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Association between Polymerization Degree of Apple Peel
Polyphenols and Inhibition of Helicobacter pylori Urease
Edgar Pastene, Miriam Troncoso, Guillermo Figueroa, Julio Alarco#n, and Herna#n Speisky
J. Agric. Food Chem., 2009, 57 (2), 416-424 • DOI: 10.1021/jf8025698 • Publication Date (Web): 07 January 2009
Downloaded from http://pubs.acs.org on January 21, 2009
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Association between Polymerization Degree of Apple
Peel Polyphenols and Inhibition of Helicobacter
EDGAR PASTENE,*,†MIRIAM TRONCOSO,‡GUILLERMO FIGUEROA,‡
JULIO ALARC ´ ON,§AND HERN ´ AN SPEISKY†
Laboratory of Antioxidants, Institute of Nutrition and Food Technology, University of Chile, Av.
Macul 5540, Santiago 138-11, Chile, Laboratory of Microbiology, Institute of Nutrition and Food
Technology, University of Chile, Av. Macul 5540, Santiago 138-11, Chile, and Laboratory of
Synthesis and Natural Products, Department of Basic Sciences, Faculty of Sciences, University of
Bı ´o-Bı ´o, Avenida Andre ´s Bello, s/n P.O. Box 447, Chilla ´n, Chile
Apple peel extracts and their fractions pooled according to their molecular size were prepared and
evaluated for their inhibitory activity against Helicobacter pylori and Jack bean ureases. Urease
Inhibitory effect of apple peel polyphenols (APPE) extracted from the Granny Smith variety was
concentration-dependent and reversible. High molecular weight polyphenols (HMW) were more active
against Helicobacter pylori and Jack bean ureases than low molecular weight polyphenols with IC50
values of 119 and 800 µg GAE/mL, respectively. The results suggest that monomeric compounds
(mainly flavan-3-ols-and quercetin-O-glycosides) will not be implicated in the antiurease effect
displayed by the apple peel polyphenolic extract. Thus, as a byproduct, apple peel is suitable for
developing functional ingredients that could be useful for neutralizing an important Helicobacter pylori
KEYWORDS: Helicobacter pylori; urease; apple; Malus domestica; polyphenols; procyanidins
Health benefits of apple polyphenols have been widely
investigated during the past decade (1). Apples are a rich source
of polyphenols, which are distributed in the pulp, seeds, and
peel (2, 3). The in ViVo protective effects of apple cloudy juices
have been demonstrated in rats treated with the colon carcino-
genic agent 1,2-dimethylhydrazine, DMH (4). Recently, an
inhibitory effect of apple polyphenols on growth was observed
on adenoma (HT29) and colon carcinoma (LT97) cells lines
(5). At a molecular level, the inhibition of HT29 cell prolifera-
tion induced by the apple polyphenols imply both an inhibition
of epithelial grow factor receptor (EGFR) autophosphorylation
and an activation of caspases (6, 7). In addition, investigations
on the epigenetic effects of apple polyphenols on cell growth,
conducted in colon cancer cells, have demonstrated an inhibition
of the expression of enzymes involved in gene methylation
(DNA methyl transferases) and a reactivation of tumor sup-
pressor genes (8).
Among several varieties, Granny Smith apples are exported
both as fresh product and as concentrate for apple juice
production. Although a great part of such exports consider the
whole fruit, during the past decade, exports of dehydrated apple
products have had a significant increase. Since apple dehydration
requires peel removal, important amounts of apple peel are
produced and classified as agro-industrial waste. Previous studies
indicated that some apple varieties could have from 40 to 50%
of the total fruit polyphenols in the peel (9). In fact, the
concentration of polyphenols in peel could be up to three times
higher than that found in pulp. The principal classes of whole
apple polyphenols include flavonoid glycosides, phenol car-
boxylic acids esters, dihydrochalcones, catechins, and procya-
nidins (10). The latter and quercetin glycosides represent up to
60% and 18% of the apple peel polyphenols, respectively (11).
Recently, various studies evaluated a possible gastrointestinal
protective role of apple polyphenols. For instance, an antiul-
cerative effect of apple polyphenol extract was reported previ-
ously in rats given the extract during ten days before inducing
gastric injury with indomethacin (12). In the same study, it was
observed in vitro, that the oxidative damage induced by either
indomethacin and/or by xanthine-xanthine oxidase (X/XO) in
MKN 28 cells (a gastric epithelium cell line) can be ameliorated
by the prior addition of the apple polyphenols to the culture.
More recently, the same group demonstrated a protective effect
of the apple polyphenol extract in rats in which gastric damage
was induced by aspirin (13). As expected, differences in the
mechanism and magnitude of the protection induced by ap-
ple polyphenols may emerge using different injury models, apple
varieties, and extract preparations. For instance, when compared
†Laboratory of Antioxidants, University of Chile.
‡Laboratory of Microbiology, University of Chile.
§University of Bı ´o-Bı ´o.
J. Agric. Food Chem. 2009, 57, 416–424
10.1021/jf8025698 CCC: $40.75
2009 American Chemical Society
Published on Web 01/07/2009
by their antiulcerative properties, Chinese quince- and apple-
polyphenols showed differences on an HCl/ethanol-induced
ulcer model (14). Although both extracts were found to be dose-
dependently effective, at the highest dose, the apple extract
preparation showed to be pro-ulcerative rather than gastro-
protective. According to the authors, the deleterious action could
be ascribed to the comparatively higher presence of chlorogenic
acid found in the apple preparation. Previously, using the same
injury model, chlorogenic acid had been reported to be pro-
ulcerogenic in rats (15). Interestingly, in varieties such as Granny
Smith, the content of chlorogenic acid in apple peel is
considerably lower than that in pulp (16).
In addition to its gastro-protective effects on chemically
induced injury, apple polyphenols may also contribute to
ameliorate chronic gastro-intestinal affections induced by
Helicobacter pylori. The latter is a Gram-negative spiral
bacterium that infects about 50% of the world’s population;
it is the only microorganism known to permanently inhabit
the human stomach (17). Many studies have established H.
pylori as an etiologic agent for gastric cancer, mucosa-
associated lymphoid tissue (MALT) lymphoma, and peptic
ulcer (18, 19). In terms of its capacity to colonize the gastric
mucosa, one of the most important features of H. pylori is
its extremely high capacity to produce urease, whose main
function is buffering H. pylori’s periplasm (20). Also, urease-
generated ammonia neutralizes gastric acidity and thereby
promotes within, the gastric lumen, a neutral microenviron-
ment surrounding the bacterium. Considering the crucial role
urease plays in H. pylori survival and gastric colonization,
we undertook a study to assess the effect of a polyphenol-
rich extract, obtained from Granny Smith apple peel wastes.
In view of its richness in procyanidins, we investigated the
existence of a relationship between the degree of polymer-
ization of the procyanidins contained in such an extract and
their possible inhibitory effect on H. pylori urease.
MATERIAL AND METHODS
Standards, Chemicals, and Solvents. Gallic acid, chlorogenic acid,
caffeic acid, (+)-catechin, (-)-epicatechin, phoridzin, quercetin, quer-
cetin 3-O-rutinoside, procyanidin B1 and B2, toluene-R-thiol, cysteam-
ine, phloroglucinol, sodium carbonate, and the Folin-Ciocalteu were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Flavonol
glycosides (quercetin 3-O-galactoside, quercetin 3-O-glucoside and
quercetin 3-O-rhamnoside were from Roth (Karlsruhe, Germany).
Procyanidin C1 was purified according to Sun and co-workers (21).
All other solvents were HPLC grade purchased from Merck (Darmstadt,
Helicobacter pylori Strains and Culture Conditions. Helicobacter
pylori (ATCC 43504) was kindly provided by Professor Apolinaria
Garcı ´a (Universidad de Concepcio ´n, Chile). Strains were incubated for
4 days in a microareobic gas environment (15% CO2, 5% O2, and 80%
N2) in Brucella broth containing 5% horse serum and 0,1%
H. pylori Urease. Urease extraction was carried out according to
the protocol suggested by Xiao and co-workers (22), with minor
modifications. Briefly, 100 mL broth cultures (optical density at 600
nm of 0.25 corresponding to 1 × 108CFU/mL) were centrifuged
(5000g, 30 min, 4 °C) to collect the bacteria, and after washing twice
with phosphate-buffered saline (pH 7.7), the H. pylori pellet was stored
at - 70 °C. The H. pylori pellet was returned to room temperature,
and after the addition of 3 mL of distilled water and protease inhibitors,
sonication was performed for 60 s. Following centrifugation (14,000g,
15 min, 4 °C), the supernatant was desalted through a Sephadex G-25
column (Sigma). The eluted crude urease was further concentrated
5-fold using a centrifugal filter device 50,000 Da NMWL (Millipore
Corporation, Bedford, MA) at 4 °C. This solution was added to an
equal volume of glycerol and stored at -20 °C until use. Total protein
was evaluated by the Bradford method (Sigma) with bovine serum
albumin as the standard. Urease activity was assessed by measuring
ammonia production using the indophenol method as described by
Weatherburn (23). One unit of urease activity was defined as the amount
of enzyme required to hydrolyze 1 µmol of urea (producing 2 µmol of
ammonia) per min per mg of total protein. The amount of ammonia
released was determined from a standard curve.
Preparation of Apple Peel Extracts and Fractionation. Apple
peels from ripe fruits of the cultivar Granny Smith (Malus domestica
cv. Granny Smith) were kindly provided by SURFRUT Ltda. (Santiago,
Chile). Briefly, frozen peels (-20 °C) were mixed with hot water (9 +
1 w/v; 5 min, 90 °C) and then pressed through a stainless steel grid
(15 µm). The cake was treated with water at 65 °C using an Ultraturrax
homogenization device (1 min). Homogenized peels were macerated
during 30 min and filtered through the stainless steel grid. Pooled
aqueous cloudy extracts were retained on absorber resin Sepabeads SP-
850 (Supelco, Bellefonte, USA) that was packed in a glass column
(50 mm i.d. × 300 mm). Water soluble ingredients such as sugars,
organic acids, and minerals were removed by washing first with five
volumes of distilled water. Retained compounds were then eluted with
70% ethanol. The ethanolic fraction was concentrated by evaporative
rotation (<40 °C) and dried under vacuum in a dessecator. Dried apple
peel polyphenol-rich extract (APPE) was stored at - 70 °C until use.
For HPLC analysis of fresh apple peel, ∼10 g of frozen peel was
weighed and then transferred to a beaker with 70% chilled aqueous
acetone, considering a 1 + 9 w/v ratio for the extraction of total
polyphenols including polymeric procyanidins. The mixtures were
homogenized using Ultraturrax and filtered first through a Whatman
no.1 filter paper and then through a 0.45 µm syringe filter (Millipore,
Bedford, MA, USA). The final filtrate was injected directly onto a liquid
To obtain low molecular weight polyphenols, APPE was suspended
in water and extracted with ethyl acetate (5 times). The ethyl acetate
extracts (EAE) were pooled and concentrated under vacuum. The
aqueous layer (AQUO) was saved. Dried EAE was further chromato-
graphed on a Toyopearl HW-40s size-exclusion chromatography column
(30 × 2.5 cm, Tosoh, Tokyo) with a linear gradient of 5% to 100% of
methanol and then to 60% acetone for elution of retained polymeric
material. Nine fractions were collected. Fractions I-VI containing low
molecular weight compounds were concentrated and dried under
Table 1. Phenolics Composition of Fresh Apple Peel and APPE Extract
Obtained fromGranny Smith Apples
compoundfresh apple phenolics (%)a
APPE phenolics (%)a
aData are the average of triplicates determined by the HPLC method and
expressed as the percentage of the total polyphenolic content measured by the
Folin-Ciocalteu method.bCalculated on the basis of rutin measured from RP-
HPLC.cEstimated by the RP-HPLC method after phloroglucinolsis.dNd ) not
Polymerization Degree of Apple Peel PolyphenolsJ. Agric. Food Chem., Vol. 57, No. 2, 2009
vacuum (LMW). The AQUO fraction was loaded on a Sephadex LH-
20 column (30 mm i.d. × 300 mm) activated previously with water
during 24 h. The column was eluted with methanol containing
decreasing proportions of water and methanol alone, and finally, the
polymeric material was recovered with 60% acetone, concentrated, and
dried under vacuum (high molecular weight compounds, HMW). TLC
was carried out on 20 × 20 cm silica gel 60 F254plates (Merck), eluted
with toluene-acetone-formic acid (3:6:1) (24).
Flavan-3-ols and procyanidins were detected by staining with
dimethylaminocinnamaldehyde (DMACA). Total polyphenolic contents
(TPC) were determinate with Folin-Ciocalteau reagent and expressed
as mg of gallic acid equivalents (GAE) per gram of dried extract. The
equation of gallic acid calibration curve was y ) 0.091x + 0.0229 (r2
High-Performance Liquid Chromatography (HPLC). Extracts and
fractions were separated by RP-HPLC using an Agilent 1100 and a
Lachrom instrument, both equipped with a 250 × 4.6 mm, 5 µm,
Kromasil KR100-5C18 column (Eka Chemicals AB, Bohus, Sweden).
The solvent system was composed of solvent A (double distilled water
containing 0.1% TFA, v/v) and solvent B (acetonitrile containing 0.1%
TFA). The following gradient system was used: 0-25 min, 10-30%
B; 25-30 min, 30-75% B; 30-35 min; 75-10% B at a flow of 1
Figure 1. Reverse-phase HPLC-DAD profiles of APPE and fresh apple peel: (A) Comparison between HPLC profiles of fresh apple peel and APPE
recorded at 280 nm showing the peaks of (1) chlorogenic acid, (2) procyanidin B1, (3) procyanidin B2, (4) (-)-epicatechin, (5) procyanidin C1, (*)
and (13) phloridzin. (B) Insert: close-up of the APPE flavan-3-ols and procyanidins region as detected by HPLC-FLD with excitation at 280 nm and
emission at 310 nm. Key for polyphenols is the same as that for the HPLC-DAD trace. (C) Chromatographic separation by TLC of the samples was
visualizedwithDMACAreagent. Lane1, (-)-epicatechin; lane2, freshapplepeel extracts; lane3, APPE; lane4, LMW; lane5, HMW; andlane6, rutin.
Figure 2. Depolymerization of APPE. RP-HPLCof APPE before (A) and after (B) degradation with phloroglucinol.
J. Agric. Food Chem., Vol. 57, No. 2, 2009 Pastene et al.
mL/min. For flavonoids, detection was at 280 nm using a diode array
detector. For procyanidins and catechins, detection was done using a
fluorescence detector with excitation at 280 nm and emission detection
at 310 nm. Known compounds were identified by matching their
retention times (tR) and online UV spectra with those of reference
substances. Although the other compounds (particularly some quercetin-
3-O-pentosides) could not be wholly identified, they were characterized
according to their class on the basis of their UV-vis spectra.
Quantification of quercetin glycosides, chalcones, phenol carboxylic
acids, epicatechin, catequin, and procianidins B1, B2, and C1 was
carried out using peak areas from external calibration curves.
Mean Degree of Polymerization of Apple Peel Extracts (mDP).
Phloroglucinol degradation (Phloroglucinolysis) was used for the
estimation of mDP and procyanidin contents according to Karonen and
co-workers (25). Briefly, 10 mg of apple extract was dissolved in 2
mL of a solution of 0.1 M HCl in methanol containing 50 mg/mL
phloroglucinol and 10 mg/mL ascorbic acid. The reaction mixture was
incubated at 50 °C for 20 min, and then 10 mL of 40 mM aqueous
sodium acetate was added to stop the reaction. For fresh apple peel
analysis, 100 mg was extracted in 1.0 mL of 70% actetone/water (v/v)
containing 10 mg/mL of ascorbic acid. Samples were vortexed to mix
thoroughly and sonicated by 20 min, before being centrifuged for 10
min (14000g). A 100 µL aliquot was evaporated to dryness under
vacuum prior to phloroglucinolysis. Additionally, mDP was obtained
by means of thiolysis with toluene-R-thiol and cysteamine (26, 27).
Phloroglucinolysis products were analyzed by the same RP-HPLC
described above. Quantitative determination of (-)-epicatechin, (+)-
catechin, and the degradation products was performed using external
standards. The calibration plots for (-)-epicatechin and (+)-catechin
showed a linear range from 2-20 µg/mL (r2) 0.9999) and 10-60
µg/mL (r2) 0.9997), respectively. The procyanidin content was
calculated by summing the mass of all subunits (excluding the
phloroglucinol portion of the phloroglucinol adducts). To calculate the
mean degree of polymerization, the sum of extension subunits was
divided by the sum of terminal subunits. The undegraded medium was
used to quantify native (-)-epicatechin and (+)-catechin in the extracts
Normal-Phase High Performance Chromatography. APPE, HMW,
and LMW procyanidins were separated by normal-phase HPLC (NP-
HPLC) according to Gu and co-workers (28) with a 250 × 4.6 mm (5
µm) Lichrospher 100 Diol column (Merck, Darmstadt, Germany). The
solvent system was composed by solvent A (acetonitrile/acetic acid,
98:2 v/v) and solvent B (methanol/water/acetic acid, 95:3:2 v/v).
Procyanidins were eluted with the following gradient system: 0-35
min, 0-40% B; 35-55 min; isocratic 40% B; 55-60; 40-0% B with
5 min of column reconditioning at a flow rate of 0.6 mL/min. Elution
was monitored by fluorescence detection with excitation at 230 nm
and emission detection at 321 nm. Peak assignment was done in
comparison with the literature (28). Oligomers up to undecamers were
estimated by NP-HPLC.
Microplate Urease Test. Evaluation of urease activity was done
according to a previously published methodology (29). H. pylori and
CanaValia ensiformis ureases (Jack bean urease; E.C.18.104.22.168; Sigma-
Aldrich), were used in the assay mixture (25 µL, 4 U) with 25 µL of
different concentrations of polyphenols. Samples were preincubated
for 0.5-4 h at room temperature in a 96-well assay plate. After
preincubation, 200 µL of 100 mM phosphate buffer at pH 6.8 containing
150 mM urea and 0,002% phenol red were added, and changes in
absorbance at 570 nm were measured by a micro plate reader Synergy
In-Gel Urease Activity. For in-gel detection of urease activity, two
procedures were used. First, 25 µL of H. pylori urease (10 µg total
proteins) was used in the assay mixture with 25 µL of different
concentrations of polyphenols. Samples were preincubated for 1 h at
room temperature. After preincubation, 10 µL were loaded and
separated by electrophoresis under nondenaturing conditions with a 7%
polyacrylamide gel in a Mini-protean III system apparatus (BioRad,
USA). Electrophoresis was performed at 100 V in a Tris-glycine buffer
system containing Tris HCl (25 mM) and glycine (250 mM) at pH
8.8. After electrophoresis, gels were washed sequentially with cold
acetate buffer (5 µM, three times) and once with bidistilled water. In-
gel activity was immediately determined according to the procedure
previously described by Mobley and co-workers (30). Briefly, after the
washing step, the gel was placed in a 0.02% cresol red-0.1% EDTA
(pH 6.7). The procedure was repeated until the gel remained yellow
and incubated with 150 mM urea at 37 °C until pink-reddish bands
With the aim to avoid incubations with the substrate and cresol red
in a liquid medium as in the original protocol, some modifications were
included. For this purpose, a thin film of agarose was prepared daily
using the casting frame of the Miniprotean III system, with the same
glass cassette used for PAGE (0.75 mm). Agarose (80 mg) was
suspended in 0.02% cresol red-0.1% EDTA (pH 6.7), containing 150
mM urea. Agarose was melted using a conventional microwave oven
and immediately loaded into the gel cassette. For this purpose, the
coomb was omitted, and the agarose thin film was cooled at room
temperature and stored at 4 °C until use. Polyphenols from apple peel
were assayed in the concentration range of 0-1000 µg/mL. Under
gentle agitation, the gels were incubated with the polyphenols (25 mL,
1 h at 25 °C). After the incubation period, samples (extracts or buffer)
were carefully drained, and the gels were carefully deposited over a
transparent plastic sheet. The agarose film was put in contact with the
polyacrylamide gel. Another plastic sheet was used to cover the agarose
film, forming a sandwich. The entire process was carried out onto the
surface of a scanner (Snapscan e20, Agfa) allowing the record of band
RESULTS AND DISCUSSION
Apple Peel Extracts Characterization. Quantitative analysis
based on the RP-HPLC polyphenolic profile indicates that
quercetin glycosides account for about 58% of the total
polyphenols present in APPE (Table 1; Figure 1A). As shown
in the APPE chromatographic profile, the major quercetin
glycosides identified were rutin, hyperoside, isoquercitrin, two
quercetin-3-O-pentosides, and quercitrin. This chromatographic
profile is remarkably similar to that obtained in the fresh peel
of Granny Smith apples (upper chromatographic profile in
Figure 1A, Table 1). The latter result suggests that the
procedures of extraction and subsequent absorption of apple peel
polyphenols did not modify the fresh apple peel profile
generating artifacts, loss of compounds, or selective enrichment.
Interestingly, the flavonoids found in this study for APPE and
fresh Granny Smith apple peel are essentially the same as those
reported previously to occur in apple peel from Granny Smith
and from other apple varieties (31).
Because of its better sensitivity and selectivity, flavan-3-ols
and procyanidins B1, B2, and C1 present in APPE were
analyzed by HPLC-FLD. As depicted in Figure 1B, flavan-3-
ols are represented mainly by epicatechin and only traces of
catechin. Total procyanidins in APPE were estimated by
phloroglucinolysis, accounting for 24.38% of the total polyphe-
nol content (Table 1). Procyanidin B1, B2, and C1 content in
Table 2. Total Polyphenol Content, Mean Degree of Polymerization and
Urease Inhibitory Activities of Fresh Apple Peel, APPE, and Fractions
Obtained by Size-Exclusion Chromatography Procedures
3.50 ( 0.8
2.9 ( 0.2
610 ( 12
480 ( 8
520 ( 10
3.0 ( 0.5
1.0 ( 0.2
9.5 ( 2
aMeanvaluesand( SD(n) 3) expressedinmilligramsof gallicacid(GAE)
equivalents per gramof dry weight (either peel or powder).bNumbers represent
theratiobetweenthemoles of phloglucinol-derivedflavan-3-ols andthemoles of
of preincubation with the extract and its fractions.dNd: not determined.
Polymerization Degree of Apple Peel PolyphenolsJ. Agric. Food Chem., Vol. 57, No. 2, 2009
APPE evaluated by RP-HPLC represented 5.38% of the total
polyphenols. These values suggest that the main procyanidins
in APPE belong to the high MW group. Indeed the analysis of
APPE by NP-HPLC showed that oligomeric material up to
undecamers represented around 19.0% of the APPE total
polyphenol content (Figure 3A). The contribution of phenol
carboxylic acids (calculated as caffeic acid) and chalcones
(calculate as phloridzin) reached 0.9 and 12%, respectively.
With the aim to fractionate APPE polyphenols according to
their molecular size, a combination of Sephadex LH-20 and
Toyopearl HW-40s column chromatography was used. As result
of this strategy, low molecular weight (LMW) and high
molecular weight (HMW) polyphenolic fractions were obtained,
which was preliminarily confirmed by TLC analysis (Figure
1C). Polymeric and oligomeric procyanidins were exclusively
concentrated in the fraction named HMW and under TLC
examination appear as a poorly separated group of compounds.
Polyphenols from LMW were visualized as better separated
spots with Rfs g0.5. It should be noted that DMACA staining
allows a selective detection of flavan-3-ol structures (monomers
and polymers), and after 15-20 min, they appear as blue to
green spots. As depicted in Figure 1C, flavan-3-ol (monomers
and some dimers) are also present in the LMW fraction along
with the quercetin glycosides (orange to brown spots) previously
identified by HPLC-DAD. Therefore, by means of the fraction-
ation procedure, polyphenols from APPE were pooled only
according to their molecular size.
Determination of the mDP of APPE, HMW, and LMW was
carried out by depolymerization via phloroglucinolysis. The
values were calculated from the HPLC-FLD chromatographic
profiles (Table 2). Although the three samples possess a
distinctive degree of polymerization, total polyphenolic contents
were very similar (500-600 mg GAE/g). Figure 2 presents the
RP-HPLC chromatographic profile of procyanidins subjected
to depolymerization with phloroglucinol. By means of this
procedure, it is possible to obtain results similar to those of the
depolymerization with toluene-R-thiol and cysteamine (data not
shown), in a shorter time and without the toxicity problems
Figure 3. Normal-phase HPLCfluorescence trace of procyanidins fromapple peel extracts. (A) APPE, (B) LMW, and (C) HMW. The numbers beside
the peaks indicate the degree of polymerization of B-type procyanidins.
J. Agric. Food Chem., Vol. 57, No. 2, 2009Pastene et al.
associated with the previous one. The epicatechin interconver-
sion to catechin is not an important reaction in this procedure.
Indeed, catechin levels detected in the samples subjected to
degradation with phloroglucinol were the same as those in the
samples without treatment (not shown). Depolymerization with
phloroglucinol generated profiles where it is clearly seen that
the main extension and terminal unit was epicatechin (Figure
2A,B). The latter eluted with a tR) 11.5 min, whereas the
nucleophile-epicatechin adduct did at tR) 6.8 min. After a
detailed analysis of the HPLC profile, it was not possible to
find the nucleophile-catechin peak. The latter usually appears
as a partially resolved peak before the epicatechin-adduct peak.
Considering all of these results, it is suggested that in the three
extracts, procyanidins would be mainly derived from epicatechin
and, in a smaller degree, from catechin just as it has been
previously informed for fresh apple peel (32). It is necessary to
notice that the depolymerization profiles corresponding to the
analysis of fresh apple peel were identical to those previously
described for APPE (Table 1).
Figure 3 shows the result of the NP-HPLC separation of
APPE procyanidin oligomers and the fractions LMW and
HMW. Profiles depicted in Figure 3A-C show procyanidin
peaks or clusters with different molecular weight. This com-
plexity would correspond to the possible rotamer combinations,
as reported by other authors (33). By means of the study of
NP-HPLC profiles, it is possible to confirm that LMW possesses
mostly monomeric units (mDP ) 1). APPE possesses an mDP
) 2-4, while for HMW mDP ) 8-10.
Effects of APPE, LMW, and HMW on Urease Activity.
The effect of APPE and its LMW and HMW fractions on urease
activity was studied using H. pylori and Jack bean (for
comparative purposes) as sources for the enzyme. Jack bean
urease has been widely used as an enzyme model for inhibitor
screening (29). Importantly, in the case of H. pylori, its R subunit
shares 48% of amino acid sequence identity with the corre-
sponding N-terminal sequences of Jack bean urease (34). As
depicted in Figure 4A-C, the whole extract and its low and
high MW fractions were all active in promoting an inhibition
Figure 4. Inhibition of urease by APPE, HMW, and LMW. (A) Semilog graphic of H. pylori urease inhibition by apple peel polyphenols. (B) Semilog
graphic of Jack bean urease inhibition by apple peel polyphenols. APPE, -O-; HMW, -3-; LMW, -0-. (C) Lineweaver-Burke plots showing 1/urease
activity and 1/substrate concentration. Data in the presence of 0 (O); 2.5 (4); and 5 (0) GAE (µg/mL) of APPE are presented as the mean of four
Polymerization Degree of Apple Peel PolyphenolsJ. Agric. Food Chem., Vol. 57, No. 2, 2009
of urease activity. Since the main feature of apple peel
polyphenols is that of acting as antioxidants, the comparison
of their effect on urease activity was done on the basis of using
micrograms of gallic acid as mass equivalent (GAE) for the
distinct tested preparation. The inhibitory effects were in all
cases concentration-dependent. Table 2 shows the IC50 for
APPE and its fractions upon H. pylori and Jack bean ureases.
For H. pylori urease, the IC50values were 119, 800, and 516
µg GAE/mL, for HMW, LMW and APPE, respectively. The
low solubility of LMW fraction precluded assaying higher
concentrations. For Jack bean urease, the IC50values were 103,
594, and 180 µg GAE/mL for HMW, LMW, and APPE,
respectively. On the basis of the IC50ranking order and level
of GAE, these results suggest that the urease-inhibitory activity
displayed by APPE is associated, primarily, with the presence
of high MW polyphenols and, to a lower degree, with the
presence of monomeric components. Furthermore, the close
similarity in both the shape and slope of the semilog curves
describing the effect of HMW and that of APPE suggests that
the inhibition of urease induced by the latter would indeed be
attributable to the presence of high MW components (Figure
The slightly smaller IC50values obtained with the Jack bean
(compared with H. pylori) urease could relate to a higher purity,
which would result in a lower nonspecific binding of polyphe-
nols to the former enzyme. Urease, extracted from H. pylori
cultures, has been reported to copurify with other proteins such
as GroEL, IlvC, HsPB, and Hsp60 (35). Indeed, SDS-PAGE
run by us for the H. pylori lysates revealed the presence of
certain bands that lacked ureolytic activity (data not shown).
Effects of APPE, LMW, and HMW on In-Gel Urease
Activity. A decrease in the urease activity was observed after
incubation with apple peel extracts. To better understand this
activity, the effect of apple peel fractions over urease electro-
phoretic mobility was evaluated. As shown in Figure 5A, HMW
concentration-dependently generated insoluble aggregates, which
resulted from the formation of procyanidin-urease complexes.
LMW also showed this kind of interaction but to a lesser degree.
These high molecular weight complexes (Figure 5A, lanes
2-4), were unable to migrate through the 7% polyacrylamide
gel under nondenaturing conditions. Although the aggregate
formation occurred, urease activity was maintained as it is
possible to evidence it after the in-gel detection with cresol red.
Apparently, this observation is not concordant with the con-
centration-dependent inhibition observed in the 96-well format
(Figure 4). This difference can be due to the methodological
difference between both assays. For instance, in the in-well assay
the urease is continually exposed to the tested polyphenols,
meanwhile in the in-gel evaluation, the polyphenols could be
removed from the aggregate during the conventional steps after
the electrophoretic run, i.e., washing the gel in a continuous
form with acetate buffer (pH 6.8), as a necessary condition to
allow the subsequent addition of cresol red. Because of this
difference, both methodologies are not comparable. Additionally,
the results in-well (enzyme kinetics) suggested a competitive
and reversible inhibition (Figures 4B); therefore, it is possible
to conclude that the in-gel activity detection would not be the
best procedure to evaluate this kind of urease inhibitors. Hence,
a modification to the original protocol was carried out, in which
the enzyme preparation (H. pylori urease) was first subjected
Figure 5. Effects of apple peel fractions upon urease activity detected in-gel. Nondenaturant gel electrophoresis of urease extracted fromH. pylori on
7% polyacrilamide. (A) Before electrophoresis, apple peel fractions LMWand HMWwere previously incubated with H. pylori urease as described in
Material andMethods. Lane1) control urease; lanes2, 3, and4) LMW) HMW) 100-400-800GAEµg/mL. (B) Effectsof applepeel crudeand
fractionated extracts upon H. pylori urease activity by a modified in-gel procedure. APPE, LMW, and HMW effect upon H. pylori urease activity in
nondenaturant gels as detected by agarose-urea-cresol red filmprocedure. The same gels stained by Coomassie blue are presented on the bottom
panel. Figures are representative of three independent experiments.
J. Agric. Food Chem., Vol. 57, No. 2, 2009Pastene et al.
to a PAGE separation (under nondenaturing conditions), then
gel strips (with urease activity previously established) were
initially incubated with increasing concentrations (expressed as
GAE) of APPE, HMW, or LMW, and removed from such
solutions afterward. Thereafter, strips were subsequently covered
with an agarose film containing urea (150 mM) and cresol red,
which allowed revealing in a semiquantitative form, the remain-
ing urease activity (Figure 5B). With this procedure, the
washing steps previously referred to as the possible cause of
urease reactivation were completely avoided. Figure 5B showed
a clear inhibitory effect of HMW against urease from a
concentration of 10 µg GAE/mL. Contrarily, APPE showed only
a slight enzyme inhibition at the same concentration, this is
consistent with the proposal that molecules (most likely pro-
cyanidin) with a higher degree of polymerization underlie the
urease-inhibitory properties of apple peel extracts. However,
when the concentration was increased up to 100 µg GAE/mL,
both preparations achieved almost a complete inhibition. The
inhibitory effect of such preparations is not attributable to
differences in the protein concentration loaded in the gels
(Figure 5B, inferior panel). These results using this method-
ological approach were consistent with the concentration-
dependence results obtained previously in the in-well assay.
Moreover, a significant restoration of urease activity was seen
when agarose films containing a higher concentration or urea
(300 mM) were used. Interestingly, in the latter assay, the
concentration needed to achieve total urease inhibition was
substantially higher than the one estimated with the improved
in-gel assay. This may suggest that electrophoretic separation
of the native urease would result in a lower nonspecific binding
of polyphenols to other H. pylori proteins.
In comparison to HMW, apple peel polyphenols, containing
low MW polyphenols, showed a 6- to 7-fold lower effectiveness
to inhibit H. pylori urease. Hitherto, only a few studies have
reported an antiurease effect of low MW polyphenols. Xiao and
co-workers (22) evaluated the H. pylori antiurease activity of
20 synthetic polyphenols based on isoflavones. The presence
of two ortho hydroxyl groups and the integrity of the C-ring
were essential for their inhibitory activity. The urease inhibition
induced by these synthetic polyphenols was time-dependent and
in some cases was totally reverted by the subsequent addition
of the thiol agents, DTT, or ?-mercaptoethanol. Contrarily in
this research, the urease-inhibitory effect displayed by APPE,
HMW, and LMW was not time-dependent and only partially
(less than 10%) reverted by DTT or ?-mercaptoethanol (data
Regarding polymerization degree, hops and red wine extracts
with demonstrated in vivo anti-H. pylori effects (36, 37) have
established a structure-activity relationship between the degree
of polymerization of the polyphenols present in these extracts
and the degree of inactivation of VacA, another important
virulence factor. Only a few studies have evaluated the effect
of some dimeric procyanidins (B1 and B2) on urease activity
(38). Unfortunately, these studies did not address the importance
of the polymerization degree with regard to the inhibitory effect
of such procyanidins. In APPE, quercetin glycosides (∼58%)
are more abundant than procyanidins (∼25%), which is
particularly interesting because of the results reported herein.
Although the HMW compounds account for a great part of H.
pylori urease inhibition, further investigation is needed to
establish if LMW (quercetin glycosides) could interact with
other molecular targets decreasing H. pylori viability. Urease
neutralization not only precludes the H. pylori colonization
ability but also the synthesis of pro-inflammatory cytokines such
as interleukin-8 (IL-8) (39). In gastric epithelial cells, IL-8
production is mediated by NF-κB. The latter activation could
be induced by H. pylori urease binding to CD74 (invariant chain
of the mayor histocompatibility complex II, MHC II). Therefore,
APPE constituents could also afford benefits by ameliorating
the inflammatory damage caused by H. pylori infection. In our
laboratory, APPE is currently investigated considering other
molecular targets relevant to the H. pylori viability and certain
pathways associated with host gastric mucosa damage.
In summary, in this work we found that APPE inhibited the
H. pylori urease in vitro. APPE effectively blocked the activity
of isolated urease, an interesting effect with respect to H. pylori
colonization capability. In this research, we successfully identi-
fied high molecular weight polyphenols as the main constituents
associated with urease inhibition.
E.P. thanks Isabel Mella (Laboratory of Microbiology,
Institute of Nutrition and Food Technology, University of Chile)
for technical assistance.
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Received for review August 20, 2008. Revised manuscript received
November 12, 2008. Accepted November 18, 2008. This study was
partially supported by Comisio ´n Nacional de Ciencia y Tecnologı ´a of
Chile (Grant No. 24080071).
J. Agric. Food Chem., Vol. 57, No. 2, 2009 Pastene et al.