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Anti-Helicobacter pylori Activity of the Methanolic Extract
of Geum iranicum
and its Main Compounds
Somayeh Shahania, Hamid R. Monsef-Esfahania, Soodabeh Saeidniab,
Parastoo Sanieec, Farideh Siavoshic, Alireza Foroumadid, Nasrin Samadie,
and Ahmad R. Goharib,*
a Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical b Medicinal Plants Research Center, Tehran University of Medical Sciences, Tehran, P. O. Box 14155-6451, Iran. Fax: +98-21-64122330. E-mail: Microbiology Department, Faculty of Sciences, University of Tehran, Tehran, Iran d Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, e Department of Drug and Food Control, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran * Author for correspondence and reprint requests Z. Naturforsch. 67 c, 172 – 180 (2012); received May 30, 2011/January 14, 2012
Geum iranicum Khatamsaz, belonging to the Rosaceae family, is an endemic plant of Iran. The methanol extract of the roots of this plant showed signifi cant activity against one of the clinical isolates of Helicobacter pylori which was resistant to metronidazole. The aim of this study was the isolation and evaluation of the major compounds of G. iranicum effective against H. pylori. The compounds were isolated using various chromatographic methods and identifi ed by spectroscopic data (1H and 13C NMR, HMQC, HMBC, EI-MS). An an-timicrobial susceptibility test was performed employing the disk diffusion method against clinical isolates of H. pylori and a micro dilution method against several Gram-positive and Gram-negative bacteria; additionally the inhibition zone diameters (IZD) and minimum inhibitory concentrations (MIC) values were recorded. Nine compounds were isolated: two triterpenoids, uvaol and niga-ichigoside F1, three sterols, β-sitosterol, β-sitosteryl acetate, and β-sitosteryl linoleate, one phenyl propanoid, eugenol, one phenolic glycoside, gein, one fl a-vanol, (+)-catechin, and sucrose. The aqueous fraction, obtained by partitioning the MeOH extract with water and chloroform, was the most effective fraction of the extract against all clinical isolates of H. pylori. Further investigation of the isolated compounds showed that eugenol was effective against H. pylori but gein, diglycosidic eugenol, did not exhibit any activity against H. pylori. The subfraction D was the effective fraction which contained tannins. It appeared that tannins were probably the active compounds responsible for the anti-H. pylori activity of G. iranicum. The aqueous fraction showed a moderate inhibitory activity against both Gram-positive and Gram-negative bacteria. The MIC values indicated that Gram-positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis are more susceptible than Gram-neagative bacteria including Escheri-chia coli and Pseudomonas aeruginosa.
Key words: Geum iranicum, Helicobacter pylori, Eugenol Introduction
infection, including antibiotics and a proton pump inhibitor, may fail for several reasons. The main Helicobacter pylori colonizes the stomachs of reason was found to be H. pylori resistance to about 50% of the world’s human population. antibiotics like clarithromycin and metronidazole This organism is the main risk factor for peptic (Mégraud, 2004). Therefore, the research for new ulceration as well as gastric mucosal-associated anti-H. pylori drugs from plant sources is ongoing lymphoid tissue (MALT) lymphoma and gastric (Nariman et al., 2004).
adenocarcinoma. The prevention of H. pylori The genus Geum, belonging to the Rosaceae colonization could potentially provide primary family, is a perennial rhizomatous herb with fi ve prevention of the mentioned diseases (Fauci et species in Iran of which G. iranicum Khatamsaz is al., 2008). The common treatments of H. pylori an endemic one (Khatamsaz, 1992; Mozaffarian, 2012 Verlag der Zeitschrift für Naturforschung, Tübingen · S. Shahani et al. · Anti-Helicobacter pylori Activity of Geum iranicum 1996). Some Geum species are used as medicinal reagent followed by heating were used for detect- plants in folk medicine (Vollmann and Schultze, ing the compounds. NMR experiments were per- 1995; Gruenwald, 2004). The roots of G. urba- formed on a Bruker Avance 500 DRX (500 MHz num and G. rivale are employed against diarrhea for 1H and 125 MHz for 13C) spectrometer (Rhein- (Gruenwald, 2004), and G. japonicum has been stetten, Germany). EI-MS spectra were measured used as diuretic and astringent in traditional Chi- on an Agilent Technology (Palo Alto CA, USA) nese medicine and Japanese folk medicine (Ming instrument with a 5973 Network mass selective et al., 2000). In an Iranian folk remedy, the infu- detector (MS model). A CO incubator (Heraeus, sion of the root of G. iranicum is employed to Hamburg, Germany) was used for the antibacte-treat gastrointestinal disorders like diarrhea, and a decoction of the whole plant is combined with wheat fl our and used as a poultice for frostbite Plant collection(Abutorabi, 2001). Previous studies on extracts of Roots of Geum iranicum were collected from Geum species have shown that they can be ef- the Gloul Sarani protected area, 75 km north of fective in the treatment of some diseases. A new Shirvan, province of Khorasan-e-Shomali, Iran, at triterpene acid, 2α,19α-dihydroxy-3-oxo-urs-12- 2460 m above sea level, during the fl owering stage en-28-oic acid, from the extract of G. japonicum in June 2009. A voucher specimen (6714 THE) showed potent inhibitory activity against HIV-1 was deposited at the herbarium of the Faculty of protease (Xu et al., 2000). In addition, the ex- Pharmacy, Tehran University of Medical Sciences, tract of G. japonicum was active against HSV-1 Tehran, Iran.
and HSV-2 (herpes simplex virus) (Kurokawa et al., 1995) and CMV (cytomegalo virus) (Yukawa et al., 1996). Some tannins isolated from the ex-tract of this plant showed potent anticoagulant Three clinical isolates of H. pylori, Is.1, Is.2, and activity (Zeng et al., 1998). Other Geum species Is.3, were obtained from patients with chronic showed antioxidant (Russo et al., 2005), antimi- gastritis who had been referred to the Endoscopy crobial (Panizzi et al., 2000), and anti-infl amma- Unit at Shariati Hospital, Tehran, Iran. Antral bi- tory (Tunón et al., 1995) effects, respectively. In opsies with positive rapid urease tests were cul-another study, antimicrobial activity was reported tured in the microbiology laboratory as described for the extract and essential oil of Geum kokani- in our previous study (Siavoshi et al., 2010). An- cum that was collected in Iran (Faramarzi et al., timicrobial susceptibility was tested by the disk 2008). Furthermore, the polar extract of this spe- diffusion method according to the guidelines of cies showed a potent inhibitory effect on matrix the NCCLS (2006). Serial dilutions of test sam- metalloproteinase activity at minimal cytotoxic ples were made in dimethyl sulfoxide (DMSO) doses (Khorramizdeh et al., 2006).
(100, 50, 25, 12.5 μg/ml). Bacterial suspensions A literature review revealed that the anti- were prepared in normal saline with the turbidity H. pylori activity of Geum species has not been of McFarland standard No. 2 (equivalent to 6 · 108 investigated. In the present study we aimed there- cell/ml). The surface of blood agar plates was in- fore to evaluate the activity of various extracts, oculated with 100 μl of each bacterial suspension. sub-fractions, and main components of G. irani- Then, plates were dried at room temperature for cum against clinical isolates of H. pylori (resistant about 10 min. The sterile blank disks (6 mm in di- to metronidazole). Furthermore, the evaluation of ameter) were placed on the surface of inoculated the antibacterial activity (against Gram-positive plates and impregnated with 10 μl of each sample and Gram-negative bacteria) of fractions, to- dilution. Control plates included blank disks im- gether with the isolation and identifi cation of the pregnated with 10 μl of DMSO. Plates were in- cubated at 37 °C under microaerobic conditions and examined after 3 – 5 d. The inhibition zone Material and Methods
Antibacterial activity of the aqueous fraction was also tested against several Gram-positive pre-coated plates (Merck, and Gram-negative bacteria including Staphylo- Darmstadt, Germany) and anisaldehyde/H SO coccus aureus ATCC 6538, Staphylococcus epider- S. Shahani et al. · Anti-Helicobacter pylori Activity of Geum iranicum midis ATCC 12228, Bacillus subtilis ATCC 6633, MeOH (9:1, 7:3, 4:6 v/v) and MeOH to yield 6 Escherichia coli ATCC 8739, and Pseudomonas fractions (A – F). Fraction A was separated by aeruginosa ATCC 9027 by a micro dilution meth- silica gel CC (2.5 × 20 cm) with CHCl /MeOH od using 96 U-shaped wells plates (NCCLS, 2006). (19:1, 9:1, 8:2 v/v) to yield 5 fractions (A – A ). A 200-μl aliquot of stock solutions of the fraction Fraction A was compound 6 (129 mg). Fraction C
(128 mg/ml) and ciprofl oxacin (100 μg/ml) as a was subjected to silica gel CC (2.5 × 20 cm) with standard antibiotic compound in Mueller-Hinton CHCl /MeOH (8:2, 7:3 v/v) to give 4 fractions broth (MHB) were transferred into the fi rst well (C – C ). Compound 7 (13 mg) was obtained from
in each row and serially diluted by mixing with C using silica gel CC (1.5 × 40 cm) with CHCl / 100 μl of MHB in subsequent wells. Then 100 μl MeOH (75:25 v/v). Fraction D was chromato- of bacterial suspension (1 · 106 CFU/ml) were graphed on a silica gel column (4 × 20 cm) with added to each well to reach the fi nal inoculum CHCl /MeOH (8:2, 6:4 v/v) and MeOH to result size of about 5 · 105 CFU/ml. After 24 h of incuba- in factions D – D of which D was compound tion at 35 °C, the microdilution trays were tested 8 (70 mg). Compound 9 (960 mg) was obtained
for the absence or presence of visible growth. from fraction E using silica gel CC (2.5 × 20 cm) The endpoint minimum inhibitory concentration (MIC) is the lowest concentration of the fraction β-Sitosteryl acetate (1): R = 0.14 in n-hexane/
at which the test strain does not demonstrate vis- CHCl (7:3 v/v). – 1H NMR (CDCl , 500 MHz): δ = 0.69 (3H, s, H-18), 0.82 (3H, d, J = 6.8 Hz, H-27), 0.84 (3H, d, J = 6.8 Hz, H-26), 0.86 (3H, t, Extraction and isolation procedure J = 7.3 Hz, H-29), 0.93 (3H, d, J = 6.5 Hz, H-21), Dried roots of G. iranicum (1 kg) were cut into 1.03 (3H, s, H-19), 2.05, (3H, s, OCH ), 4.61 (1H, small pieces and extracted with EtOAc and MeOH m, H-3), 5.38 (1H, brd, J = 5 Hz, H-6). – 13C NMR (3 × 4 l for each solvent), successively, by percola- (CDCl , 500 MHz): δ = 11.8 (C-18), 11.9 (C-29), tion at room temperature to obtain EtOAc (6 g) 18.8 (C-21), 19.0 (C-27), 19.3 (C-19), 19.8 (C-26), and MeOH (75 g) extracts. The EtOAc extract 21.0 (C-11), 21.4 (COOCH ), 23.1 (C-28), 24.3 was subjected to silica gel column chromatogra- (C-15), 26.1 (C-23), 27.7 (C-2), 28.2 (C-16), 29.1 phy (CC) (4 × 20 cm) using n-hexane/CHCl (7:3, (C-25), 31.8 (C-8), 31.9 (C-7), 33.9 (C-22), 36.1 3:7 v/v), EtOAc, and MeOH as eluents to give (C-20), 36.6 (C-10), 36.9 (C-1), 38.1 (C-4), 39.7 7 fractions (A – G). Fraction A was further sepa- (C-12), 42.3 (C-13), 45.8 (C-24), 50.0 (C-9), 56.0 rated by silica gel CC (3 × 20 cm) with n-hexane, (C-17), 56.7 (C-14), 73.9 (C-3), 122.6 (C-6), 139.6 n-hexane/CHCl (7:3, 3:7 v/v) and CHCl to give 5 (C-5), 170.5 (C=O). – EI-MS: m/z (%) = 396 [M– fractions (A – A ) of which fraction A contained HOAc]+ (100), 381 (15), 288 (9), 275 (6), 255 (18), compound 1 (15 mg). Silica gel CC (2 × 20 cm) 213 (21).
was used for separation of fraction B to yield
β-Sitosteryl linoleate (2): R = 0.37 in n-hexane/
7 fractions (B –B ) with n-hexane/CHCl (7:3, 6:4, CHCl (7:3 v/v). – 1H NMR (CDCl , 500 MHz): 4:6 v/v) and CHCl . Fraction B was 2 (22 mg). δ
= 0.68 (3H, s, H-18), 0.81 (3H, d, J = 7 Hz, H-27), Fraction C was fractionated with n-hexane/CHCl 0.83 (3H, d, J = 7 Hz, H-26), 0.84 (3H, m, H-29), (9:1, 5:5 v/v) by silica gel CC (2 × 20 cm) to give 0.88 (3H, t, J = 6.5 Hz, H-18’), 0.92 (3H, d, J = fractions C – C ; compound 3 (141 mg) was iso-
7 Hz, H-21), 1.03 (3H, s, H-19), 1.25 – 1.38 (14H, lated from fraction C . Compound 4 (5 mg) was
m, H-4’, 5’, 6’, 7’, 15’, 16’, 17’), 1.59 – 1.65 (2H, m, obtained from fraction D using silica gel CC H-3’), 2.03 – 2.08 (4H, m, H-8’, 14’), 2.29 (2H, t, J = (2.5 × 20 cm) with n-hexane/EtOAc (8:2 v/v).
7.5 Hz, H-2’), 2.76 (2H, t, J = 6.5 Hz, H-11’), 4.6 The MeOH extract (75 g) was suspended in wa- (1H, m, H-3), 5.30 – 5.40 (5H, m, olefi nic protons). ter and extracted with CHCl to obtain CHCl and – 13C NMR (CDCl , 500 MHz): δ = 11.8 (C-18), aqueous fractions. The CHCl fraction was further 11.9 (C-29), 14.1 (C-18’), 18.8 (C-21), 19.0 (C-27), chromatographed with n-hexane/CHCl (7:3, 3:7 19.3 (C-19), 19.8 (C-26), 21.0 (C-11), 22.7 (C-17’), v/v) and CHCl to give 6 fractions of which frac- 23.1 (C-28), 24.3 (C-15), 25.1 (C-3’), 25.6 (C-11’), tion 5 was compound 5 (17 mg). The aqueous por-
26.0 (C-23), 27.2 (C-8’,14’), 27.8 (C-2), 28.3 (C- tion of the MeOH extract (70 g) was subjected to 16), 29.1 (C-25), 29.2 (C-4’), 29.3 (C-5’), 29.4 (C- silica gel CC (10 × 20 cm) with EtOAc, EtOAc/ 15’), 29.6 (C-6’), 29.7 (C-7’), 31.6 (C-16’), 31.8 S. Shahani et al. · Anti-Helicobacter pylori Activity of Geum iranicum (C-7), 31.9 (C-8), 33.9 (C-22), 34.4 (C-2’), 36.2 Catechin (6): R = 0.37 in CHCl /MeOH (8:2
(C-20), 36.5 (C-10), 37.0 (C-1), 38.2 (C-4), 39.7 v/v). – 1H NMR (CD OD, 500 MHz): δ = 2.51 (C-12), 42.3 (C-13), 45.8 (C-24), 50.0 (C-9), 56.0 (1H, dd, J = 16.1, 8.2 Hz, H-4a), 2.86 (1H, dd, J = (C-17), 56.7 (C-14), 73.7 (C-3), 122.6 (C-6), 127.9 16.1, 5.4 Hz, H-4b), 3.97 (1H, m, H-3), 4.57 (1H, (C-10’), 128.0 (C-12’), 130.0 (C-9’), 130.2 (C-13’), d, J = 7.5 Hz, H-2), 5.86 (1H, d, J = 2.2 Hz, H-8), 139.7 (5), 173.3 (C=O). – EI-MS: m/z (%) = 676 5.93 (1H, d, J = 2.2 Hz, H-6), 6.72 (1H, dd, J = 8.1, [M]+ (1), 396 (100), 381 (15), 288 (12), 275 (14), 2 Hz, H-6’), 6.76 (1H, d, J = 8.1, H-5’), 6.84 (1H, d, J = 2 Hz, H-2’). – 13C NMR (CD OD, 500 MHz): β-Sitosterol (3): R = 0.13 in n-hexane/EtOAc
δ = 28.4 (C-4), 68.6 (C-3), 82.7 (C-2), 95.4 (C-8), (9:1 v/v). – 1H NMR (CDCl , 500 MHz): δ = 0.68 96.2 (C-6), 100.7 (C-10), 115.1 (C-2’), 116.0 (C-5’), (3H, s, H-18), 0.79 (3H, d, J = 6.5 Hz, H-27), 0.82 119.9 (C-6’), 132.0 (C-1’), 146.1 (C-3’), 146.1 (C- (3H, d, J = 6.5 Hz, H-26), 0.83 (3H, t, J = 7.5 Hz, 4’), 156.8 (C-9), 157.4 (C-5), 157.6 (C-7).
H-29), 0.91 (3H, d, J = 6.5 Hz, H-21), 0.99 (3H, s, Niga-ichigoside F1 (2α,3β,19α,23-tetrahydroxy- H-19), 3.5 (1H, m, H-3), 5.3 (1H, brd, J = 5 Hz, urs-12-en-28-oic acid β- H-6). – 13C NMR (CDCl , 500 MHz): δ = 11.8 (C-18), 11.9 (C-29), 18.8 (C-21), 19.0 (C-27), 19.4 (7): R = 0.47 in CHCl /MeOH (75:25 v/v). – 1H
(C-19), 19.8 (C-26), 21.1 (C-11), 23.0 (C-28), 24.3 NMR (CD OD, 500 MHz): δ = 0.7 (3H, s, H-24), (C-15), 26.0 (C-23), 28.3 (C-16), 29.1 (C-25), 31.6 0.78 (3H, s, H-26), 0.93 (3H, d, J = 6.5 Hz, H-30), (C-2), 31.8 (C-7), 31.9 (C-8), 33.9 (C-22), 36.1 (C- 1.04 (3H, s, H-25), 1.21 (3H, s, H-29), 1.34 (3H, s, 20), 36.5 (C-10), 37.2 (C-1), 39.7 (C-12), 42.3 (C- H-27), 2.52 (1H, s, H-18), 3.25 (1H, d, J = 11 Hz, 13), 45.8 (C-24), 50.1 (C-9), 56.0 (C-17), 56.8 (C- H-23a), 3.49 (1H, d, J = 11 Hz, H-23b), 3.67 (1H, 14), 71.8 (C-3), 121.7 (C-6), 140.7 (C-5). – EI-MS: m, H-2), 3.35 – 3.82 (5H, H -2 – 6), 5.31 (2H, d, m/z (%) = 414 [M]+ (100), 399 (38), 396 (32), 381 J = 8.1 Hz, H -1, H-12). – 13C NMR (CD OD, (40), 303 (75), 273 (37), 231 (41), 213 (67).
500 MHz): δ = 13.9 (C-24), 16.6 (C-30), 17.6 Uvaol (12-ursen-3β,28-diol) (4): R = 0.23 in (C-26), 17.7 (C-25), 19.2 (C-6), 24.7 (C-27), 24.8
n-hexane/EtOAc (8:2 v/v). – 1H NMR (CDCl , (C-11), 26.5 (C-16), 27.1 (C-29), 27.2 (C-21), 29.6 500 MHz): δ = 0.80 (3H, s, H-25), 0.81 (3H, d, (C-15), 33.5 (C-7), 38.3 (C-22), 38.9 (C-10), 40.8 J = 5.8 Hz, H-30), 0.94 (3H, d, H-29), 0.95 (3H, s, (C-8), 42.8 (C-14), 42.9 (C-20), 44.1 (C-4), 48.0 (C- H-24), 0.99 (3H, s, H-26), 1.00 (3H, s, H-27), 1.11 1), 48.2 (C-9), 48.4 (C-5), 54.9 (C-18), 66.4 (C-23), (3H, s, H-23), 3.19 (1H, d, J = 11 Hz, H-28a), 3.23 69.7 (C-2), 73.6 (C-19), 78.3 (C-3), 129.7 (C-12), (1H, dd, J = 4.9, 11.3 Hz, H-3), 3.53 (1H, d, J = 139.5 (C-13), 178.5 (C=O); glucose: 62.4 (C-6’), 11 Hz, H-28b), 5.14 (1H, t, J = 3.5, H-12). – 13C 71.1 (C-4’), 73.8 (C-2’) 78.3 (C-5’), 78.5 (C-3’), NMR (CDCl , 500 MHz): δ = 15.6 (C-24), 15.7 95.8 (C-1’). – EI-MS: m/z (%) = 504 [M–C H O ] (C-25), 16.8 (C-29), 17.3 (C-26), 18.3 (C-6), 21.3 (4), 426 (23), 344 (100), 264 (15), 246 (18), 239 (C-30), 23.3 (C-27), 23.4 (C-11), 26.0 (C-16), 27.2 (C-2), 28.1 (C-23), 29.7 (C-15), 30.6 (C-21), 32.8 Gein (eugenyl vicianoside) (8): R = 0.2 in
(C-7), 35.2 (C-22), 36.8 (C-17), 38.0 (C-10), 38.8 CHCl /MeOH (8:2 v/v). – 1H NMR, 13C NMR, (C-1), 38.8 (C-4), 39.3 (C-19), 39.4 (C-20), 40.0 (C- and HMBC (CD OD, 500 MHz): see Table I.
8), 42.8 (C-14), 47.6 (C-9), 54.0 (C-18), 55.1 (C-5), 69.9 (C-28), 79.0 (C-3), 125.0 (C-12), 138.7 (C-13). Sucrose (9): R = 0.28 in EtOAc/MeOH (6:4
– EI-MS: m/z (%) = 442 [M]+ (5), 411 (35), 234 v/v). – 1H NMR (D O, 500 MHz): δ = 3.31 (1H, (42), 203 (100), 189 (42), 133 (29).
t, J = 9.5 Hz, H -4), 3.4 (1H, dd, J = 10, 3.8 Hz, Eugenol (5): R = 0.26 in n-hexane/CHCl (3:7
H -2), 3.51 (2H, s, H -1), 3.59 (1H, t, J = 9.5 Hz, v/v). – 1H NMR (CDCl , 500 MHz): δ = 3.32 (2H, H -3), 3.66 (4H, m, H -6 and H -6), 3.69 (1H, d, J = 6.65 Hz, H-7), 3.87 (3H, s, OCH ), 5.05 (2H, m, H -5), 3.72 (1H, m, H -5), 3.89 (1H, t, J = m, H-9), 5.48 (1H, brs, OH), 5.95 (1H, m, H-8), 8.5 Hz, H -4), 4.04 (1H, d, J = 8.7 Hz, H -3), 6.69 (2H, m, H-3 and H-5), 6.84 (1H, d, J = 8.5 Hz, 5.25 (1H, d, J = 3.8 Hz, H -1). – 13C NMR (D O, H-6). – 13C NMR (CDCl , 500 MHz): δ = 39.9 (C- 500 MHz): δ = 60.1 (C -6), 61.3 (C -1), 62.3 7), 55.8 (OCH ), 111.1 (C-3), 114.2 (C-6), 115.5 (C -6), 69.2 (C -4), 71.0 (C -2), 72.4 (C -5), (C-9), 121.2 (C-5), 131.9 (C-4), 137.8 (C-8), 143.9 72.5 (C -3), 73.9 (C -4), 76.3 (C -3), 81.3 (C - S. Shahani et al. · Anti-Helicobacter pylori Activity of Geum iranicum Table I. NMR data of compound 8 in CD OD.
H-6’a, H- 6’b, H-2”, H-5”a, H-5”b and 13C NMR, HMQC, HMBC, 1H-1H COSY and EI-MS were employed for identifi cation of the The inhibition zone diameters (IZD) of test isolated compounds. In the previous study, the samples and antibiotics against H. pylori are NMR data of gein were reported in CDCl and summarized in Tables II and III. No inhibition pyridine-d (Shigenaga et al., 1985). To the best zone was observed for DMSO in control plates. of our knowledge, there is no report on the 2D- The MIC values of the aqueous fraction and cip- NMR correlations of this compound, so that this rofl oxacin (as a positive control) against several is the fi rst report on the NMR data of gein in Gram-positive and Gram-negative bacteria are CD OD and HSQC and HMBC correlations (Ta- roots, nine compounds including two triterpe- Discussion
noids, uvaol (4) (Mahato and Kundu, 1994) and
niga-ichigoside F1 (7) (Bowen-Forbes et al., 2009),
The MeOH extract of G. iranicum was consid- three sterols, β-sitosterol (3), β-sitosteryl acetate ered an effective extract against one of the clini-
(1) (Goad and Akihisa, 1997), and β-sitosteryl li-
cal isolates of H. pylori which was resistant to noleate (2) (Huh et al., 2010; Dyas et al., 1991), metronidazole. As shown in Table II, the chloro-
one phenyl propanoid, eugenol (5), one phenolic
form fraction, obtained from the MeOH extract, glycoside, gein (8) (Takeda et al., 1998; Shimoda
was not effective against H. pylori, but the aque- et al., 2007; Shigenaga et al., 1985), one fl avanol, ous fraction showed a potent activity (Table II). (+)-catechin (6) (Banavides et al., 2006), and su-
Subfractions of the aqueous part were evaluated crose (9) (Agrawal, 1992) were isolated by col-
for their antibacterial activity against all isolates. umn chromatography and identifi ed by compari- Fraction D was the only one that displayed an- son of their spectroscopic data with those in the tibacterial activity (MIC = 25 μg/ml). Therefore, literature (Fig. 1). Spectroscopic data such as 1H the fractions obtained from D fractionation were S. Shahani et al. · Anti-Helicobacter pylori Activity of Geum iranicum Table II. Inhibition zone diameters (mm) of some extracts of G. iranicum. 35  0.5 30  0.5 24  0.5 28  0 26  0.5 18  0.25 24  0.57 24  0.5 16  0 20  0.5 19  0.5 14  0 22  0.5 21  0.25 12  0.5 15  0.5 12  0.57 12  0.5 10  0.5 10  0.5 The results are shown as mean  SD (n = 3).
Is, clinical isolate of H. pylori.
Table III. Inhibition zone diameters (mm) of some an- Table IV. Minimum inhibitory concentrations of the tibiotics against clinical isolates of H. pylori.
aqueous fraction of G. iranicum against several Gram-positive and Gram-negative bacteria.
a The bacterial strains are Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027.
evaluated again and resulted in one active frac- tion D (Table II). Finally, the isolated compounds from the chloroform and aqueous fractions (eugenol, gein, catechin, and niga-ichigoside F1) were examined for anti-H. pylori activity. Among the tested compounds, eugenol (5), isolated from
the chloroform fraction, showed an antibacterial effect at a concentration of 100 μg/ml with inhibi- tion zones of 20, 22, and 10 mm against the iso- lates 1, 2, and 3 respectively. Eugenol is a major compound in the root oil of G. iranicum (Sha- a Is, clinical isolate of H. pylori.
hani et al., 2011) and is employed as a fl avour- S. Shahani et al. · Anti-Helicobacter pylori Activity of Geum iranicum 2: R= Linoleate
8: R= Į-L-Ara (1ĺ6)-ȕ-D-Glc
Fig. 1. Chemical structures of the isolated compounds.
Fig. 2. HMBC correlations of compound 8 (H  C).
ing agent in cosmetics and food products, as well al., 2005), anti-infl ammatory (Yogalakshmi et al., as a cement material in dentistry (Atsumi et al., 2010), anti nociceptive (Daniel et al., 2009), and 2005). Furthermore, eugenol has been found to antidepressant activity (Tao et al., 2005). A litera-exhibit a broad range of biological activities in- ture review shows that the minimal bactericidal cluding antibacterial (Devi et al., 2010), antifun- concentration (MBC) of eugenol (obtained from gal (Campaniello et al., 2010), antiviral (Benen- essential oils) was 100 μg/ml against one strain cia and Courreges, 2000), antioxidant (Atsumi et of H. pylori, which had been isolated from a pa- S. Shahani et al. · Anti-Helicobacter pylori Activity of Geum iranicum tient with non-ulcer dyspepsia (Bergonzelli et al., be one of the important groups of compounds ac- 2003). Elsewhere it was reported that eugenol tive against H. pylori.
(commercial preparation) inhibited the growth of The aqueous fraction exhibited a moderate in- 30 strains of H. pylori at a concentration of 2 μg/ hibitory activity against both Gram-positive and ml (Ali et al., 2005). Our antibacterial data for Gram-negative bacteria. The Gram-positive bac-eugenol agree with the results reported by Ber- teria were inhibited at 16 mg/ml, while Gram-neg- ative bacteria were inhibited at about 32 mg/ml.
Diglycosylated eugenol, named gein (8), pro-
In conclusion, considering the increasing resist- duced no inhibition zone. Gein has been isolated ance of H. pylori to antibiotics like metronidazole from other Geum species (Shigenaga et al., 1985), and clarithromycin, there is great interest in fi nd- and no biological activity has been reported for ing new drugs from natural sources. The results of this compound. It seems that glycosylation of this study show that G. iranicum has a signifi cant eugenol causes a decrease in anti-H. pylori activ- antibacterial activity against resistant clinical iso- ity. Also other purifi ed compounds like catechin lates of H. pylori. Eugenol is one of the effective (6) and niga-ichigoside F1 (7) did not display any
compounds, whereas other purifi ed compounds antibacterial activity at all concentrations.
(like gein, catechin, and niga-ichigoside F1) were Fraction D exhibited antibacterial activity as not effective. Finally, the tannin-containing frac- shown in Table II. Further investigation of frac- tion of G. iranicum showed considerable anti- tion D indicated that this fraction can precipi- H. pylori activity. Further investigation is required tate a solution of gelatin (1%), containing sodium to purify the active tannins from this plant.
chloride (10%), and give a blue-black precipitate with ferric chloride. Therefore, this fraction should include polar compounds, especially hydrolysable tannins (Evans, 2009). There is a report in the lit- This research has been supported by a grant erature on the antibacterial activity of hydrolysa- from Tehran University of Medical Sciences and ble tannins derived from medicinal plants against Health Services. The authors thank Mr. Yousef H. pylori (Funatogawa et al., 2004). For this rea- Ajani from the Institute of Medicinal Plants for son, it seems that tannins from G. iranicum might plant collection and identifi cation.
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