Food Chemistry 132 (2012) 2081–2088 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem New flavonol glycosides from Barbeya oleoides Schweinfurth Mai M. Al-Oqail a, Adnan J. Al-Rehaily a,⇑, Wafaa H.B. Hassan a,c, Taghreed A. Ibrahim a,d, Mohammad S. Ahmad a, Sherif S. Ebada b, Peter Proksch b a Pharmacognosy Department, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia Institute of Pharmaceutical Biology, Heinrich Heine University, Düsseldorf, Germany c Pharmacognosy Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt d Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt b a r t i c l e i n f o Article history: Received 15 October 2011 Received in revised form 7 December 2011 Accepted 15 December 2011 Available online 30 December 2011 Keywords: Barbeya oleoides Flavonol glycosides Antimicrobial Spasmolytic a b s t r a c t Three new flavonol glycosides, 30 ,50 dimethoxymyricetin-400 -O-a-L-rhamnopyranosyl (1–4) b-D-glucopyranoside (1), 30 -methoxyquercetin-400 -O-a-L-rhamnopyranosyl (1–4) b-D-glucopyranoside (2) and 30 methoxyqurecetin-600 -O-a-L-rhamnopyranosyl (1–6) b-D-glucopyranoside (3), have been isolated from the aerial part of Barbeya oleoides Schweinf., along with twelve known compounds, uvaol (4), ursolic acid (5), corosolic acid (6), arjunolic acid (7), b-sitosterol-3-O-b-D-glucoside (8), (+)–catechin (9), (-)-epicatechin (10), isorhamnetin-40 -O-glucoside (11), arjunglucoside I (12), D-(-)-bornesitol (13), gallocatechin (14) and epigallocatechin (15). Compounds 4–15 were isolated for the first time from Barbeyaceae. Structure elucidation of compounds 1–3 was based on MS and NMR data. The ethyl acetate extract of the stems as well as compounds 5, 6, 14 and 15 showed significant antimicrobial activity, while the ethanol extracts of leaves, stems and compounds 4, 7, 8, 13–15 have dose-dependent spasmolytic action. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction 2. Experimental section Barbeya oleoides Schweinf. (B. oleoides) belongs to the family Barbeyaceae, which is one of the smallest families in the plant kingdom and is represented by only this species (Dickison & Sweitzer, 1970). Barbeya oleoides is a bushy shrub or small tree up to 5 m high. The plant is widely distributed in the mountainous regions of northeast Africa, particularly in Ethiopia and Somalia and adjacent parts of the Arabian Peninsula, which includes the southwest of Saudi Arabia and Yemen (Chaudhary & Al-Jowaid, 1999; Dickison & Sweitzer, 1970). It is used as a folkloric remedy for treatment of fever, infection, oedema or related inflammatory diseases (Yesilada et al., 1997). According to a literature survey only barbeyol has been isolated from B. oleoides (Ahmed, Al-Rehaily, & Mossa, 2002). Recently, the plant was reported to reduce mycelia growth and inhibit spore germination of five pathogenic fungi causing serious diseases of vegetable crops (Baka, 2010). This paper describes the isolation and characterisation of three new flavonol glycosides along with twelve known secondary metabolites and the antimicrobial and spasmolytic activities of its extracts and several isolates. 2.1. General experimental section ⇑ Corresponding author. Tel.: +966 14673740; fax: +966 14677245. E-mail address: ajalreha@ksu.edu.sa (A.J. Al-Rehaily). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.12.055 Evaporation of solvents was done at 40 °C under reduced pressure, using a BuchiÒ rotary evaporator, Model 011; ultraviolet absorption spectra were obtained using a Hewlett–Packard HP845 UV–Vis spectrometer; the ultraviolet lamp used in visualising TLC plates was a MineralightÒ device, multiband UV, 254/ 366 nm, obtained from UVP, Inc., San Gabriel CA; melting points were determined on a Mettler FP 80 Central Processor supplied with a Mettler FP 81 MBC Cell Apparatus, and were uncorrected; specific rotations were measured as solutions in methanol or chloroform, unless otherwise specified, on a Perkin–Elmer 241 Mc polarimeter, using a one-decimetre tube (Perkin–Elmer, Waltham, MA); infra-red spectra were recorded on a Perkin–Elmer FTIR model 1600 spectrophotometer; 1H and 13C NMR spectra were recorded in CDCl3, CD3OD and DMSO-d6 on a Bruker Avance DRX500 instrument (Central Lab. at the College of Pharmacy, King Saud University; Bruker Biospin GmbH, Rheinstetten, Germany) at 500 MHz for protons and 125 MHz for carbons using the residual solvent signal as an internal standard and/or NMR measurements done at Heinrich-Heine University, Düsseldorf. ROESY experiments were recorded at 300 K on Bruker DPX 300, ARX 400, 500 or AVANCE DMX 600 NMR spectrometers. All 1D and 2D spectra were obtained using the standard Bruker software; mass spectra were 2082 M.M. Al-Oqail et al. / Food Chemistry 132 (2012) 2081–2088 obtained by LC-MS (API Quattro micro) equipped with direct probe and a Z-spray electrospray ion source (MicromassÒ, Quattro Micro™; Waters, Milford, MA). Purification of compounds was performed by preparative C-18 HPLC. 2.2. Biological materials 2.2.1. Plant materials The plant was collected in March 2006 from south of Baljouraji, southern part of Saudi Arabia and identified by Dr. M. Atiqur Rahman, Professor of Taxonomy, College of Pharmacy, King Saud University (KSU), and a voucher specimen (# 15023) was deposited at the local herbarium. The aerial parts and roots were air-dried indoors at room temperature then grounded to coarse powder using Condux Mill. 2.2.2. Microorganisms American Type of Culture Collection (ATCC) standard against various microorganisms namely: Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Mycobacterium smegmatis and Candida albicans were used. 2.2.3. Animals Male albino guinea-pigs (500 g body weight) and white male New Zealand rabbits (2.5 kg) were used for spasmolytic action. All animals were provided by the Experimental Animals Care Center, KSU, Riyadh. 2.3. Extraction and isolation Powders of air dried leaves (2.48 kg) and stems (1.35 kg) were first extracted via percolation with petroleum ether (40–60 °C). Both petroleum ether extracts were concentrated in vacuo to yield fractions A (leaves 26.1 g) and B (stems 4.2 g). The dried marc of both parts was extracted with 96% EtOH by cold maceration, and the solvent evaporated to furnish dark brown fractions C (leaves 95.7 g) and D (stems 55.3 g). The ethanol extracts of leaves (90.0 g) and stems (52.3 g) were separately suspended in water and subsequently extracted with n-hexane, chloroform, ethyl acetate and n-butanol. Each fraction was dried over anhydrous sodium sulphate and evaporated to dryness to yield fraction E (n-hexane, 12.3 g), fraction F (chloroform, 5.3 g), fraction G (ethyl acetate, 10 g), fraction H (n-butanol, 9.1 g) and fraction I (aqueous, 50.7 g) from leaves extract and fractions, J (n-hexane, 2.3 g), K (chloroform, 3.9 g), L (ethyl acetate, 9.3 g), M (n-butanol, 7.1 g) and N (aqueous, 22.9 g) from stems extract. Fraction H (7.5 g) was applied to a Sephadex LH-20 column (50 g) and eluted with MeOH. Twenty fractions (200 mL) were collected and evaporated to dryness in vacuo. Fractions 13–15 (240.4 mg), containing flavonoid constituents were combined, owing to their TLC similarities, and subsequently purified by semipreparative HPLC (C18 reversed phase column) using 15% water:acetonitrile as eluent, yielding 1 (7 mg) and an inseparable mixture of 2 and 3 (5 mg). Fraction A (5 g) was subjected to chromatography on a silica gel column. The column was eluted using increasing concentrations of ethyl acetate in petroleum ether to end up with nine pooled fractions. b-Amyrin was the main component of Fraction 4 and was detected through comparison of TLC behaviour with authentic standard. Fraction 9 was acetylated using pyridine and acetic anhydride at room temperature producing a reaction mixture of acetylated products, which was then separated on chromatotron (1 mm plate) using 5% ethyl acetate in n-hexane to afford compound 4 (95 mg). Fractions E, F, J and K were combined together (23.8 g) based on TLC behaviour and 22 g was chromatographed over a silica gel column. The column was eluted using chloroform, 5% methanol in chloroform and 10% methanol in chloroform, successively. The 5% methanol in chloroform elution resulted in three fractions 1– 3. Repeated column chromatography of these fractions resulted in the isolation of compounds 5 (13 mg), 6 (215 mg), 7 (355 mg) and 8 (35 mg). Fraction L (7.1 g) was chromatographed over silica gel column using chloroform as eluent and the polarity was increased by methanol. Ten fractions were collected (1–10). Fraction 4 (200 mg) was applied to a reversed-phase silica gel column using 70% water/methanol as eluent and 7 sub-fractions (a–g) were collected. Sub-fractions d and f yielded compound 9 (27.2 mg) and compound 10 (5.2 mg), respectively. Fraction G (9 g) was chromatographed on silica gel column and eluted successively with increasing polarity of methanol in chloroform and 150 fractions were collected. Fraction 3 (270 mg) was chromatographed on silica gel column (10 g) using ethyl acetate: methanol: water (30:5:4) as solvent system and was further purified using preparative TLC to give compound 11 (7 mg) as a yellow residue. Fraction 7 (64.2 mg) was loaded on a C18 reversed phase column (20 g) by using 30% acetonitrile /water as eluent to yield 5 sub-fractions (I–V). From sub-fraction (III) compound 12 (15 mg) was obtained as a white residue. Fraction I (10 g) was dissolved in methanol and the insoluble part was filtered off. The solid material was identified as compound 13 (290 mg). Powder of air dried roots of B. oleoides (200 g) was extracted via percolation with water. The water extract was then extracted with ethyl acetate and n-butanol to afford 5.1 g and 10.3 g, respectively. Ethyl acetate fraction (1.5 g) was chromatographed on chromatotron (4 mm plate) using 20% methanol in chloroform with acetic acid (0.03%) to yield the inseparable gummy mixture of 14 and 15 (120 mg). The structures of all isolated compounds were shown in Fig. 1. 2.3.1. Compounds 1–15 Compound (1): yellow residue {Rf 0.45; ethyl acetate:methanol:water (30:5:4)}; UV (MeOH) kmax 254 and 362 nm; ESI-MS [M + H] + at m/z 655 in positive ion mode for C29H34O17 in addition to fragments at m/z 509 and 347 accounted for the loss of two sugar moieties; 1H and 13C NMR (MeOD, 500 and 125 MHz): Tables 1 and 2; HMBC and ROESY: Fig. 2. Compounds (2 and 3): brown gummy solid mixture of 2 and 3 (3:2); ESI-MS: m/z 625. [M + H] + for C28H32O16 along with fragments at m/z 477 and 315 accounted for the loss of two sugar moieties; UV spectrum (MeOH) kmax 254 and 358 nm; 1H and 13C NMR (MeOD, 500 and 125 MHz): Tables 1 and 2; HMBC and ROESY: Fig. 2. Compound (4): viscous mass; FTIR (mmax, KBr): 1750, 1250– 1027 cm1; EI-MS: m/z 526 for C34H54O4; 1H and 13C NMR data were consistent with other literature (Mahato & Kundu, 1994). Compound (5): white amorphous powder; m.p. 260–262 °C; [a]D + 62.0 (c. 1.0, MeOH); FTIR (mmax, CHCl3): 3400, 1680, 1185 cm1; EI-MS: m/z 456 [M]+ for C30H48O3; 1H and 13C NMR data were consistent with other literatures (Seebacher, Simic, Weis, Safand, & Kunert, 2003). Compound (6): white powder; m.p. 240–242 °C; [a]D + 49.9 (c. 0.3, CHCl3). UV (CH3OH) kmax 260 nm; FTIR (mmax, KBr): 3430, 1692 cm1; EI-MS: m/z 472 [M]+ for C30H48O4; 1H and 13C NMR data were consistent with other literatures (Dat et al., 2005). Compound (7): yellowish white powder, [a]D + 58.1(c. 0.40, EtOH); m.p. 296–297 °C; FTIR (mmax, KBr): 3417, 1697, 1045 cm1; EI-MS: m/z 488 for C30H48O5; 1H and 13C NMR data were consistent with other literature (Ping, Jie, Cong, Cheng, & Tosho, 1992). 2083 M.M. Al-Oqail et al. / Food Chemistry 132 (2012) 2081–2088 OCH3 HO O 7 2` 3` 4` 1` 5` 6` 1 8 6 9 2 10 3 5 OR1 R2 4 OH OH O Compound R1 1 2 3 R2 Glu (4-1) Rha Glu (4-1) Rha Glu (6-1) Rha OCH3 H H R7 R6 R7 4 5 6 7 H H OH OH OCOCH3 OH OH OH R2 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH2OH CH2OCOCH3 COOH COOH COOH CH3 CH3 CH3 H H H H CH3 12 OH OH CH3 CH2OH COO-Glu. OH CH3 No.: R1 R6 R5 R1 27 R3 R4 R5 R2 R3 R4 OH HO O OH OH OH 9 OH HO O O HO 8 HO OH OCH3 OH OH O O HO OH O OH OH HO O HO 11 10 OH OH OH O OH OH OH HO OCH3 HO O OH HO OH OH OH 13 14 OH OH OH HO O OH 15 OH OH Fig. 1. Structures of compounds 1–15. Compound (8): white crystals; m.p. 289–290 °C; FTIR (mmax, KBr): 3500–3200, 1026, 2920, 2820, 1460 cm1; UV (CH3OH) kmax 243 nm; EI-MS: m/z 576 [M]+ for C35H60O6, m/z 414 [M+-glucose], m/z 162 (sugar unit); 1H and 13C NMR data were consistent with other literature (Good & Akihisa, 1997). Compound (9): brown powder; [a]D + 8 (c. 0.23, MeOH); UV (CH3OH) kmax 277 and 220 nm; FTIR (mmax, KBr): 2600–3400, 1620 cm1; EI-MS: m/z 290 [M]+ C15H14O6, m/z 139, 138, 110, 152, 151, and 123; 1H and 13C NMR data were consistent with other literature (Kofink, Papagiannopoulos, & Galensa, 2007). Compound (10): brown crystals; [a]D –29.6 (c. 0.23, MeOH); UV (MeOH) kmax 217 and 280 nm; 1H and 13C NMR data were consistent with other literature (De Souza, Cipriani, Iacomini, Gorin, & Sassaki, 2008). 2084 M.M. Al-Oqail et al. / Food Chemistry 132 (2012) 2081–2088 Table 1 1 H NMR data of compounds 1–3 in CD3OD. Carbon no. 1 dH (J Hz) 2 dH (J Hz) 3 dH (J Hz) 6 8 2‘ 30 -OCH3 50 60 100 200 300 400 500 600 6.24 (1H, d, J = 2.3 Hz) 6.45 (1H, d, J = 2.3 Hz) 7.62 (1H, s) 3.99 (3H, s) 3.99 (3H, s) 7.62 (1H, s) 5.30 (1H, d, J = 7.5 Hz) 3.50 3.55 3.60 (1H, m) 3.55 (1H, m) Ha 3.82 (1H, m) Hb 3.41 (1H, m) 4.50 (1H, br s) 3.61 (1H, m) 3.35 (1H, m) 3.35 (1H, m) 3.55 (1H, m) 1.21 (3H, d, J = 6.0 Hz) 6.25 (s) 6.45 (s) 7.96 (1H, d, J = 2 Hz) 4.02 (3H, s) 6.96 (1H, d, J = 8.2 Hz) 7.66 (1H, dd, J = 8.2, 2 Hz) 5.26 (1H, d, J = 6.5 Hz) 3.40 (1H, m) 3.50 (1H, m) 3.50 (1H, m) 3.75 (1H, m) Ha 3.80 (1H, m) Hb 3.44 (1H, m) 4.57 (1H, br s) 3.65 (1H, m) 3.60 (1H, m) 3.60 (1H, m) 3.60 (1H, m) 1.12 (3H, d, J = 6.0 Hz) 6.25 (s) 6.45 (s) 8.05 (1H, d, J = 2 Hz) 3.99 (3H, s) 6.94 (1H, d, J = 8.2 Hz) 7.64 (1H, dd, J = 8.2, 2 Hz) 5.28 (1H, d, J = 6.5 Hz) 3.40 (1H, m) 3.50 (1H, m) 3.50 (1H, m) 3.75 (1H, m) Ha 3.75 (1H, m) Hb 3.55 (1H, m) 4.57 (1H, br s) 3.65 (1H, m) 3.60 (1H, m) 3.60 (1H, m) 3.60 (1H, m) 1.21 (3H, d, J = 6.0 Hz) 1000 2000 3000 40 5000 6000 Table 2 13 C NMR data of compounds 1–3 in CD3OD. Carbon no. 1 dC 2 dC 3 dC Carbon No. 1 dC 2 dC 3 dC 2 158.5 s 156.8 s 156.8 s 3 4 5 6 7 8 9 10 10 20 30 30 -OCH3 40 135.8 s 175.7 s 160.6 s 98.1 d 163.8 s 95.1 d 158.5 s 103.0 s 122.8 s 107.2 d 146.7 s 57.3 q 138.8 s 135.6 s 175.7 s 160.5 s 99.9 d 166.1 s 94.8 d 158.8 s 104.4 s 123.8 s 116.0 d 146.0 s 56.9 q 148.1 s 135.6 s 175.7 s 160.5 s 99.9 d 166.1 s 94.8 d 158.8 s 104.4 s 123.8 s 116.2 d 146.0 s 57.2 q 148.1 s 50 50 -OCH3 60 100 200 300 400 500 600 1000 2000 3000 4000 5000 6000 146.7 s 57.3 q 107.2 d 103.3 d 73.8 d 75.5 d 79.0 d 76.0 d 64.8 t 101.9 d 69.5 d 74.5 d 74.5 d 75.5 d 17.9 q 114.5 d – 121.5 d 102.5 d 73.1 d 75.0 d 78.5 d 77.4 d 64.5 t 102.5 d 70.0 d 75.5 d 75.0 d 75.0 d 18.0 q 114.5 d – 121.5 d 102.5 d 72.3 d 75.5 d 70.8 d 78.0 d 68.5 t 101.9 d 69.5 d 75.7 d 75.7 d 75.0 d 17.9 q Compound (11): yellow residue; FTIR: 3450, 1654, 1560 cm1; EI-MS: m/z 316 for aglycone (C22H22O11); 1H and 13C NMR data were consistent with other literatures (Fossen & Pedersen, 1998). Compound (12): amorphous powder; [a]D + 4.6 (c. 0.75, MeOH); FTIR (mmax, KBr): 3400, 1731, 1645 cm1; EI-MS: m/z 666 for C36H58O11. 1H and 13C NMR data were consistent with other literature (Abe & Yamauchi, 1987). Compound (13): white brown powder; m.p. 183–185°C, [a]D 65.5 (c. 0.05, H2O); FTIR: 3333, 2927 cm1; EI-MS [M]+ at m/z 194 for C7H14O6; 1H and 13C NMR data were consistent with other literatures (Angyal, 1983). Compounds (14 and 15): yellow amorphous powders; UV (MeOH) kmax 271.5 nm; FTIR spectra (mmax, KBr): 3400–3267, 1610 cm1; EI-MS: m/z 278 for C15H14O7; 1H and 13C NMR data were comparable with those of gallocatechin and epigallocatechin (Someya, Yoshiki, & Okubo, 2002). 2.4. Antimicrobial assay 2.4.1. Antimicrobial screening by determination of the inhibition zone The antimicrobial activity of petroleum ether, chloroform, ethyl acetate, n-butanol and aqueous extracts of leaves and stems and the isolated compounds were tested according to the National Committee of Clinical Laboratory Standards (National committee for clinical laboratory standards, 2002) against various microorganisms, Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 15036), Pseudomonas aeruginosa, Candida albicans and Mycobacterium smegmatis (ATCC 10231). The positive antibacterial and antifungal activities were established by the presence of inhibition zones after 24 h for bacteria and 48 h for fungi (NCCLS, 2002). 2.4.2. Determination of minimum inhibitory concentration (MIC) Antimicrobial activities of petroleum ether, chloroform, ethyl acetate, n-butanol and aqueous extracts of leaves and stems as well as isolated compounds of B. oleoides were evaluated based on the agar dilution method against the sensitive microorganisms. Mueller–Hinton agar medium was sterilised by autoclaving at 121 °C for 15 minutes and used at a concentration of 38 g/L. After cooling to 50 °C, the medium was impregnated with different tested solutions in different concentrations dissolved in 0.2 mL DMSO/10 mL medium. Plates made of the impregnated medium were inoculated with tested microorganisms and incubated at 37 °C for 48 h. The used concentrations are considered active when complete growth 2085 M.M. Al-Oqail et al. / Food Chemistry 132 (2012) 2081–2088 CH3 H OCH3 1''' H H HO O O 1'' HO OH OH HO O OH HO O OCH3 H OH 1 OH O CH3 H HO OCH3 H H HO O O OH OH 1''' O OH 1'' HO HO O H H OH OH 2 O CH3 H O O OCH3 H O H HO O 1'' HO HO OH OH OH OH O H H OH OH 3 O Fig. 2. Important HMBC (?) and ROESY ($) correlations for compounds 1–3. inhibition of the organisms (Mitscher, Leu, Bathla, Wu, & Beal, 1972) is observed. 2.5. Spasmolytic activity Male albino guinea-pigs (500 g body weight) were killed and the abdomens opened. Segments (2.5 cm) of the ileum were cut out and suspended in a 10-mL water-jacketed organ bath containing an oxygenated Tyrod’s solution at 37 °C. Each tissue was attached to a myograph F-60 transducer (tension range 0.5–5 g) and attached to a Narco Biosystems (Austin, TX) physiograph. Each tissue was allowed to equilibrate for 20 minutes under 0.5 g tension before the start of the experiments. Dose-response curves of several spasmogens Ach (10–200 ng/ml), histamine (10–300 ng/ ml) and barium chloride (1–20 l/ml) and the respective submaximum doses were chosen to investigate the effect of the test extracts and compounds on the spasmogens induced contraction. Initial pilot experiments were performed to obtain initial data about the effective doses and then a suitable dose e.g. 100 lg/ml bathing fluid was selected for testing against the spasmogens using a contact time of 5 min. To test the influence of increasing the concentration of Ca++ on the antagonistic effect of the extracts or compounds against the spasmogens a procedure similar to that described (Bakheet, ElTahir, Al-Sayed, El-Obeid, & Al-Rashood, 1999) was used by adding an increasing small concentrations of CaCl2 (1% aqueous small portion of solution) to the tissue bathing fluid (normal Tyrode’s solution). White male New Zealand rabbits (2.5 kg) were killed and pieces of the jejunum (2.5 cm long) were cut and suspended in oxygenated Kerb’s solution at 37 °C as described previously (El-Tahir et al., 1987). The setting of tissue for recording of the spontaneous activity was similar to that described for the isolated guinea-pig above. To test the effect of the different extracts on the spontaneously contracting jejuneum separate different volumes of each suspended extract were added individually to the tissue and allowed to contact it for 4 min to record effect if any. Extract was washed out and the tissue was allowed to rest for 10 min then another dose was tested. Fresh tissue was used to test each extract (El-Tahir et al., 1987). 3. Results and discussion 3.1. Phytochemical result The UV spectrum of 1 showed absorption bands at kmax 254.0 and 362.0 nm that suggested the flavonol nature of the compound (Chen & Zuo, 2007). When sodium methoxide shifting reagent was added band I showed an absence of bathochromic shift which indicated the occupation of the C-40 while the bathochromic shift 2086 M.M. Al-Oqail et al. / Food Chemistry 132 (2012) 2081–2088 of +52 nm with aluminium chloride confirmed the existence of a free hydroxyl group at C-5 and/or C-3. There was no shift in band I with boric acid providing evidence of involvement in favour of C30 /40 and C-40 /50 . While on the other hand, appearance of bathochromic shift in band II by 12 nm with sodium acetate indicated the presence of a free hydroxyl group at C-7. Acid hydrolysis of 1 released glucose, rhamnose and 30 ,50 dimethoxymyricetin. This was confirmed by the ESI-MS spectrum which showed a molecular ion peak at m/z 655 [M + H]+ which is in agreement with the molecular formula C29H34O17; in addition a fragment at m/z 347 resulted from loss of two sugar moieties. 1H NMR values in Table 1 showed two meta coupled protons at dH 6.24 (d, 2.3 Hz) and 6.45 (d, 2.3 Hz) for H-6 and H-8 respectively, suggesting a 5, 7-substituted A ring; a singlet signal at dH 7.62 integrated for two protons was assigned to H-20 and H-60 in ring B; another sharp singlet was observed at dH 3.99 (6H) for two methoxy groups. The above data imply that 1 is a disaccharide of 30 ,50 dimethoxymyricetin (syringetin) (Minkyun, Byung, & Kihwan, 2009). Two signals in the sugar region at dH 5.30 (1H, d, J = 7.5 Hz, dC 103.3) and 4.50 (1H, br s, dC 101.9) corresponding to anomeric protons of b-D-glucose and a-L-rhamnose units, respectively, were also observed. Chemical shifts of proton and carbon of glucose and rhamnose moieties are comparable with the reported values for rutinoside (Gohar, 2002). The HMBC spectrum (Fig. 2) exhibited long range correlation between the anomeric proton H-100 (dH 5.30) and C-40 (dC 138.8), which established the location of the sugar moiety at C40 . This was further proved by the long range correlation between 7.62 (20 /60 ) and C-40 and this key observation differentiates it from the known compound syringetin 3-rutinoside. Furthermore, the long-range correlations between the anomeric proton of rhamnose (dH 4.50, H  1000 ) and the C-400 of glucose (dC 79.0) revealed the 1–4 linkage between glucose and rhamnose. The proton signal at dH 3.99 (6H, 2  OCH3) was correlated in HMBC experiments with C-30 and C-50 at dC 146.7, which confirmed the positions of the two methoxy groups. Further support of the above data was extracted from the ROESY data (Fig. 2) in which the two methoxy groups at dH 3.99 correlate with H-20 /60 at dH 7.62, which confirmed the location of two methoxy group at C-30 and 50 . The ROESY spectrum assigned the position of sugar moiety at C-40 through appearance of a cross peak; between the anomeric proton at dH 5.30 (H-100 ) for glucosyl unit and H-20 /60 at dH 7.62, glucose protons and H-20 /60 ; between the anomeric proton at dH 4.50 ðH  1000 Þ for rhamnosyl unit with H-20 /60 at (dH 7.62) and with methoxy groups at dH 3.99. Moreover, the attachment between the two sugars unit was also confirmed by the ROESY correlation which was represented as a cross peak between H  1000 of rhamnose unit at dH 4.50 with H-400 of glucose at dH 3.60. The EI-MS fragments at m/z 509 [M+ – rhamnose] and 347 [509 – glucose] and the support of UV data confirmed the glycosidic substitution sequence at C-40 (dC 138.8), in which the glucose is the inner sugar and rhamnose is the outer unit. The structure of compound 1 is therefore established as 30 ,50 dimethoxymyricetin-400 -O-a-L-rhamnopyranosyl (1–4) -b-D glucopyranoside. Compounds 2 and 3 were obtained as a brown gummy solid mixture from butanol-soluble fraction H. The 1H NMR data (Table 1) have showed a mixture of two compounds (2 and 3). Flavone glycoside nature of the mixture is suspected when it produced yellow colour on treatment with alkali/aluminium chloride and showed positive Molish’s test (Evans, 2002; Mabry, Markham, & Thomas, 1970). Two absorption bands exhibited at kmax 254 and 358 nm in its UV spectrum indicated the presence of typical chromophore of flavonol (Chen, Zuo, & Deng, 2001). Addition of NaOCH3, AlCl3 and NaOAc shift reagents suggested the likely presence of 30 , 40 and disubstituted flavonol glycoside with free hydroxyl groups at 3, 5 and 7-positions. The ESI-MS of the mixture showed a molecular ion peak at m/z 625 [M + H]+, which was attributable to a molecular formula of C28H32O16, 477 [M+ – rhamnose], 315 [477 – glucose]+. The 1H NMR data of both compounds 2 and 3 were similar to those of 1, except for the absence of one methoxy group signal arising from the B ring. The 1H NMR data of the glycone moiety was quite similar to those of isorhamnetin (30 -methoxyquercetin) (Fossen & Pedersen, 1998; Hyun et al., 2005). Interestingly, the 1H NMR spectrum for the mixture of compounds 2 and 3 has showed two common proton singlets at dH 6.25 and 6.45 for H-6 and H-8. The other diagnostic proton signals for compound 2 resonated at dH 7.96 (d, J = 2.0 Hz), dH 6.96 (d, J = 8.2 Hz) and 7.66 (dd, J = 8.2, 2.0 Hz) for H-20 , H-50 and H-60 , respectively. Significant proton signals for compound 3 have appeared at dH 8.05 (d, J = 2.0 Hz), 6.94 (d, J = 8.2 Hz) and 7.64 (d, J = 8.2, 2.0 Hz) for H-20 , H-50 and H-60 , respectively. In addition, two methoxy groups’ protons for compounds 2 and 3 also appeared separately at dH 4.02 and dH 3.99. Two pairs of anomeric proton signals were recorded at dH 5.26 (d, 6.5 Hz) and dH 4.57 (br s) for H-100 and H  1000 of compound 2 and at dH 5.28 (d, 6.5 Hz) and dH 4.57 (br s) for H-100 and H  1000 of compound 3. Acid hydrolysis of the mixture yielded glucose and rhamnose sugars and one aglycone unit. The above data indicate the presence of two different compounds differing only in the sugar linkage. Glucosidation of both compounds 2 and 3 at C-40 was concluded from the HMBC correlation (Fig. 2) of the two anomeric protons of glucose moieties at dH 5.26 and 5.28 with C-40 at dC 148.1. Moreover, there were three bond correlations between protons at dH 7.96, 7.66 (20 , 60 ), 8.05, 7.64 (20 , 60 ) for compounds 2 and 3 with C40 , which confirmed the position of sugar moieties at C-40 . ROESY spectrum confirmed the above suggestion by showing correlations of each H-100 with H-20 , H-50 and H-60 from both compounds of the mixture. The positions of rhamnose moieties of 2 and 3 were at C-400 (78.5) and C-600 (68.5), respectively. In addition, long-range correlations were also detected between the two anomeric protons of both rhamnose (dH 4.57, H  1000 ) and C-400 and C-600 of glucose moieties at dC 78.5 and 68.5. The rhamnose sugars of 2 and 3 also showed correlations (via ROSEY) for H  1000 with H-400 and H  6000 Table 3 Results of antimicrobial screening of successive extracts (1 mg/mL) of B. oleoides. Micro-organism Bacillus subtilis Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Mycobacterium smegmatis Candida albicans Leaves extract Stems extract Pet. ether Chloroform Ethyl acetate Butanol Aqueous Pet. ether Ethyl acetate Butanol Gentamycin (10 lg/mL) Amphotericin B (10 lg/mL) + +   +    + +  +  +  +  +   + +   + + + + + +  + + + + +       + + +  + + +    + + + + + +  + M.M. Al-Oqail et al. / Food Chemistry 132 (2012) 2081–2088 Inhibition % Histamine D-(-)-Bornesitol (13) seemed to be non-selective and non-specific in its antagonism to spasmogens. Uvaol diacetate (4) appeared to be more active against acetylcholine with some blocking activity against histamine. Compounds 7, 8 and 13, and the mixture 14 and 15 were more active against BaCl2. These results point to the inherent ability of these compounds to block the intestinal calcium channels. Addition of 10 mg CaCl2/mL bathing fluid reversed the inhibitory effects of each of the above compounds. Inhibition %BaCl2 Acknowledgements 90 80 70 60 Inhibition % Acetylcholine 50 40 2087 30 20 10 0 4 7 8 13 14 &15 Fig. 3. % Inhibition of the tested compounds (4, 7, 8, 13, 14 and 15) at a dose of 100 lg/mL on the spasmogens-induced contractions on isolated guinea-pig ileum. confirming the attachment of glucose units of compounds 2 and 3 at H-400 and 600 . Thus, 2 and 3 were identified as a mixture of 30 methoxyquercetin-400 -O-a-L-rhamnopyranosyl (1–4) b-D glucopyranoside (2) and 30 -methoxyquercetin-400 -O-a-L-rhamnopyranosyl (1–6) b-D glucopyranoside (3). 3.2. Pharmacological results 3.2.1. Antimicrobial activity 3.2.1.1. Antimicrobial screening by determination of zone of inhibition. The petroleum ether, chloroform, ethyl acetate, n-butanol and aqueous extracts of leaves and petroleum ether, ethyl acetate and n-butanol extracts of stems (Table 3), as well as compounds 1–15 were subjected to antimicrobial screening. Besides the extracts, only compounds 5, 6 and mixture of 14 and 15 showed activity. 3.2.1.1.1. Determination of minimum inhibitory concentration (MIC). The MIC value of the isolated compounds, ursolic acid (5), corsolic acid (6), and the mixture 14 and 15 were 50, 25 and 25 lg/mL, respectively. 3.2.2. Spasmolytic activity 3.2.2.1. Effect of the different extracts on the spontaneously contracting rabbit jejunum. Addition of total ethanol extract of stems and leaves and successive fractions of stems ethanol extract (n-hexane, chloroform, ethyl acetate, n-butanol and aqueous) of B. oleoides to the spontaneously contracting tissue to produce a final concentration of 100–1000 lg/ml bathing fluid induced dose-dependent inhibition. The percentages of inhibition induced by these extracts at the maximum dose tested (1000 lg/mL) were 76%, 65%, 68.2%, 92%, 80%, 77% and 55%, respectively. The maximum inhibition of the contracting rabbit jejunum was observed with the chloroform extract and the minimum inhibition was observed with the aqueous stems extract. 3.2.3. Effect of the test compounds on the spasmogens-induced contractions in isolated guinea-pig ileum The pure compounds uvaol diacetate (4), arjuonlic acid (7), bsitosterol-3-O-b-D-glucoside (8), D-(-)-bornesitol (13) and the mixture of 14 and 15 were tested against acetylcholine, histamine and BaCl2-induced contractions, each at a concentration of 100 lg/mL bathing fluid and a contact time of 5 min. These compounds showed variations in their ability to block the actions of the different spasmogens. These effects are depicted in Fig. 3. The authors are thankful to Prof. Ghada Shaker, Department of Pharmaceutics, College of Pharmacy, KSU, for assisting in some of microbiology work and Dr. V. Wray at the Helmholtz center for infection research, Braunschweig, Germany for running ROESY experiments. 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