Pharmaceutical Biology 2006, Vol. 44, No. 4, pp.

292296

Antiulcerogenic Activity of Alhagi maurorum

A.S. Awaad Amani,1 D.J. Maitland,2 and G.A. Soliman3 Aromatic and Medicinal Plant Department, Desert Research Center, Cairo, Egypt; 2Chemistry and Forensic Medicine Department, School of Life Science, Bradford University, Bradford, United Kingdom; 3Pharmacology Department, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt
1

Abstract
Six main flavonoid glycosides were isolated, for the first time, from the ethanol extract of Alhagi maurorum Boiss (Leguminosae). They were identified as kaempferol, chrysoeriol, isorhamnetin, chrysoeriol-7-O-xylosoid, kaempferol-3-galactorhamnoside, and isorhamnetin 3-O-b-D-apio-furanosyl (1-2) b-D-galactopyranoside. Their identities were established by m.p., UV, EI-mass, Fab-mass, 600 MHz 1H and 13C NMR. The total extract (300 and 400 mg=kg) and two of the isolated compounds (chrysoeriol 7-O-xylosoid and kaempferol-3-galactorhamnoside, 100 mg=kg each) showed a very promising antiulcerogenic activity with curative ratios 66.31%, 69.57%, 75.49%, and 77.93%, respectively. Keywords: Alhagi maurorum, antiulcerogenic activity, chrysoeriol-7-O-xylosoidflavonoids, isorhamnetin, kaempferol-3-galactorhamnoside. (El-Saayed et al. 1993; Singh et al., 1999) were isolated from Alhagi graecorum Boiss. These flavonoids were identified as tamarixtin 3-O-dirhamnoside, isorhamnetin 3-O-glucosylneo-hesperidoside, isorhamnetine 3-O-robinoside, isorhamnetin 3-O- rotinoside, quercetin 3-O-rhamnoside, kampferol 3-O-galactoside, quercetin 0 3, 7-diglycoside, isorhamnetin 3-rutinoside, daidzein 7, 4 -di0 hydroxyisoflavone, calycisin 3 -hydroxyformononetin, and isorhamnetin and tamarxtin aglycones. Alhagi maurorum Boiss is customarily used in folk medicine as a remedy for rheumatic pains, bilharziasis, liver disorders, and for various types of gastrointestinal discomfort (Bolus, 1983), but there is no scientific background that supports this use. The aim of the current work was to study the antiulcerogenic effect of Alhagi maurorum Boiss to reveal its possible use for the treatment of gastric ulcer and to discover the compounds responsible for this activity.

Introduction
The family Leguminosae is comprised of about 550 genera and more than 13,000 species (Bolus, 2000), including several members that are used in folklore medicine (Lewis & Lewis 1977). The family provides us with many edible plants as well as a variety of medicinal plants that constitute an important source of raw materials used in the pharmaceutical industries. Chemical investigation of the Alhagi species revealed the presence of several contents such as fatty acids and sterols (Ghosal et al., 1974; Kudliki et al., 1991; Kalhoro et al., 1997), flavonoids (AlYahya et al., 1987; El-Saayed et al., 1993; Singh et al., 1999), coumarins (Behari & Gupt, 1980), alkaloids (Behari & Gupt, 1980), and vitamins. Twelve flavonoids

Materials and Methods

Plant Material The aerial parts of Alhagi maurorum Boiss were collected from the Sewa Oasis during summer 2002. Identification of the plant was verified by the late Prof. N. El-Hadidi, Professor of Taxonomy, Botany Department, Faculty of Science, Cairo University, and by comparison with plant description in Flora of Egypt (Tackholm, 1974; Bolus 2000). A voucher specimen was kept in the herbarium of the Desert Research Center. Plant sample was air-dried in shade, reduced to fine powder, packed in tightly closed containers, and stored for phytochemical and pharmacological studies.

Accepted: 1 March 2006 Address correspondence to: S. Awaad Amani, Desert Research Center, 1 Mathaf Elmatariah Street, Cairo, Egypt. Tel.: 00202-6335529; Fax: 00202-6357858; E-mail: amaniawaad@hotmail.com DOI: 10.1080/13880200600714160 # 2006 Taylor & Francis Group, LLC

Antiulcerogenic activity of A. maurorum Solvent Systems The solvent systems (a) chloroform:methanol (95:5), (b) ethyl acetate:methanol:water (30:5:4), (c) butanol:acetic acid:water (4:1:5), (d) ethyl acetate:methanol:acetic acid:water (65:15:10:10), (e) acetic acid:water (15:85) were used for developing the chromatoplates. Visualization of chromatograms was achieved under UV before and after exposure to ammonia vapor or by spraying with aluminum chloride (Stahl, 1969). Apparatus Melting points were determined on a Kofler hot-stage apparatus (UK) and are uncorrected; mass spectra (electrospray negative ion) sample was dissolved in acetonitrile on a Micromass Quattro spectrometer (Germany). 1 H and 13C NMR spectra, using external electronic referencing through the deuterium resonance frequency of the solvent, were determined at 600.17 or 150.91 MHz, respectively, with a JEOL ECA 600 spectrometer fitted with an auto 5 mm X=H probe, Mannheim Hitachi 717 automatic analyzer (Japan). Carbon atom types were established in the 13C NMR spectrum by employing a combination of broad- and proton-decoupled and distortionless enhancement by polarization transfer (DEPT) experiments with 64 K data points over a spectrum width of 17,605.6 Hz. [1JCH] and [2JC-H and 3JC-H]. 1H-13C correlations were established by using heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bound correlation (HMBC) pulse sequences, respectively. 1H1H correlations were by double quantum filtered correlation spectroscopy (COSY). Phytochemical study Extraction and isolation Defatted powder (1 kg) of the aerial parts of the plant was extracted in a Soxhlet apparatus with 95% ethanol. The ethanol extract was concentrated under reduced pressure (150 g) and diluted with water (200 ml), filtered over a piece of cotton, then successively extracted with ether, chloroform, ethyl acetate, and n-butanol. Each extract was dried over anhydrous sodium sulfate and concentrated to yield 4, 10, 18.5, and 30 g dry extracts, respectively. TLC examination (systems a and b) revealed the presence of the same spots in ether and chloroform extracts. Accordingly, both extracts were combined together (14 g) and applied on column chromatography packed with silica gel G (420 g) and eluted gradually with chloroformmethanol, where compounds 13 were isolated. The ethyl acetate and n-butanol extracts were found to have the same spots when chromatographic tested on TLC (system b). Both extracts were collected together and subjected to preparative TLC (system b) followed with repeated PPC (systems c and f). Bands corresponding

293

Figure 1. Compounds isolated from Alhagi maurorum.

with each flavonoid were separately extracted with methanol, concentrated, and resubmitted to a column Silica gel G and eluted with the same system (b) where compounds 47 were isolated (Figure 1). Compound 1: (20 mg) yellow crystals, Rf 0.91 (system b), m.p. 277C, UV, k max in MeOH: nm 367, 268; (AlCl3): 265, 350, 420; (AlCl3=HCl): 265, 350, 420; (NaOA): 275, 300 (sh), 380; (NaOAc=H3BO3): 267, 319 (sh), 380; (NaOMe): 285, 322, 430. 1H-NMR (MSO-d6): 0 0 d 8.0 (2H, d, J 8 Hz, H2 and H6 ), d 6.9 (2H, d, 0 0 J 8 Hz, H3 and H5 ), d 6.4 (1H, d, J 2.5 Hz, H8) and d 6.2 (1H, d, J 2.5 Hz, H6). EI-MS m=z (% re. int): 285 (M) (100), 258 (15), 229 (16), 184 (8), 121 (22) and 93 (10). From the previous data and by comparison with authentic and published data (Mabry et al., 1970), this compound is identified as kaempferol. Compound 2: (30 mg) yellow crystals, Rf 0.91 (system b), m.p. 256C, UV, k max in MeOH: nm 345, 270 (sh), 351; (AlCl3): 272, 303, 371, 391; (AlCl3=HCl): 272, 303, 355, 391; (NaOAc): 276, 356, 402; (NaOAc=H3BO3): 275, 350; (NaOMe): 266, 333, 402. 1H-NMR (DMSO-d6): 0 0 d 7.64 (dd, J 2.8 Hz, H6 ), d 7.57 (d, J 2 Hz, H2 ), d 0 6.97 (d, J 8 Hz, H5 ), d 6.95 (s, H3), d 6.87 (d, J 2.1 Hz, H8), 6.153 (d, J 2.1 Hz, H6), and 3.86 (s, OCH3). EI-MS m=z (% re. int): 300 (M) (100), 285 (50), 272 (16), 269 (8), 152 (22), 152 (12), 148 (20). From the previous data and by comparison with published data (Markham, 1982), this compound is identified as chrysoeriol. Compound 3: (30 mg) yellow crystals, Rf 0.83 (system b), m.p. 256C, UV, k max in MeOH: nm 365, 304

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S.A. Amani et al. (DMSO-d6) showed signals at d 7.9 (1H, dd, J 2.2, 8.8 Hz, H-60 ), d 7.53 (1H, d, J 2.2 Hz, H-20 ), d 6.20 (1H, d, J 8.8 Hz, H-50 ), d 5.62 (1H, d, J 7.5 Hz, H100 ), d 5.32 (1H, d, J 1.0 Hz, H-1000 ), d 3.82 (3H, S, OCH3), d 3.203.90 (m, remaining sugar protons).13C NMR (DMSO) 155.4 (C-2), 133.3 (C-3), 177.2 (C-4), 161.1 (C-5), 98.5 (C-6), 169.1 (C-7), 93.3 (C-8), 155.9 0 0 (C-9), 104.2 (C-10), 130.1 (C-1 ), 110.8 (C-2 ), 145.8 0 0 0 0 (C-3 ), 150.3 (C-4 ), 115.6 (C-5 ), 122.4 (C-6 ), 99.1 (C-100 ), 74.8 (C-200 ), 73.8 (C-300 ), 68.3 (C-400 ), 75.7 (C-500 ), 60.1 (C-600 ), 108.8 (C-1000 ), 76.1 (C-2000 ), 79.3 (C-3000 ), 73.9 (C-4000 ), 64.4 (C-5000 ), 55.5 (OCH3). From the previous data and by comparison with published data (Mabry et al., 1970) this compound is identified as isorhamnetin 3-O-bD-apio-furanosyl (1-2) b-D-galactopyranoside. Pharmacological study Preparation of the plant extract Dried aerial parts of Alhagi maurorum were extracted in a Soxhlet apparatus with ethanol 95%. The ethanol extract was completely dried under vacuum, weighed, and the residue was used in testing. The dried plant extract was freshly suspended in distilled water just before administration. Determination of median lethal dose (LD50) LD50 of the ethanol extract was determined as described before (Finny, 1964). For this purpose, five groups of five mature albino mice (2025 g body weight) each were used. The tested extract was administered orally in doses of 20004000 mg=kg body weight in addition to a group used as a control (given the solvent). Rats were kept under observation for 24 h during which time the number of dead animals in each group was recorded. Antiulcerogenic effect Forty male albino rats of 180200 g body weight were obtained from the Laboratory Animal Colonies (Helwan, Egypt). Animals were kept under good hygienic conditions and fed on standard diet and watered ad libitum. They were divided into eight equal groups and starved for 48 h before use to ensure an empty stomach (Galvin & Mikhail 1976). To avoid dehydration during the period of fasting, rats were supplied with sucrose (BDH) 8% (w=v) solution in NaCl (BDH) 0.2% (w=v), which was removed 1 h before experimentation. The first group was kept as a normal control, and the second one was kept as a positive control. Three treatment groups received the ethanol extract of Alhagi maurorum Boiss in doses of 200, 300, and 400 mg=Kg.b.wt. and another two treatment groups were administered compounds 4 and 5, respectively, in a dose

(sh), 266, 254; (AlCl3): 422, 363, 305 (sh), 262; (AlCl3=HCl): 419, 355, 303 (sh), 264; (NaOAc): 395, 317, 274, 264; (NaOAc=H3BO3): 366, 305, 269, 257; (NaOMe): 417, 238, 272, 242. 1H-NMR (DMSO-d6): d 0 7.96 (d, J 2.1 Hz, H2 ), d 7.50 (dd, J 2.1,8.5 Hz, 0 0 H6 ), d 6.92 (d, J 8.5 Hz, H5 ) d 6.37 (d, J 2 Hz, H8) and 6.17(d, J 2 Hz, H6). EI-MS m=z (% re. int): 316 (M) (100), 301 (50), 285 (16), 164 (30), 152 (22) and 124 (10). From the previous data and by comparison with published data (Markham, 1982), this compound is identified as isorhamnetin. Compound 4: (500 mg) yellow crystals, Rf 0.432 (system b), m.p. 350C, UV, k max in MeOH: nm 250, 270 (sh), 350; (AlCl3): 276, 297 (sh), 356, 392; (AlCl3= HCl): 276, 297 (sh), 392; (NaOA): 253, 355, 410; (NaOAc=H3BO3): 253, 265 (sh), 350; (NaOMe): 260, 0 390. 1H-NMR(DMSO-d6): d 7.66 (dd, J 2.8 Hz, H6 ), 0 0 d 7.59 (d, J 2 Hz, H2 ), d 6.98(d, J 8 Hz, H5 ), d 6.96 (s, H3), d 6.90 (d, J 2.1 Hz, H8), d 6.20 (d, 0 J 2.1 Hz, H6), d 5.06 (d, J 6.9 Hz H1 ), d 3.87 (s, OCH3) and d 3.73.2 (m, rest of sugar proton).13C NMR (DMSO) 154.8 (C-2), 103.3 (C-3), 176.9 (C-4), 162.1 (C-5), 98.8 (C-6), 168.9 (C-7), 94.3 (C-8), 156.5 0 0 (C-9), 103.9 (C-10), 131.1 (C-1 ), 111.8 (C-2 ), 146.8 (C0 0 0 0 3 ), 149.8.3 (C-4 ), 116.5 (C-5 ), 121.9 (C-6 ), 106.2 (C100 ), 73.8 (C-200 ), 76.8 (C-300 ), 67.8 (C-400 ), 66.7 (C-500 ), 55.5 (OCH3). EI-MS m=z (% re. int): 432 (M) (100), 301 (50), 273 (36), 269 (8), 152 (22), 152 (12), 148 (20). From the previous data and by comparison with published data (Mabry et al., 1970), this compound is identified as chrysoeriol-7-O-xylosoid. Compound 5: (600 mg) yellow crystals, Rf 0.40 (system b). UV k max in MeOH: (nm) 265, 350;(AlCl3) 265, 300 (sh), 410(AlCl3=HCl) 265, 350, 410(NaOAc) 275, 300, 380(NaOAc=H3BO3) 270, 310, 375 (NaOMe) 275, 330, 400. 1H-NMR (DMSO-d6): d 7.9 (2H, d, 0 0 0 J 8 Hz, H2 and H6 ), d 6.8 (2H, d, J 8 Hz, H3 0 and H5 ), d 5.8 (1H, d, J 2.5 Hz, H8), d 5.6 (1H, d, J 2.5 Hz, H6), d 5.4 (1 H, d, anomeric proton), d 5.2 (1 H, d, J 2 Hz, H1000 rhamnose), d 34 (m, remaining sugar protons) and d 1.2 (3 H, d, J 6 Hz, CH3 rhamnose). 13C NMR (DMSO): d 153.2 (C-2); 132.6 (C-3); 174.3 (C-4); 159.6 (C-5); 98.8 (C-6); 160.4 (C-7); 93.9 (C-8); 153.2 (C-9); 103.4 (C-10); 121.1 (C-10 ); 130.3 (C-20 ); 114.8 (C-30 ); 157.4 (C-40 ), 114.7 (C-50 ); 130.3 (C-60 ); 102.5 (C-100 ); 71.2 (200 ); 73.2 (300 ); 68.5 (400 ); 75.5 (500 ); 59.8 (600 ); 101.3 (1000 ) d 70.6 (C-2000 ), 70.4 (C-3000 ), 71.9 (C-4000 ), 70 (C-5000 ) and d 17.6 (C-6000 ). From the previous data and by comparison with published data (Markham, 1982), this compound is identified as kaempferol-3-galactorhamnoside. Compound 6: (15 mg) yellow crystals, Rf 2.35 (system b), UV k max in MeOH: (nm) 260, 271 sh, 358: (AlCl3):273, 305, 367, 409; (AlCl3=HCl): 274, 300 sh, 364, 411; (NaOAc): 279, 325, 375; (NaOAc=H3BO3) 260, 271 sh, 358; (NaOMe) 277, 388 d 1H NMR

Antiulcerogenic activity of A. maurorum of 100 mg=Kg.b.wt. In addition, a group of rats was given ranitidine as a reference drug in a dose of 100 mg kg=Kg.b.wt. The ethanol extract, compounds 4, 5, and ranitidine were given orally via a stomach tube. Two doses were given in the first day at 0800 and 1600 h; a third dose was given on the second day 1.5 h before induction of gastric ulceration. All rats except the normal control were given ethanol (Merck) 50% (v=v) (in distilled water) in a dose of 10 ml=kg orally to induce gastric ulceration. Normal control rats received equivolumes of distilled water only at the same time intervals. One hour after ethanol administration, all rats were sacrificed by an overdose of chloroform, and the stomachs were rapidly removed, opened along their greater curvature, and gently rinsed under running tap water. Lesions in the glandular part of the stomach were measured under an illuminated magnifying microscope (10). Long lesions were counted and their lengths were measured. Petechial lesions were counted, and then each five petechial lesions were taken as 1 mm of ulcer (Cho & Ogle, 1979). To calculate the ulcer index (mm), the sum of the total length of long ulcers and petechial lesions in each group of rats was divided by its number. The curative ratio was determined according to the formula: The results obtained were statistically analyzed using t-test (Snedecor & Cochran, 1976).

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Results and Discussion

Compounds 13 were identified as kaempferol, chrysoeriol, and isorhamnetin by comparing their EI-MS, 1H NMR, 13C NMR, UV spectrum in methanol and different shift reagents with published data. Acid hydrolysis of compound 4 revealed the sugar xylose that was identified by HPLC, PC, and TLC (systems e and f), and an aglycone that was found to be identical with compound 2 when compared with its TLC, UV shift reagents. It is substituted at position 7 as indicated by their UV spectra upon addition of diag-

nostic shift reagent. From the obtained data and by their comparison with other published data (Mabry et al., 1970; Markham, 1982), these compounds were identified as chrysoeriol 7-O-xylosoid. Compound 5 revealed the sugars galactose and rhamnose after acid hydrolysis, which were identified by PC and TLC (systems c and d), and an aglycone that was found to be identical with compound 8 when compared with its TLC, UV shift reagents. It is substituted at position 3 as indicated by its UV spectra upon addition of diagnostic shift reagent, so from data given and by comparison with published data, these compounds were identified as kaempferol-3galactorhamnoside. Acid hydrolysis for compound 6 gave galactose and isorhamnetin, a milder hydrolysis with 0.1 N H2SO4 afforded galactose, apiose, and isorhamnetin. The two sugars and the aglycone were identified by TLC comparison with authentic samples. The presence of two doublets at d 5.62 and 5.32 with J 7.5 Hz demonstrated the b-configuration of both galactosyl and apiosyl residues. 13C NMR, C-200 was shifted downfield (3.00 ppm), which indicated the position of attachment of apiosyl moiety at the 200 hydroxyl. From all the above data, this compound was identified as isorhamnetin-3-O-b-D-apiofuranosyl (1-2) b-D-galactopyranoside (Mabry et al., 1970). Gastric damage induced by ethanol was characterized by both long ulcers and petechial lesions. The number of ulcers and the ulcer index in control rats (received ethanol) were highly significant (p < 0.001) when compared with normal untreated animals (receiving distilled water). Repeated oral administration of the ethanol extract of Alhagi maurorum in doses of 300 and 400 mg=kg reduced the severity of gastric damage, as the ulcer index was significantly decreased to 2.90 and 2.62 mm, respectively, compared with 8.61 mm in the positive control group. The curative ratio was 66.31% and 69.57% after administration of both doses (Table 1). Compounds 4 and 5 in a dose of 100 mg=kg showed the best results. They significantly decreased the ulcer index into 2.11 and 1.90 mm, respectively, compared with 8.61 mm in the positive control group and elevated the curative ratio

Table 1. Antiulcerogenic effect of the ethanol extract of Alhagi maurorum Boiss and its isolated compounds in rats (n 5). Treatment group Normal control Positive control Total extract Dose (mg=kg b.wt) 00 00 200 300 400 100 100 100 Number of ulcers (M SE) 0 6.8 0.48a 6.1 0.24 4.7 0.35 3.9 0.20 2.8 0.17 2.2 0.10 6.2 0.30 Ulcer index (mm) 0 8.61 0.46a 7.54 0.32 2.90 0.25 2.62 0.21 2.11 0.12 1.90 0.10 8.40 0.44 Curative ratio (%) 12.42 66.31 69.57 75.49 77.93 2.43

Compound 4 Compound 5 Ranitidine

a

Compared with normal control (p < 0.001). p < 0.01 compared with positive control. p < 0.001 compared with positive control.

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S.A. Amani et al.

El-Saayed NH, Inshak MS, Kandil FI, Mabry TJ (1993): Flavonoids of Alhagi graecolum. Pharmazie 48: 6889. Finny DJ (1964): Statistical Methods in Biological Assay. London, Charles Griffin and Company Ltd., p. 597. Galvin GB, Mikhail AA (1976): Stress and ulcer etiology in the rat. Physil Beh 16: 135139. Ghosal S, Srivastara RS, Battacharya SK, Debanath PK (1974): The active principles of Alhagi pseuddalhagi Bphenethylamine and tetrahydroisoquinoline bases. Planta Med 26: 318326. Kalhoro MA, Kapadia Z, Badar Y (1997):Physicochemical studies of indigenous medicinal plants. Bangladesh J Sci Ind Res 32: 418421. Kudliki WP, William D, Kramer SK, Makhamed BG, Iskakov BK (1991):Eukaryotic protein synthesis initiation factor 2. Eur J Biochem 197: 623629. Lewis HW, Lewis EM (1977): Medical Botany Plants Affecting Mans Health. New York, Wiley-Interscience, pp. 312368. Mabry TJ, Markham KR, Thomas MB (1970): The Systematic Identification of Flavonoids. Berlin, Springer-verlag, p. 398. Markham KR (1982): Techniques of Flavonoid Identification. New York, Academic Press, p. 235. Singh VP, Bineeta Y, Pandey VB (1999): Flavanone glycosides from Alhagi pseudalhagi. Phytochemistry 51: 587590. Snedecor GW, Cochran WG (1976): Statistical Methods, 6th ed. New York, Wiley-Interscience, pp. 502503. Stahl E (1969): Thin-Layer Chromatography, 2nd ed. London, George Allon and Unwirid, Berlin, Springer, p. 980. Tackholm V (1974): Students Flora of Egypt, 2nd ed. Cairo, Egypt, The Cooperative Printing Company, p. 657.

to 75.49% and 77.93%, respectively. The curative ratio after ranitidine administration was only 2.43%. Failure of ranitidine to decrease gastric damage induced by ethanol could be attributed to its mechanism of action, as it blocks the histaminergic receptors, so prevents the stomach from producing excess acid. This mechanism cannot protect the gastric mucosa against the irritant and damaging effects of ethanol. It could be concluded that the ethanol extract of Alhagi maurorum and the isolated compounds are highly safe for human use. Due to their antiulcerogenic activity, they could be used orally either for prophylaxis or for treatment of gastric ulcer.

References
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