Phytochemical composition, protective and therapeutic effect on gastric ulcer and α-amylase inhibitory activity of Achillea biebersteinii Afan.

Arch. Pharm. Res. DOI 10.1007/s12272-014-0544-9

RESEARCH ARTICLE

Phytochemical composition, protective and therapeutic effect on gastric ulcer and a-amylase inhibitory activity of Achillea biebersteinii Afan. Howaida I. Abd-Alla • Nagwa M. M. Shalaby Manal A. Hamed • Nagy Saba El-Rigal • Samira N. Al-Ghamdi • Jalloul Bouajila

•

Received: 10 September 2014 / Accepted: 24 December 2014 The Pharmaceutical Society of Korea 2015

Abstract Three sesquiterpene lactones [two germacranolides (micranthin and sintenin) and one guaianolide (4b,10adihydroxy-5b,7b,8bH-guaia-1,11(13)dien-12,8a-olide)] and four derivatives of 3-methoxy flavones (santin, quercetagetin3,6,30 -trimethyl ether, quercetagetin-3,6-dimethyl ether, and 5,7 dihydroxy 3,30 ,40 -trimethoxy flavone) were isolated from the ethyl acetate extract (EAE) of the aerial parts of Achillea biebersteinii Afan. (Asteraceae). Evaluation of protective and therapeutic effects of EAE against ethanol-induced gastric ulcer in rats was carried. Antiulcer activity evaluation was done through measuring ulcer indices, stomach acidity, gastric volume and lesion counts. Oxidative stress markers; malondialdehyde, glutathione and superoxide dismutase were also estimated. The work was extended to determine the histopathological assessment of the stomach. Gastric ulcer exhibited a significant elevation of the ulcer index and oxidative stress markers. The extract attenuated these increments and recorded protective and therapeutic effects against gastric

H. I. Abd-Alla (&) N. M. M. Shalaby Department of Natural Compounds Chemistry, National Research Centre, Dokki, 12622 Giza, Egypt e-mail: [emailprotected] M. A. Hamed N. S. El-Rigal Department of Therapeutic Chemistry, National Research Centre, Dokki, 12622 Giza, Egypt S. N. Al-Ghamdi Faculty of Science, Branch of Girls, King Abdul-Aziz University, 21589 Jeddah, Saudi Arabia J. Bouajila Universite´ de Toulouse, Faculte´ de pharmacie de Toulouse, Laboratoire des Interactions Mole´culaires et Re´activite´ Chimique et Photochimique UMR CNRS 5623, Universite´ PaulSabatier, 118 route de Narbonne, 31062 Toulouse, France

ulcer. Hyperglycaemia increases the mucosal susceptibility to ulcerogenic stimuli and predisposes gastric ulceration. In vitro a-amylase inhibitory assay was applied to evaluate the post prandial antihyperglycaemia activity. The result showing that the EAE has the ability to reduce starch-induced postprandial glycaemic excursions by virtue of potent intestinal a-amylase inhibitory activity. These findings demonstrated the remarkable potential of A. biebersteinii as valuable source of antiulcer agent with post prandial hyperglycaemia lowering effect. Keywords Achillea biebersteinii Germacranolides Guaianolides 3-Methoxy flavones Gastric ulcer a-Amylase inhibition

Introduction The genus Achillea is one of the most important genera of the Asteraceae family and comprises about 85 species, widespread throughout the world. The name of the genus originates from the ancient use as a wound-healing remedy by the Trojan hero Achilles (Saeidnia et al. 2011). Achillea species have been used in folk medicine and sold in herbal shops. Herbal teas prepared from some Achillea species are traditionally used for abdominal pain and flatulence in different countries. An infusion of the dry or fresh flowering herb is used by the Bedouin for the treatment of coughs, aromatic bitter stomachic and anthelmintic (Elgamal et al. 1991). In folk medicine, yarrow species (Achillea spp.) were used for toothache and teeth hypersensitivity as a gum or a gargle. It is also used as a tea bag for kidney inflammation as well as a carminative. The constituents of Achillea are mainly terpenes (essential oil) (So¨kmen et al. 2004), flavonoids (Saeidnia

123

H. I. Abd-Alla et al.

et al. 2011), phenolic acids, sesquiterpene lactones, ionone glucosides and terpenoids (Mahmoud and Al-Shihry 2006). A. biebersteinii Afan was reported to have wound healing (Akkol et al. 2011), anti-gout (Hudaib et al. 2011), anticancer, antioxidant, antimicrobial, anti-epimastigote, and herbicidal potential (Saeidnia et al. 2011). Gastric ulcer is a major health hazard in terms of both morbidity and mortality (Jeong 2002; Chaturvedi et al. 2009) and one of the most common gastrointestinal diseases. In recent years, a widespread search has been launched to identify new antiulcer drugs from natural sources. Plant extracts are some of the most attractive sources of new drugs and have been shown to produce promising results for the treatment of gastric ulcer (Mard et al. 2008; Motawi et al. 2012). Hyperglycaemia has been reported to increase propensity of gastric ulceration (Chaturvedi et al. 2009). Many Achillea species are well known in traditional herbal medicine for hyperglycaemia treatment (Saeidnia et al. 2011) or had been established by animal experimentations viz. A. millefolium, A. santolina, and A. Ligustica (Saeidnia et al. 2011; Conforti et al. 2005). The present study was conducted to investigate the phytochemical composition of A. biebersteinii collected from Saudi Arabia and to assess protective and therapeutic efficacy of extract against ethanol-induced gastric ulcer in rats.

Materials and methods Plant materials The aerial part of A. biebersteinii Afan. was collected from mountains of Surrah, Almandaq Valley, Al-Baha, Saudi Arabia at altitude of 800–1,200 m height, in April 2010. They were identified by Dr. Farag Abd-Allah Alghamdi, Botany Department, King Abdul-Aziz University, Jeddah, Saudi Arabia. Voucher specimen was deposited in herbarium of National Research Centre, Cairo (Egypt). General Electrothermal digital and Gallenkamp electrothermal melting point Apparatus were used. UV–Vis analyses for pure samples were recorded, separately, on methanol. UV– Vis spectra were measured using a Shimadzu UV 240 spectrophotometer (200–600 nm). The NMR spectra were recorded at 300 (1H) and 75 (13C) MHz on Varian Mercury 300 (Varian, UK) or Bruker Avance 500 NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) and operating frequency was 125.75 MHz for 13C NMR spectra. For NMR spectrometer, d values are reported as ppm relative to tetramethylsilane (TMS) in the convenient solvent. EI-

123

MS spectra were recorded on a Finnigan MAT 95 spectrometer (70 eV) (Finnigan MAT, San Jose, CA). Column chromatography (CC) was carried using silica gel (Si) 60 mesh of 35–60 and 60–120 (E. Merck, Darmstadt, Germany) or Sephadex LH-20 (Pharmacia Fine Chemicals AB, Uppsala, Sweden). Thin-layer chromatography (TLC) was performed on silica gel GF254 pre-coated plates (Fluka). The chromatograms were visualized under UV light at 365 nm before and after exposure to ammonia vapour and AlCl3 (1 g powder of AlCl3 in 100 mL of ethanol, R2) for the flavones or spraying with sulfuric acid (30 % in ethanol, R1) reagent for the terpenes. The solvent systems (v/v) were as follows: S1, toluene/EtOAc (7:3); S2, benzene/EtOAc (8:2); S3, CHCl3/MeOH (9:1) were used for TLC, while S4, n-butanol/acetic acid/H2O (4:1:5, top layer); S5, 30 % acetic acid were used for the paper chromatography (PC). After spraying, heat the dry chromatogram at 105 C for 10 min. The reagents and chemicals used were of analytical grade. Phytochemical study Extraction, fractionation and isolation Dried powdered aerial part (2.2 kg) was successively extracted with petroleum ether (60–80 C) and ethyl acetate in a Soxhlet. The ethyl acetate was evaporated in vacuo at 40 C to yield brown oily crude extract (EAE, 27.3 g). EAE was chromatographed on a column (4 9 120 cm) of Si (450 g) using toluene, toluene/ethyl acetate (1:1), and ethyl acetate to give three major collective fractions; Fc I, Fc II, and Fc III, respectively. The major fraction (Fc I, 6.1 g) was subjected to CC (3 9 65 cm) using Si flash (250 g) and eluted with increasing polarities of petroleum ether/toluene mixtures. Two subfractions were obtained. The first was crystallized from ethyl alcohol giving white precipitate (270 mg) and was subjected further to repeated Si CC (2 9 10 cm, 20 g) using toluene/ethyl acetate (8:2) as an eluent to give compound 1 (26 mg, 1.18 %) and compound 2 (18 mg, 0.81 %). The second was eluted with toluene/ ethyl acetate (9.5:0.5) giving a semi pure compound, followed by application on Sephadex LH-20 (methanol) to give compound 5 (11 mg, 0.5 %). Fc II (5.4 g) was subjected to column chromatography (2 9 40 cm, 150 g) using Si 60 and elution with toluene/ethyl acetate (9.5:0.5) provided compound 6 (15 mg, 0.68 %) after crystallization from chloroform. The Fc III (3.1 g) was applied on Si CC (2 9 10 cm, 90 g) using toluene/ethyl acetate in increasing polarity giving three semi pure sub fractions (Fc III-1–Fc III-3). Subfractions Fc III-1 and Fc III-3 were, separately, applied on Si CC (2 9 10 cm, 20 g) using n-hexane/ethyl acetate gradient giving semi pure compounds, followed by application, separately, on Sephadex LH-20 (ethanol) to

Phytochemical composition and anti-ulcer and a-amylase inhibitory activity of Achillea biebersteinii

give compound 3 (12 mg, 0.54 %) and compound 4 (10 mg, 0.45 %). Subfraction Fc III-2 was applied on Si CC (2 9 10 cm, 30 g) and elution was made with toluene/ethyl acetate (7:3) and the yellow precipitate obtained was subjected to Sephadex LH-20 (ethanol) affording compound 7 (22 mg, 1 %). The purity of compounds 1–3 were checked by TLC using S1–S3 solvent systems and spray reagent R1. While PC using, the solvent systems S4 and S5 were used for checking the purity of compounds 5–7 and sprayed with R2. All compounds were characterized mainly by spectroscopic methods; UV, MS, 1H NMR, 13C NMR and through comparison of physical and spectral data with that in the literature. Their spectral data of isolated compounds were illustrated as the following: Micranthin (1): C19H26O7; white crystals; 288–290 C; ?ve EIMS: m/z 367 [M ? H]?; UV spectral data: kmax, nm (MeOH): 241 nm; 1H NMR (DMSO-d6, 300 MHz): d ppm 5.48 (d, J = 9.9 Hz, H-5a), 5.34 (dd, J = 11.3, 5.7 Hz, H-3a), 5.10 (t, J = 10 Hz, H-6b), 4.26 (d, J = 9 Hz, H-9a), 3.04 (d, J = 11.4 Hz, H-1a), 2.62 (quintet, J = 7.8 Hz, H-11a), 2.44 (t, J = 9 Hz, H-7a), 2.19 (m, H-2a), 2.05, 2.01 (s, 2 OCOCH3), 1.85 (m, H-8a), 1.74 (s, CH3-15), 1.66 (m, H-8b), 1.56 (m, H-2b), 1.18 (s, CH3-14), 1.13 (d, J = 7.5 Hz, CH3-13); 13C NMR (DMSO-d6, 75 MHz): d ppm 178.8 (C-12), 169.5, 169.2 (2 OCOCH3), 140.7 (C-4), 123.1 (C-5), 78.8 (C-9), 78.2 (C6), 75.2 (C-3), 61.5 (C-1), 61.2 (C-10), 43.9 (C-7), 40.3 (C11), 29.4 (C-8), 28.3 (C-2), 20.9, 20.6 (2 OCOCH3), 12.2 (CH3-15), 11.7 (CH3-14), 10.2 (CH3-13). Sintenin (2): C19H26O6; white crystals; 214–216 C; ?ve EIMS: m/z 351 [M ? H]?; UV spectral data: kmax, nm (MeOH): 242 nm; 1H NMR (DMSO-d6, 300 MHz): d ppm 5.24 (m, 1a, H-1), 5.03 (m, H-3a), 5.02 (m, H-9a), 4.82 (d, J = 9.9 Hz, H-5a/6b), 2.64 (quintet, J = 7.5 Hz, H-11a), 2.35 (m, H-2/7a); 2.01, 1.96 (s, 2 OCOCH3), 1.91 (m, H-8b), 1.71 (m, H-8a), 1.62 (s, CH3-15), 1.43 (s, CH314), 1.13 (d, J = 7.8 Hz, CH3-13); 13C NMR (DMSO-d6, 75 MHz): d ppm 178.9 (C-12), 169.6, 169.0 (2 OCOCH3), 136.8 (C-4), 136.5 (C-10), 127.0 (C-1), 125.7 (C-5), 80.2 (C-9), 79.0 (C-6), 78.4 (C-3), 44.5 (C-7), 40.3 (C-11), 30.5 (C-8), 30.4 (C-2), 20.9, 20.7 (2 OCOCH3) 11.7 (CH3-15), 11.2 (CH3-14), 10.5 (CH3-13). 4b,10a-dihydroxy-5b,7b,8bH-guaia-1,11(13)dien-12,8aolide (3): C15H20O4, colourless needles, EIMS m/z (100 %): [(M-H2O)?, 26]; 228 [(M- 2H2O)?, 12]; 203 [(228-CH3), 14]; 1H NMR (DMSO-d6, 500 MHz): d ppm 6.22 (d, J = 2.7 Hz, H-13Z), 5.76 (d, J = 2.4 Hz, H-13E), 5.66 (q, J = 2.1 Hz, H-2), 4.87 (ddd, J = 12.2, 8.2, 4.5, H-8b), 3.34 (d, J = 8.1, H-7b), 2.51 (dt, J = 12.2, 1.7, H-5b), 2.39 (dt, J = 16.3, 2.0, H-3b), 2.32 (ddd, J = 16.4, 2.2, 1.5, H-3a),

2.13 (dd, J = 13.0, 4.5, H-9b), 1.99 (dd, J = 13.1, 12.2, H-9a), 1.97 (dt, J = 13.7, 2.1, H-6b), 1.47 (dt, J = 13.6, 12.3, H-6a), 1.35 (s, H-14b), 1.26 (s, H-15a); 13C NMR (DMSO-d6, 125 MHz): d ppm 172.0 (C-12), 153.3 (C-1), 142.4 (C-11), 123.5 (C-13), 121.5 (C-2), 82.7 (C-4), 79.4 (C-8), 71.8 (C-10), 56.9 (C-5), 47.3 (C-3), 44.7 (C-9), 43.2 (C-7), 33.1 (C-6), 27.4 (C-14), 24.8 (C-15). 5,7 Dihydroxy 3,30 ,40 -trimethoxy flavone (4): C18H16O7, yellow amorphous powder, EI-MS m/z (100 %): 344 [M?, 100], 329 [M?-15, 48.2], 301 [(M-3x CH2), 45.5], 258 [8.3], 167 [10.6], 158 [15.4], 151 [7.8]; UV spectral data: kmax, nm (MeOH): 254, 267, 251; (?NaOMe): 275, 313, 280; (?AlCl3): 270, 276, 297, 356, 401; (?AlCl3/HCl): 262, 275, 300sh, 352, 399; (?NaOAc): 276, 314, 392; (?NaOAc/H3BO3): 259, 267, 351; 1H NMR (CDCl3-d6, 300 MHz): d ppm 12.65 (1H, s, OH-5), 7.70 (1H, d, J = 2.1 Hz, H-20 ), 7.68 (1H, dd, J = 2.1, 8.4 Hz, H-60 ), 7.05 (1H, d, J = 8.4 Hz, H-50 ), 6.45 (1H, d, J = 2.2 Hz, H-8), 6.36 (1H, d, J = 2.2 Hz, H-6), 3.99, 3.88, 3.86 (3H, s each, 3OMe-3,30 ,40 ). Santin (5): C18H16O7; yellow crystals, 161–162 C; MS m/z ( %): 344 [M?, 100], 345 [(M ? H)?, 35.6], 330 [M?– CH3/? H, 19.4], 329 [M?–CH3, 58.3], 301 [M?–CO/CH3, 48.2], 283 [301-H2O, 25.0], 258 [301-CO/CH3, 11.0], 135 [B2?, 18.5], 132 [B1?, 6.4]; UV spectral data: kmax, nm (MeOH): 273, 343; (?NaOMe): 278, 297, 371; (?AlCl3): 281, 305 sh, 360, 409; (?AlCl3/HCl): 283, 305 sh, 354, 409 sh; (?NaOAc): 273, 299 sh, 367; (?NaOAc/H3BO3): 273, 343; 1H NMR (DMSO-d6, 300 MHz): d ppm 12.70 (1H, s, OH-5), 7.93 (2H, d, J = 8.7 Hz, H-20 /60 ), 7.03 (2H, d, J = 8.7 Hz, H-30 /50 ), 6.49 (1H, s, H-8), 3.81 (3H, s, OCH3-6), 3.76 (3H, s, OCH3-3), 3.72 (3H, s, OCH3-40 ); 13C NMR (DMSO-d6, 75 MHz): d ppm 178.1 (C-4), 161.2 (C40 ), 157.2 (C-7), 155.0 (C-2), 152.3 (C-5), 151.5 (C-9), 137.5 (C-3), 131.1 (C-6), 129.7 (C-20 /6), 122.2 (C-10 ), 114.0 (C-30 /50 ), 104.6 (C-10), 93.9 (C-8), 59.8 (OCH3-6), 59.6 (OCH3-3), 55.3 (OCH3-40 ). Quercetagetin-3,6,30 -trimethyl ether (6): C18H16O8; yellow powder; 127–128 C; MS m/z (100 %): 360 [M?, 49.1], 361 [(M ? H)?, 15.2], 345 [M?–CH3, 23.2], 342 [M?–H2O, 17.0], 330 [M?–HCHO, 6.3], 317 [M?–CH3CO, 37.5], 316 [330-CH3/? H, 17.0], 303 [317-CH3/? H, 3.6], 299 [330-OCH3, 17.9], 151 [B2?, 20.5], 148 [B1?, 9.8]; UV spectral data: kmax, nm (MeOH): 257, 274, 352; (?NaOMe): 275, 336, 415; (?AlCl3): 267, 284sh, 379; (?AlCl3/HCl): 267, 284, 366; (?NaOAc): 373, 310sh, 376; (?NaOAc/H3BO3): 256, 273, 353. 1H NMR (DMSOd6, 300 MHz): d ppm 12.74 (s, OH-5), 7.55 (1H, d, J = 2.7 Hz, H-20 ), 7.53 (1H, dd, J = 2.7, 8.7 Hz, H-60 ), 6.94 (1H, d, J = 8.7 Hz, H-50 ), 6.54 (1H, s, H-8), 3.85 (3H, s, OCH3-6), 3.84 (3H, s, OCH3-3), 3.78 (3H, s, OCH3-30 );

123

H. I. Abd-Alla et al. 13

C NMR (DMSO-d6, 75 MHz): d ppm 178.0 (C-4), 157.2 (C-7), 155.4 (C-2), 152.2 (C-5), 151.4 (C-9), 149.6 (C-40 ), 147.3 (C-30 ), 137.3 (C-3), 131.0 (C-6), 122.1 (C-60 ), 120.8 (C-10 ), 115.5 (C-50 ), 111.9 (C-20 ), 104.5 (C-10), 94.0 (C-8), 59.9 (OCH3-6), 59.6 (OCH3-3), 55.6 (OCH3-40 ). Quercetagetin-3,6-dimethyl ether (7): yellow needle crystals; 131–132 C; C17H14O8; MS m/z (100 %): 346 [M?, 43.3], 331 [M?–CH3, 26.7], 328 [(M-H2O)?, 36.7], 317 [M?–CHO, 3.3], 315 [M?–OCH3, 23.3], 300 [M?– CO–H2O, 40.0], 287 [317-HCHO, 13.3], 273 [331-COHCHO, 40.0], 153 [(A1 ? H)?, 26.7], 152 [A1?, 43.3]; UV spectral data: kmax, nm (MeOH): 259, 272sh, 356; (?NaOMe): 272, 339, 409; (?AlCl3): 278, 338sh, 436; (?AlCl3/ HCl): 266, 277sh, 369; (?NaOAc): 272, 404; (?NaOAc/ H3BO3): 265, 384; 1H NMR (DMSO-d6, 300 MHz): d ppm 12.77 (s, OH-5), 7.53 (1H, d, J = 2.1 Hz, H-20 ), 7.43 (1H, dd, J = 2.1, 8.4 Hz, H-60 ), 6.90 (1H, d, J = 8.4 Hz, H-50 ), 6.51 (1H, s, H-8), 3.77 (3H, s, OCH3-6), 3.74 (3H, s, OCH3-3); 13C NMR (DMSO-d6, 75 MHz): d ppm 178.1 (C-4), 157.2 (C-7), 155.6 (C-2), 152.3 (C-5), 151.4 (C-9), 148.6 (C-40 ), 145.1 (C-30 ), 137.3 (C-3), 131.0 (C-6), 120.5 (C-60 ), 120.8 (C-10 ), 115.3 (C-50 ), 115.7 (C-20 ), 104.5 (C10), 93.8 (C-8), 59.9 (OCH3-6), 59.6 (OCH3-3). Animals and euthanasia Forty two male Wistar albino rats (8 weeks aged; 100–120 g) were selected for this study. They were obtained from the Animal House, National Research Center, Dokki, Egypt. All animals were kept in a controlled environment of air and temperature with access to water and diet ad libitum. Animals were anesthetized with diethyl ether through inhalation and dissected through abdominal midline incision. Ethics Anesthetic procedures and handling with animals complied with the ethical guidelines of the Ethical Committee of the Federal Legislation and National Institutes of Health Guidelines in USA were approved by the Medical Ethical Committee of the National Research Centre in Egypt (Approval No.: 09210). Doses and route of administration Absolute ethanol was orally administered at a dose of 0.5 mL/100 g body weight (BW) on 24 h empty stomachs (Motawi et al. 2012). A. biebersteinii extract was orally given at a dose of 200 mg/kg BW daily for a week. Ranitidine, a reference antiulcer drug, was orally administered at a dose of 100 mg/kg BW for a week (Motawi et al. 2012).

123

Experimental groups The rats were divided into seven equal groups (six rats each). Group 1 consisted of untreated control rats. Groups 2 and 3 (protective groups) have administered either of the plant extract or ranitidine daily for 7 days prior to an oral dose of absolute ethanol on 24 h empty stomachs and sacrificed 1 h later. Groups 4 and 5 (therapeutic effect) received one oral dose of absolute ethanol on 24 h empty stomachs, and were treated with either of the plant extract or ranitidine daily for 7 days. Group 6, received the ethanol dose on 24 h empty stomachs, sacrificed after 1 h and served as the gastric ulcerative rats for the protective groups. Group 7, received the ethanol dose, was not further treated for 7 days and served as the gastric ulcerative rats for the therapeutic groups. Sample preparations and biochemical assays Gastric lesion counts Abdomen was removed carefully to avoid discharge of its content. It was removed, opened from the long curvature, washed with normal saline, expanded and fixed on the dissection plate, and lesion numbers were counted using a magnifying lens (Szelenyi and Thiemer 1978). Gastric total acidity The gastric contents were collected and centrifuged at 3,000 9 g for 15 min. The supernatant volumes (in lL) were measured and the total acidity was determined by titration with 0.1 M NaOH using 2 % phenolphthalein as an indicator. The results were expressed as molar equivalent (mEq)/L (Guedes et al. 2008). Oxidative stress markers Stomach tissue was homogenized in normal physiological saline solution (0.9 % NaCl) (1:5 w/v). The homogenate was centrifuged at 4 C for 5 min at 3,000 9 g and the supernatant was used for the determination of oxidative stress markers. Malondialdehyde (MDA), a product of polyunsaturated fatty acids oxidation, was determined as an indicator of lipid peroxidation according to Buege and Aust method (Buege and Aust 1978). Its concentration was calculated using the extinction coefficient value 1.56 9 105 M-1 cm-1 and the reading at 535 nm. Glutathione (GSH) was assayed according to Moron et al. (1979) using dithiobis-2-nitrobenzoic acid (DTNB) in PBS. The reaction colour was read at 412 nm. Superoxide dismutase (SOD) was assayed according to Nishikimi et al. (1972) where the

Phytochemical composition and anti-ulcer and a-amylase inhibitory activity of Achillea biebersteinii

increase in NADH oxidation was measured at 560 nm using its molar extinction coefficient 6.22 9 103 M-1 cm-1. Histopathological study Stomach portions were cut, fixed in 10 % paraformaldehyde, and embedded in paraffin wax blocks. Tissue sections of 5 lm thickness were stained with hematoxylin and eosin (H&E) and Masson’s trichrome, then examined under a light microscope for determination of pathological changes (Hirsch et al. 1997). Determination of a-amylase inhibitory activity Determination of a-amylase inhibitory activity was carried according to Conforti et al. (2005). A starch solution (0.5 % w/v) was obtained by stirring 0.125 g of potato starch in 25 mL of sodium phosphate buffer (20 mM) with sodium chloride (6.7 mM, pH 6.9) at 65 C for 15 min. The enzyme solution was prepared by mixing 25.3 mg of a-amylase in 100 mL of cold distilled water. A. biebersteinii EAE was dissolved in buffer to give final concentrations from 10 to 1,000 lg/mL. The colorimetric reagent was prepared by mixing a sodium potassium tartrate solution (12.0 g of sodium potassium tartrate-tetrahydrate in 8.0 mL of NaOH 2 M) and 3,5-dinitrosalicylic acid solution (96 mM). Both control and plant extract were added with starch solution and left to react with a-amylase solution under alkaline conditions at 25 C. The reaction was measured over 3 min. The generation of maltose was quantified by the reduction of 3,5-dinitrosalicylic acid to 3amino-5-nitrosalicylic acid. This reaction (corresponding to the color change from orange-yellow to red) is detectable at 540 nm. The a-amylase inhibition was expressed as a percentage of inhibition and calculated from the equation:

consecutive purification steps on Si columns to yield two germacranolides and one guaianolide sesquiterpene lactones in addition to four derivatives of 3-methoxy flavones (Fig. 1). The structures of isolates were identified on the basis of interpretation of their physicochemical analyses (UV, MS, 1D and 2D NMR), comparison with the corresponding published data. They were identified as the sesquiterpene lactones, micranthin (1) (Hatam et al. 1992), sintenin (2) (Hatam et al. 1992) and 4b,10a-dihydroxy5b,7b,8bH-guaia-1,11(13)dien-12,8a-olide (3) (Ahmed et al. 1993). Structures of four methoxy flavones; 5,7 dihydroxy 3,30 ,40 -trimethoxy flavone (4) (Urbatsch et al. 1976), santin (5) (Horie et al. 1998), quercetagetin-3,6,30 trimethyl ether (6) (Markham 1982), quercetagetin-3,6dimethyl ether (7) (Markham 1982) were also identified. The isolated compounds were identified for the first time from this species except sintenin (2) and quercetagetin3,6,30 -trimethyl ether (6). The 1H NMR spectra of 1 and 2 showed that these two compounds are sesquiterpene lactones of the germacranolide type and compound 3 of the guaianolide type. The assignments were made by chemical shifts, coupling patterns and two dimensional homocorrelated NMR spectra. Compounds 4–7 exhibited a chromatographic properties and response towards spraying reagents characteristic for 3-methoxylated flavones. This evidence was supported by MS analyses, on the basis of their UV spectra with diagnostic shift reagents, and NMR spectra (Markham 1982).

OAc

AcO

½ðA540 control A540 sampleÞ=A540 control 100 Statistical analysis All data were expressed as mean ± SD of six rats in each group. Statistical analysis was carried by one-way analysis of variance (ANOVA). Costat Software Computer Program using least significance difference between groups accompanied with post hoc test, was applied at p \ 0.05.

OAc

R3 O

CH2 OH

OMe OH

4: R1=R3=OCH3; R2=H 5: R1=H; R2=R3=OCH3

Results and discussion

6: R1=R2=OCH3; R3=OH

Phytochemical investigation

7: R1= OCH3; R2= R3=OH

The EAE from the aerial part of A. biebersteinii Afan. (Asteraceae) was fractionated on a Si column, followed by

Fig. 1 Chemical structures of the isolated compounds from Achillea biebersteinii Afan

123

H. I. Abd-Alla et al.

Protective and therapeutic effect of EAE of A. biebersteinii aerial parts

The present study revealed a significant increase in MDA, SOD activity, and GSH. Demir et al. (2003) postulated that high gastric mucosal MDA level in patients with peptic ulcer and gastritis are thought to reflect free radical mediated gastric mucosal damage. In agreement with our results, Tandon et al. (2004) observed significant elevation of SOD in gastric ulcer. Stress causes stimulation of the stomach leading to local hypoxia or actual ‘‘ischemia’’. The ischemic condition causes an increase in the level of H2O2 by the action of SOD, which, in conjunction with O2, generates OH•. Thus, hydroxyl radicals oxidize important cellular constituents such as structural and functional proteins and membrane lipids. Lipid peroxidation MDA causes loss of membrane fluidity, impairs ion transport and membrane integrity and finally loss of cellular functions (Tandon et al. 2004; Shaaban et al. 2012). In agreement with our results, Motawi et al. (2012) recorded an increase in GSH level after ethanol induced gastric ulcer in rats. This result was in contrast to many investigations who recorded significant depletion of glutathione level after ulcer induction (Sathisha et al. 2011). This was attributed to the glutathione status that is dependent on the relative activity of many enzymes. The increased activity of enzymes involved in GSH synthesis (c-glutamyl-cysteine synthetase) and GSH reduction (glutathione reductase) can lead to an increased GSH concentration (Sathisha et al. 2011). Conversely, increased activities of GSH peroxidase and GSH transferase (the enzyme responsible for the conjugation of toxic compounds with GSH) leads to decreased GSH concentration. This result was in accordance with the observed decrease of glutathione peroxidase and glutathione transferase in indomethacin-induced gastric ulcer (Koc et al. 2008), ethanol-induced mucosal injury (Motawi et al. 2012) and in stress ulcer (Liu et al. 2011) which may give additional support to our results. In contrast to this finding, Halabi et al. (2014) observed a decrease in SOD after ethanol induced gastric ulcer to rats. They attributed this decrease to the high utilization of

The protection of stomach with EAE of A. biebersteinii (200 mg/kg) for one week before ulcer induction recorded significant decrease in gastric volume by 45.11 %, as compared with the one hour ulcerative group (group 6). Total gastric acidity showed a significant decrease by 38.73 %, while lesion counts decreased by 47.05 %. Ranitidine (100 mg/kg) drug showed significant decrease in gastric volume, total gastric acidity and lesion counts by 25.30, 27.43 and 29.41 %, respectively (Table 1). Gastric ulcer protected for one week with the ethyl acetate extract (EAE) recorded significant decrease in GSH, MDA and SOD by 61.96, 43.25 and 65.96 %, respectively. Ranitidine showed a significant decrease in all oxidative stress markers by 37.55, 32.32 and 56.12 %, respectively, comparing with one hour ulcer group (Table 2). The treatment of stomach with the extract after ulcer induction recorded significant decrease in gastric volume by 34.14 %, as compared to the one week ulcerative group (group 7). Total gastric acidity showed significant decrease by 42.37 %, while lesion counts decreased by 45.45 %. The treatment with ranitidine after ulcer induction showed significant decrease in gastric volume, total gastric acidity and lesion counts by 28.69, 33.89 and 27.27 %, respectively (Table 3). The treatment of gastric ulcer by the EAE showed significant decrease in oxidative stress markers by 84.61, 54.43 and 64.80 % for GSH, MDA and SOD, respectively, while ranitidine drug exhibited significant decrease in the oxidative stress markers by 79.80, 33.54, and 52.51 % (Table 4). Gastric ulceration by ethanol resulted in an increase in gastric volume and total acidity. This result was in line with the results of Gracioso et al. (2002) who attributed these increases to the effect of hydrochloric acid released from the surface of parietal cells. The same authors added that mucus and HCO3 released from the surface of epithelial cells are part of the mucosal defensive mechanisms and play an important role in protecting the gastroduodenal mucosa.

Table 1 Protective effect of EAE of A. biebersteinii aerial parts on gastric ulcer markers in rats Parameters Gastric volume Total acidity Lesion counts

Normal control 147.00 ± 5.43

19.20 ± 1.92d –

Ulcer (U)

U ? treatment with EAE a

1,552.00 ± 31.14 (?955.78)

44.40 ± 2.30 (?131.25)a 17.00 ± 1.30 (–)

U ? treatment with Ranitidine c

1,159.20 ± 63.19 (-25.30)b

27.20 ± 1.92 (-38.73)c

32.22 ± 1.94 (-27.43)b

12.00 ± 1.14 (-29.41)b

851.80 ± 42.80 (-45.11)

9.00 ± 1.98 (-47.05)

Data are mean ± SD of six rats in each group. Gastric volume is expressed as lL, total acidity as mEq/L. Unshared letters between groups are the significance values at p \ 0.0001. Values between brackets are percentage change over control group, while values between parentheses are percentage change over ulcer group

123

Phytochemical composition and anti-ulcer and a-amylase inhibitory activity of Achillea biebersteinii Table 2 Protective effect of EAE of A. biebersteinii aerial parts on oxidative stress markers in rats Parameters

Normal control

Ulcer (U)

GSH

23.00 ± 1.58d d

MDA SOD

0.91 ± 0.15

U ? treatment with EAE

U ? treatment with Ranitidine

83.60 ± 2.70 (?263.47)a

31.80 ± 2.48 (-61.96)c

52.20 ± 1.92 (-37.55)b

2.44 ± 0.30 (-43.25)

2.91 ± 0.15 (-32.32)b

13.00 ± 1.58 (-65.96)

16.76 ± 0.83 (-56.12)b

4.30 ± 0.39 (?372.52)

9.85 ± 0.75

38.20 ± 2.86 (?287.81)

Data are mean ± SD of six rats in each group. Data are expressed as lg/mg protein for glutathione, lmol/mg protein for lipid peroxides and superoxide dismutase. Unshared letters between groups are the significance values at p \ 0.0001. Values between brackets are percentage change over control group, while values between parentheses are percentage change over ulcer group

Table 3 Therapeutic effect of EAE of A. biebersteinii aerial parts on gastric ulcer markers in rats Parameters

Normal control

Ulcer (U)

U ? treatment with EAE

U ? treatment with Ranitidine

Gastric volume

147.00 ± 5.43bc

212.60 ± 9.15 (?44.12)a

140.00 ± 2.12 (-34.14)c

151.60 ± 1.14 (-28.69)b

19.20 ± 1.92b

23.60 ± 2.40 (?22.91)a

13.60 ± 1.14 (-42.37)c

15.60 ± 1.15 (-33.89)c

8.00 ± 0.76 (-27.27)b

Total acidity Lesion counts

–

11.00 ± 1.22 (–)

6.00 ± 0.83 (-45.45)

Table 4 Therapeutic effect of EAE of A. biebersteinii aerial parts on oxidative stress markers in rats Parameters

Normal control

Ulcer (U)

GSH

23.00 ± 1.58d

228.80 ± 2.77 (?894.78)a

35.21 ± 2.38 (-84.61)c

46.20 ± 2.86 (-79.80)b

0.91 ± 0.15

1.44 ± 0.21 (-54.43)

2.10 ± 0.12 (-33.54)b

9.85 ± 0.74

12.60 ± 1.14 (-64.80)

17.00 ± 1.00 (-52.51)b

MDA SOD

U ? treatment with EAE

3.16 ± 0.10 (?247.25)

35.80 ± 1.92 (?263.45)

U ? treatment with Ranitidine

SOD in the decomposition of superoxide anion generated by lipid peroxidation. Regarding to the histopathological figures of stomach, normal gastric mucosa showed crypts of overlying gastric glands lined by mucus secreting cells with rounded nuclei. The Lamina propria was intact, infiltrated by scattered lymphocytes, blood vessels and fibrous tissue (Fig. 2a). Collagen deposition was within normal limits (Fig. 2b). Figure 2c and d displayed the ulcers form after one hour of absolute alcohol induction. The gastric mucosa showed deep ulcer reached to the basement membrane lined the L. propria. The thickened ulcer base showed some polymorphous lymphocytes fibrin. The gastric glands were hyperplastic and surrounded the ulcer. The L. propria showed few lymphocytes and polymorphonuclear leucocytes. Marked fibrous tissue was observed. After one week of ulcer induction, narrow ulcers reached to the basement

membrane were recorded. Mild hyperplasia of the gastric glands was noticed. The L. propria was infiltrated by mild number of chronic inflammatory cells. Fibrotic tissue was observed (Fig. 2e, f). Protection with plant extract before ulcer induction showed superficial gastric erosion in the mucosa. Shallow superficial erosions were noticed and the L. propria showed mild infiltrations by lymphocytes and polymorphonuclear leucocytes (Fig. 3a, b). Scattered fibrosis after protection by plant extract was observed (Fig. 3b). The protection by ranitidine drug recorded hyperplastic mucosa with superficial erosions. The L. propria was widening by mild-moderate chronic inflammatory cells, lymphocytes and polymorphonuclear leucocytes (Fig. 3c, d). Treatment of ulcer with plant extract showed intact mucosa and focal superficial erosions with healed base ulcer membrane. The L. propria was infiltrated by a mild

123

H. I. Abd-Alla et al. Fig. 2 Photomicrograph of rat gastric mucosa stained with H& E (a, c, e) and Masson’s trichrom (b, d, f) (9100). Normal control stomach (a, b), 1 h ulcerative gastric mucosa (c, d), 1 week ulcerative gastric mucosa (e, f). Arrows indicate deep ulcer reached to the basement membrane lined the Lamina propria. Small arrows indicate narrow ulcers reached to the basement membrane

number of chronic inflammatory cells (Fig. 3e, f). Minimal degree of fibrosis was recorded (Fig. 3f). Treatment with ranitidine showed healed mucosa with scanty superficial erosions and hyperplasia of the gastric glands. The L. propria was infiltrated by a mild number of chronic inflammatory cells with fibrosis reached to 12 % (Fig. 3g, h). Therefore, treatment with EAE of A. biebersteinii recorded improvement in gastric volume, total acidity, lesion counts and the oxidative stress markers. Treatment with plant extract recorded more potent effect than its protective action. The results reinforced the presence of antisecretory, antioxidant, and antiulcerogenic effects of A. biebersteinii extract which was confirmed by the observed histopathological changes of the gastric mucosa.

123

Phytochemical investigation of A. biebersteinii in the current study resulted in the isolation of four methoxy flavones. Several mechanisms have been proposed to explain the gastroprotective effect of methoxy flavones (Chaturvedi et al. 2009). In addition, they have been found to be free radical scavengers; free radicals play an important role in ulcerative and erosive lesions of the gastrointestinal tract. All these biological activities are well correlated with the terpenes and phenolics content of EAE (Guedes et al. 2008). Previously, it was shown that infusions prepared from 15 Achillea species have an antioxidant capacity and protective effects against H2O2-induced oxidative damage in human erythrocytes and leukocytes (Konyalioglu and Karamenderes 2005).

Phytochemical composition and anti-ulcer and a-amylase inhibitory activity of Achillea biebersteinii Fig. 3 Photomicrograph of protected ulcerative gastric mucosa stained with H& E (a, c) and Masson’s trichrom (b, d) (9100). Mucosa protected by plant extract (a, b), and Ranitidine drug (c, d). Arrows indicate shallow erosions. Treated ulcerative gastric mucosa stained with H& E (e, g) and Masson’s trichrom (f, h) (9100). Mucosa treated by plant extract (e, f) and Ranitidine drug (g, h). Arrows indicate small and very narrow superficial erosions with healed base ulcer membrane

Diabetic state has deleterious effect on various functions of gastrointestinal tract. Recent studies indicated that peptic ulcers related to diabetic state are more severe and often associated with complications such as gastrointestinal bleeding (Chung et al. 2001; Chaturvedi et al. 2009). An antidiabetes herbal preparation, including A. millefolium extract, showed significantly decreasing for the level of glucose and fructosamine in alloxan-induced non-obese diabetic (Mustafa et al. 2012). a-Amylases have been targets for the suppression of postprandial hyperglycaemia. We investigated extract from A. biebersteinii to determine

the inhibitory effect on a-amylase. In vitro enzymatic starch digestion with the EAE from plant at concentrations of 10–1,000 lg/mL was noticed in Table 5. In the current study, the EAE showed appreciable carbohydrate inhibitory activity. It recorded biologically active inhibition of aamylase enzyme activity at the concentration of inhibitor 10–1,000 lg/mL when compared to the standard acarbose. A significant increase in reducing power with the increase in concentrations of the extract (linear-relationship) and at low doses the reducing activity showed insignificant change.

123

H. I. Abd-Alla et al. Table 5 a-Amylase inhibition (%) of EAE of A. biebersteinii aerial parts Concentrations (lg/mL)

Acarbose (positive control)

EAE

22.18 ± 1.27a

17.50 ± 1.11e

24.75 ± 1.87b

25.20 ± 3.39

100 500

37.38 ± 4.15 42.49 ± 4.51c

31.16 ± 1.98f 39.50 ± 2.45b

1,000

61.29 ± 2.62d

58.10 ± 1.60b

LSD 5 %

4.86

3.69

a-Amylase is expressed as %; data are mean ± SD of triplicates Unshared superscript letters between treatments are significance values at p \ 0.05

These findings demonstrated the remarkable potential of A. biebersteinii as valuable source of antiulcerogenic agent with post prandial hyperglycaemia lowering effect. The ulcer protective effect of the EAE of A. biebersteinii may be due to its antidiabetic and gastric antisecretory effects. Further studies are needed to identify the molecules responsible for the pharmacological effects and for clinical and pharmaceutical applications. Conflict of interest

There are no conflicts of interest to report.

References Ahmed, A.A., A.A. Mahmoud, H.J. Williams, A.I. Scott, J.H. Reibenspies, and T.J. Mabry. 1993. New sesquiterpene alphamethylene lactones from the Egyptian plant Jasonia candicans. Journal of Natural Products 56: 1276–1280. Akkol, E.K., U. Koca, I. Pesin, and D. Yilmazer. 2011. Evaluation of the wound healing potential of Achillea biebersteinii Afan. (Asteraceae) by in vivo excision and incision models. Evidence Based Complementarty and Alternative Medicine 2011: 474026. Buege, J.A., and S.D. Aust. 1978. Microsomal lipid peroxidation. Methods in Enzymology 52: 302–310. Chaturvedi, A., G. Bhawani, P.K. Agarwal, S. Goel, A. Singh, and R.K. Goel. 2009. Antidiabetic and antiulcer effects of extract of Eugenia jambolana seed in mild diabetic rats: study on gastric mucosal offensive acid-pepsin secretion. Indian Journal of Physiology and Pharmacology 53: 137–146. Chung, S.H., C.G. Choi, and S.H. Park. 2001. Comparisons between white Ginseng Radix and rootlet for antidiabetic activity and mechanism in KKAy mice. Archives of Pharmacal Research 24: 214–218. Conforti, F., M.R. Loizzo, G.A. Statti, and F. Menichini. 2005. Comparative radical scavenging and antidiabetic activities of methanolic extract and fractions from Achillea ligustica All. Biological and Pharmaceutical Bulletin 28: 1791–1794. Demir, S., M. Yilmaz, M. Koseoglu, N. Akalin, D. Aslan, and A. Aydin. 2003. Role of free radicals in peptic ulcer and gastritis. The Turkish Journal of Gastroenterology 14(1): 39–43. Elgamal, M.H.A., S. Abdelwahab, and H. Duddeck. 1991. Constituents of Achillea fragrantissima. Fitoterapia 62: 362. Gracioso, J.D.S., W. Vilegas, C.A. Hiruma-Lima, and A.R.M. Souza Brito. 2002. Effects of tea from Turnera ulmifolia L. on mouse

123

gastric mucosa support the turneraceae as a new source of antiulcerogenic drugs. Biological and Pharmaceutical Bulletin 25: 487–491. Guedes, M.M., A.C. da Silva Carvalho, A.F. Lima, S.R. de Sousa Lira, S.S. de Queiroz, E.R. Edilberto Rocha Silveira, F.A. Almeida Santos, and V.S. Rao. 2008. Gastroprotective mechanisms of centipedic acid, a natural diterpene from Egletes viscosa LESS. Biological and Pharmaceutical Bulletin 31: 1351–1355. Halabi, M.F., R.M. Shakir, D.A. Bardi, N.S. Al-Wajeeh, A. Ablat, P. Hassandarvish, M. Hajrezaie, A. Norazit, and M.A. Abdulla. 2014. Gastroprotective activity of ethyl-4-[(3,5-di-tert-butyl-2hydroxybenzylidene) amino]benzoate against ethanol-induced gastric mucosal ulcer in rats. PLoS ONE 9: e95908. Hatam, N.A.R., N.J. Yousif, A. Porzel, and K. Seifert. 1992. Sesquiterpene lactones from Achillea micrantha. Phytochemistry 31: 2160–2162. Hirsch, C., C.S. Zouain, J.B. Alves, and A.M. Goes. 1997. Induction of protective immunity and modulation of granulaomatous hypersensitivity in mice using PIII, an anionic fraction of Schistosoma mansoni adult worm. Parasitology 115: 21–28. Horie, T., Y. Ohtsurs, K. Shibata, K. Yamashita, M. Tsukayama, and Y. Kawamura. 1998. 13C NMR special assignment of the a-ring of polyoxgenated flavones. Phytochemistry 47: 865–874. Hudaib, M.M., K.A. Tawaha, M.K. Mohammad, A.M. Assaf, A.Y. Issa, F.Q. Alali, T.A. Aburjai, and Y.K. Bustanji. 2011. Xanthine oxidase inhibitory activity of the methanolic extracts of selected Jordanian medicinal plants. Pharmacognosy Magazine 7: 320–324. Jeong, C.S. 2002. Effect of butanol fraction of Panax ginseng head on gastric lesion and ulcer. Archives of Pharmacal Research 25: 61–66. Koc, M., H. Imik, and F. Odabasoglu. 2008. Gastroprotective and anti-oxidative properties of ascorbic acid on indomethacininduced gastric injuries in rats. Biological Trace Element Research 126: 222–236. Konyalioglu, S., and C. Karamenderes. 2005. The protective effects of Achillea L. species native in Turkey against H2O2-induced oxidative damage in human erythrocytes and leucocytes. Journal of Ethnopharmacology 102: 221–227. Liu, Y., Z. Qi, S. Guo, Z. Wang, X. Yu, and S. Ma. 2011. Role of corticotrophin releasing hormone in cerebral infarction-related gastrointestinal barrier dysfunct ion. World Journal of Emergency Medicine 1: 59–65. Mahmoud, A.A., and S.S. Al-Shihry. 2006. A new ionone glucoside and terpenoid constituents from Achillea biebersteinii and their antifungal activity. Natural Product Communications 1: 697–703. Mard, S.A., Z. Bahari, N. Eshaghi, and Y. Farbood. 2008. Antiulcerogenic effect of Securigera securidaca L. seed extract on various experimental gastric ulcer models in rats. Pakistan Journal of Biological Sciences 11: 2619–2623. Markham, K.R. 1982. Techniques of flavonoid identification. London: Academic Press. Moron, M.S., J.W. Depierre, and B. Mannervik. 1979. Level of glutathione, glutathione reductase and glutathone-S-transferase activities in rat lung and liver. Biochimica et Biophysica Acta 582: 67–78. Motawi, T.K., M.A. Hamed, R.M. Hashem, M.H. Shabana, and Y.R. Ahmed. 2012. Protective and therapeutic effects of Argyreia speciosa against ethanol induced gastric ulcer in rats. Zeitschrift fu¨r Naturforschung C 67: 47–57. Mustafa, K.G., B.A. Ganai, S. Akbar, M.Y. Dar, and A. Masood. 2012. b-Cell protective efficacy, hypoglycemic and hypolipidemic effects of extracts of Achillea millifolium in diabetic rats. Chinese Journal of Natural Medicines 10: 185–189.

Phytochemical composition and anti-ulcer and a-amylase inhibitory activity of Achillea biebersteinii Nishikimi, M., N.A. Rae, and K. Yagi. 1972. The occurrence of superoxide anion in the action of reduced phenazine methosulphate and molecular oxygen. Biochemical and Biophysical Research Communications 46: 849–853. Saeidnia, S., A.R. Gohari, N. Mokhber-Dezfuli, and F. Kiuchi. 2011. A review on phytochemistry and medicinal properties of the genus Achillea. DARU 19: 173–186. Sathisha, R., B. Vyawaharea, and K. Natarajanb. 2011. Antiulcerogenic activity of Lantana camara leaves on gastric and duodenal ulcers in experimental rats. Journal of Ethnopharmacology 134: 195–197. Shaaban, M., H.I. Abd-Alla, A.Z. Hassan, H.F. Aly, and M.A. Ghani. 2012. Chemical characterization, antioxidant and inhibitory effects of some marine sponges against carbohydrate metabolizing enzymes. Organic and Medicinal Chemistry Letters 2: 1–12.

So¨kmen, A., M. So¨kmen, D. Daferera, M. Polissiou, F. Candan, M. ¨ nlu¨, and H.A. Akpulat. 2004. The in vitro antioxidant and U antimicrobial activities of the essential oil and methanol extracts of Achillea biebersteini Afan. (Asteraceae). Phytotherapy Research 18: 451–456. Szelenyi, I., and K. Thiemer. 1978. Distention ulcer as a model for testing of drugs for ulcerogenic side effects. Archives of Toxicology 41: 99–105. Tandon, R., H.D. Khanna, M. Dorababu, and R.K. Goel. 2004. Oxidative stress and antioxidants status in peptic ulcer and gastric carcinoma. Indian Journal of Physiology and Pharmacology 48: 115–118. Urbatsch, L.E., T.J. Mabry, M. Miyakado, N. Ohno, and H. Yoshioka. 1976. Flavonol methyl ethers from Ericameria diffusa. Phytochemistry 15: 440–441.

123

Phytochemical composition, protective and therapeutic effect on gastric ulcer and α-amylase inhibitory activity of Achillea biebersteinii Afan. - PDF Download Free (2024)