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PROFESSOR ALEJANDRO F. BARRERO
Department of Organic Chemistry, Prof. Viqar Uddin Ahmad Prof. Niel A. Koorbanally
University of Granada, Karachi, Pakistan Durban, South Africa
Campus de Fuente Nueva, s/n, 18071, Granada, Spain
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afbarre@ugr.es
Novara, Italy Tokyo, Japan
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Dipartimento di Chimica Bioorganicae Biofarmacia, Prof. Yoshinori Asakawa Prof. Hartmut Laatsch
Universita di Pisa, Tokushima, Japan Gottingen, Germany
via Bonanno 33, 56126 Pisa, Italy Prof. Roberto G. S. Berlinck Prof. Marie Lacaille-Dubois
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State Key Laboratory of Natural and Biomimetic Drugs, Taipei, Taiwan
Florence, Italy
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Tokyo University of Pharmacy and Life Sciences, Barcelona, Spain Antwerp, Belgium
Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan
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Chicago, IL, USA Düsseldorf, Germany
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Department of Chemistry Prof. Fatih Demirci Prof. Phila Raharivelomanana
University of Wollongong Eskişehir, Turkey Tahiti, French Polynesia
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Department of Chemistry, Milano, Italy
Murcia, Spain
Texas Christian University,
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m.reinecke@tcu.edu Saint John, NB, Canada Richmond, UK
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Department of Chemistry Reims, France Palampur, India
The University of Alabama in Huntsville Prof. John L. Sorensen
Prof. Duvvuru Gunasekar
Huntsville, AL 35809, USA
Tirupati, India Manitoba, Canada
wsetzer@chemistry.uah.edu
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Faculty of Pharmaceutical Sciences Niigata, Japan Scottsville, South Africa
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y-tezuka@hokuriku-u.ac.jp
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2015
NPC Natural Product Communications Vol. 10
No. 8
Anti-Helicobacter pylori Activity of Four Alchemilla Species (Rosaceae) 1369 - 1371
Marija Krivokućab, Marjan Niketićc, Marina Milenkovićd, Nataša Goliće, Carla Masiaf,
Maria Maddalena Scaltritof, Francesca Sistof and Tatjana Kundakovića*
a
Department of Pharmacognosy, University of Belgrade, Faculty of Pharmacy, Vojvode Stepe 450,
11221 Belgrade, Serbia
b
Pharmacy Department,University of Banja Luka, Faculty of Medicine, Save Mrkalja 14, Banja Luka,
Bosnia and Herzegovina
c
Natural Hystory Museum, Njegoševa 51, 11000 Belgrade, Serbia
d
Department of Microbiology and Immunology, University of Belgrade, Faculty of Pharmacy, Vojvode Stepe 450,
11221 Belgrade, Serbia
e
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, 11010 Belgrade, Serbia
f
Department of Biomedical, Surgical and Dental Sciences, University of Milan, Via Festa del Perdono 7,
20122 Milan, Italy
ktatjana@pharmacy.bg.ac.rs
The present study evaluated the anti-Helicobacter pylori activity of Alchemilla glabra Neygenf. (A. sect. Alchemilla), A. monticola Opiz (A. sect. Plicatae S.E.
Fröhner), A. fissa Günther & Schummel (A. sect. Calycinae (Buser) Buser) and A. viridiflora Rothm. (A. sect. Calycinae), and identified ellagic acid and
quercetin-3-O-β-D-glucoside. Anti-H. pylori activity was tested against ten clinical isolates and one reference strain (ATCC 43504). The methanol extracts
were more active than the dichloromethane and cyclohexane extracts. The ranges of concentrations were between 4 μg/mL for methanol extracts of
A. viridiflora, A. glabra and A. monticola, and 256 μg/mL for cyclohexane extracts of A. viridiflora, A. glabra and A. fissa. The best overall activity was
obtained with A. monticola extracts. No significant difference was found in the ellagic acid contents of the methanol extracts of the tested Alchemilla species
(0.2-0.3 mg/mL), and anti-H. pylori activity was similar (4-32 μg/mL). Ellagic acid exhibited strong activity at very low concentrations (0.125-0.5 μg/mL),
while the second identified compound, quercetin-3-O-β-D-glucoside, was also very active in concentration of 2-16 μg/mL.
Helicobacter pylori (H. pylori), a ubiquitous human pathogen, is a action of A. glabra Neygenf. (A. sect. Alchemilla), A. monticola
Gram-negative bacterium associated with the development of Opiz (A. sect. Plicatae S.E. Fröhner), A. fissa Günther & Schummel
chronic gastritis, peptic ulcer disease, and gastric cancer [1,2]. (A. sect. Calycinae (Buser) Buser) and A. viridiflora Rothm. (A.
Standard antibiotic therapy is often not effective due to the high sect. Calycinae), and identified compounds.
resistance of H. pylori [3]. Plants are important sources of
biologically active compounds with significant antimicrobial The total tannin content in the methanol extracts of the tested
properties. In particular, tannin containing plants have been used in Alchemilla species was 4.8-12.3%, calculated as pyrogallol, while
the treatment of gastric diseases not only because of their astringent the quantity of ellagic acid was between 0.2-0.3 mg/mL, which was
effect on gastric mucosa, but also because of anti-H. pylori lower than the results of other authors for A. vulgaris s.l. extract
activity. Water soluble tannins, catechins and proanthocyanidins, as (Table 1) [17,18]. Besides ellagic acid, quercetin-3-O-β-D-glucoside
well as ellagitannins, have been shown previously to have anti-H. was identified in the methanol extracts of A. monticola and A.
pylori activity at low concentration [4-7]. Ellagic acid is a potent viridiflora.
competitive inhibitor of gastric H+, K+-ATPase, and thus markedly
inhibits acid secretion, and stress-induced gastric lesions. Enzyme Table 1: The content of tannins (%) and ellagic acid (mg/mL) in methanol extracts.
inhibition was also evident for tannic acid [8,9]. Plant species Tannins* (%) Ellagic acid (mg/mL)
A. glabra 8.2 0.3
A. fissa 12.3 0.2
Various species of Alchemilla L. (Rosaceae) are used in traditional A. viridiflora 4.8 0.2
medicine to treat the symptoms of sore throat, for wound healing, A. monticola 8.5 0.2
against bleeding, nausea and vomiting [10]. Alchemilla species have *
The results were expressed as the percentage of pyrogallol.
potent free radical scavenging activity, attributed to the phenolic
compounds, tannins, and flavonoid glycosides [11-13]. A. vulgaris Anti-H. pylori activity was tested against ten clinical isolates and
s.l. has been reported to possess ellagitannins (agrimoniin, one reference strain (ATCC 43504). The activity of the extracts is
laevigatin F, pedunculagin), flavonoids, essential oil, phytosterols presented in Table 2. The methanol extracts were more active
and aliphatic hydrocarbons [13-15]. than the dichloromethane and cyclohexane extracts. The range
of concentrations was from 4 μg/mL for methanolic extracts of
While the antioxidant, antimicrobial and neutrophil-modulating A. viridiflora, A. glabra and A. monticola to 256 μg/mL for
activities of A. glabra have been studied, anti-Helicobacter and cyclohexane extracts of A. viridiflora, A. glabra and A. fissa. The
other biological activities have not been described previously [16]. best overall activity was obtained with A. monticola extracts.
The present study was carried out to evaluate the anti-H. pylori
1370 Natural Product Communications Vol. 10 (8) 2015 Krivokuća et al.
Table 2: Anti-Helicobacter pylori activity of methanol, dichlormethane and cyclohexane extract of selected Alchemilla species (MIC, μg/mL).
Strains A. viridiflora A. fissa A. glabra A. monticola M* C**
MeOH CH2Cl2 C6H6 MeOH CH2Cl2 C6H6 MeOH CH2Cl2 C6H6 MeOH CH2Cl2 C6H6
1. 4 128 128 8 64 128 4 64 64 4 16 8 S R
ATCC 43504 16 64 128 16 128 128 16 128 128 8 64 64 R S
2. 8 64 128 32 128 256 32 128 128 4 64 32 S S
3. 4 64 64 4 64 64 4 64 64 4 32 32 S S
4. 4 64 128 4 64 128 4 64 256 4 32 64 S R
5. 4 128 128 8 128 256 4 128 256 8 64 64 R S
6. 4 64 128 16 128 256 16 128 128 8 64 64 S S
7. 4 16 128 4 64 256 4 64 128 16 32 32 S R
8. 4 64 64 4 64 64 4 64 64 8 32 32 R R
9. 8 128 128 8 256 256 32 256 128 8 64 64 R R
10. 8 128 256 16 256 256 8 256 256 16 64 64 S S
* ** ***
MIC-minimal inhibitory concentration, Metronidazole, Claritromycin; 1-10 – clinical isolates of Helicobacter pylori; MeOH – methanol, CH2Cl2 –dichloromethane, C6H6 –
cyclohexane.
Table 3: Anti-Helicobacter pylori activity of ellagic acid and quercetin-3-O-β-D- Quantification of ellagic acid: Ellagic acid content was determined
glucoside (MIC μg/mL).
in methanol extracts (2 mg/mL). HPLC separation was performed
Strains Ellagic acid Quercetin-3-O-β-D- M* C** using an Agilent 1200 Series system equipped with a G1311A
glucoside
ATCC 43504 0.5 8 R S quaternary pump, a G1329A injector and G1315D DAD detector.
1 0.5 16 S R The following conditions were used: column Zorbax Eclipse XDB-
0.5 16 S S
2
3 0.25 4 S S
C18 (4.6 x 250 mm, 5 µm); column temperature 25oC; injection
4 0.125 4 S R volume 5 µL; a flow rate 0.350 mL/min; detection at 256 and 367
5 0.25 16 R S nm. The mobile phase (A) water + 0.1% formic acid and (B)
6 0.5 16 S S
7 0.125 4 S R acetonitrile was used with a linear gradient: 0-2 min from 10 to 20%
8 0.25 4 R R (B), 2-4.5 min from 20 to 90% (B), 4.5-4.8 min 90% (B), 4.8-4.9
9 0.125 8 R R min from 90 to 10% (B), 4.9-12.0 min 10% (B). The calibration
10 0.25 2 S S
1-10 – clinical isolates of Helicobacter pylori; *Metronidazole, **Claritromycin.
curve was constructed from standard solutions of ellagic acid in
methanol in the concentration range 0.017-0.55 mg/mL. The
Ellagic acid exhibited strong activity in very low concentrations relationship between the peak area and concentration of ellagic acid
(0.125-0.5 μg/mL), while the second identified compound, was linear with a regression equation y=73579.63x+1232.5472. The
quercetin-3-O-β-D-glucoside was also very active in concentrations correlation coefficient value corresponding to the calibration curve
of 2-16 μg/mL (Table 3). Previously, ellagic acid has shown activity was r2=0.998. Each measurement was performed in triplicate. The
against H. pylori ATCC 10231 with MIC values of 0.125 mg/mL, same chromatographic conditions were used for qualitative analyses
which was in accordance with our results, and MBC values against of quercetin-3-O-β-D-glucoside.
H. pylori G21 and 10K of 2 μg/mL and 10 μg/mL, respectively
[5,19]. In the same experiment, quercetin-3-O-β-D-glucoside
Anti-Helicobacter pylori activity: Ten clinical isolates of H. pylori,
possessed very low activity against H. pylori G21 and 10K with including one highly resistant to metronidazole (M) (MIC > 16
MBC 480 μg/mL and 240 μg/mL, respectively [19]. ng/ml), 3 resistant to claritromycin (C) (MIC > 1 ng/ml), 2 resistant
to both M and C, and 4 C and M susceptible, were tested. A
reference strain of H. pylori (ATCC 43504) was used as a control.
The high activity of the methanol extracts could be explained by the
The strains were maintained at -80° C in Wilkins Chalgren Broth
presence of tannins, especially ellagic acid and ellagitannins. Also,
(Difco) with 10%, v/v, horse serum (Seromed) and 20%, v/v,
the high activity of quercetin-glucoside could not be disregarded.
glycerol (Merck) until required for experiments. Before being used,
the strains were sub-cultured twice on Columbia agar base (Difco)
Experimental
supplemented with 10% horse serum and 0.25% bacto yeast extract
Plant extract preparation: Aerial parts of A. fissa and A. viridiflora (Difco). Plates were incubated for 72 h at 37°C in an atmosphere of
were collected in July 2013 at Mt Suva Planina (Serbia), while 10% CO2 in a gas incubator. Before inoculation, the shape and
A. monticola and A. glabra were collected in August 2013 from motility of the organisms were controlled by Gram staining and
Mt Stara Planina (Serbia). Voucher specimens (20130708/1-2, phase-contrast microscopy. MICs were determined by a modified
20130708/1-2) were deposited in the Natural History Museum broth dilution method, as previously described [21]. Briefly,
(Belgrade, Serbia) and identification of plant material was made by twofold serial dilution of the test compounds were prepared in a 96-
Dr Marjan Niketić. The air-dried, powdered material (380 g of A. well microtiter plate containing 100 µL of MegaCellTM RPMI-
fissa; 320 g of A. viridiflora; 53 g of A. glabra; 8.27 g A. monticola) 1640 medium with 3% fetal calf serum (FCS). An inoculum
were extracted with cyclohexane, dichlormethane and methanol, equivalent to 1 McFarland standard was prepared in Wilkins
successively. The solvents were evaporated under low pressure to Chalgren broth and diluted in MegaCellTM RPMI-1640 medium
yield extracts. with 3% FCS. Each well was inoculated with H. pylori at a final
concentration of approximately 5 x 105 CFU/well. The plates were
Quantification of tannins: The official spectrometric method from incubated at 37°C under microaerophilic conditions (10% CO2 in a
Ph. Eur. 7.0 was used for quantification of tannins in the methanol gas incubator) and examined after 72 h of incubation.
extracts [20].
Acknowledgments - The authors are grateful to the Ministry of
Science (Grant Nos. 173021 and 34012) for financial support.
Anti-Helicobacter pylori activity of four Alchemilla species Natural Product Communications Vol. 10 (8) 2015 1371
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