKNOWLEDGMENTS
This study was supported by CONACYT 214286, ‘‘Re-
scate de especies subvaloradas tradicionales de la dieta
mexicana y su contribución para el mejoramiento de la
nutrición en México,’’ and by DGAPA-UNAM (PAPIIT
IN214317).
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
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