《有机化学》课程教学资源（参考书籍）Nature pruduct commucations，An International Journal for Communications and Reviews Covering all Aspects of Natural Products Research，Pawan K. Agrawal.pdf_P26-P30

Flavonoid glycosides from Chrozophora senegalensis Natural Product Communications Vol. 1 (12) 2006 1091 RO O OH O OH OR2 OR1 1 R = H R1 = (6''-caffeoyl)glc R2 = glc 2 R = (2''-p-coumaroyl)-glc-(6-1)rha R1 = Me R2 = H 3 R= (6''-p-coumaroyl)glc RO O OH O OMe Figure 1: Structures of compounds 1-3. Table 2: 1 H- and 13C-NMR data of compounds 2-3 (CD3OD, 600 MHz)a . position 2 3 δH δC δH δC 2 157.9 164.5 3 139.9 6.70 s 103.7 4 180.0 184.0 5 164.3 164.0 6 6.54 d (2.0) 101.2 6.56 d (2.0) 100.2 7 164.5 165.3 8 6.73 d (2.0) 95.8 6.77 d (2.0) 95.6 9 158.8 158.6 10 107.1 106.0 1' 123.6 122.8 2' 7.71 d (1.5) 116.2 7.94 d (8.5) 129.4 3' 145.0 7.06 d (8.5) 116.3 4' 149.6 159.0 5' 6.90 d (8.0) 117.3 7.06 d (8.5) 116.3 6' 7.65 dd (1.5, 8.0) 123.3 7.94 d (8.5) 129.4 OMe 3.90 s 56.1 3.92 s 56.3 7-O-Glc 1'' 5.06 d (7.5) 100.0 5.05 d (7.5) 100.3 2'' 4.74 dd (7.5, 9.0) 74.5 3.55 dd (7.5, 9.0) 74.0 3'' 3.47 t (9.0) 77.0 3.47 t (9.0) 77.5 4'' 3.45 t (9.0) 71.0 3.43 t (9.0) 71.5 5'' 3.30 m 77.7 3.61 m 75.8 6''a 6''b 4.00 dd (5.0, 12.0) 3.60 dd (3.0, 12.0) 67.5 4.64 dd (4.5, 12.0) 4.25 dd (2.5, 12.0) 64.3 Rha 1''' 4.80 d (1.5) 101.9 2''' 3.94 dd (1.5, 3.4) 72.2 3''' 3.88 dd (3.4, 9.5) 71.8 4''' 3.55 t (9.0) 74.5 5''' 4.20 m 69.6 6''' 1.12 d (6.5) 17.6 p-coumaroyl 1 124.9 127.0 2,6 7.45 d (8.5) 130.2 7.45 d (8.5) 129.5 3,5 6.73 d (8.5) 116.5 6.75 d (8.5) 116.0 4 161.0 150.1 α 6.38 d (16.0) 118.0 6.38 d (16.0) 118.0 β 7.41 d (16.0) 146.8 7.43 d (16.0) 147.0 COO 168.7 168.8 a Coupling pattern and coupling constants (J in Hertz) are in parentheses. (C-7), δ 4.74 (H-2'') and 168.7 (COO), and δ 4.80 (H-1''') and 67.5 (C-6''). Therefore, the structure quercetin 3-methyl ether-7-O-α-L-rhamnopyranosyl- (1→6)-(2''-p-coumaroyl)-β-D-glucopyranoside was assigned to compound 2. Compound 3 was obtained as a yellow amorphous powder and its ESI-MS showed an [M-H]- ion peak at m/z 591. The molecular formula C31H28O12 was confirmed by elemental analysis. In the 1 H-NMR spectrum (Table 2) two singlets at δ 6.70 and 3.92, two doublets at δ 6.77 and 6.56 each (1H, d, J = 2.0 Hz), and two o-coupled protons at δ 7.94 and 7.06 each (2H, d, J = 8.5 Hz) were present permitting the identification of the aglycon as apigenin 4'-methyl ether or acacetin [5]. Additionally for 3, resonances of one anomeric proton and one p-coumaroyl residue were observed in the 1 H-NMR spectrum at δ 5.05

1092 Natural Product Communications Vol. 1 (12) 2006 Vassallo et al. (1H, d, J = 7.5 Hz), 7.45 and 6.75 each (2H, d, J = 8.5 Hz) and δ 7.43 and 6.38 each (1H, d, J = 16.0 Hz), respectively. 1D-TOCSY, DQF-COSY, and HSQC NMR experiments showed the presence of one β-D-glucopyranosyl unit characterized by an acylation shift at H2-6 (δ 4.64 and 4.25). The configuration of the glucose unit was determined as reported for compound 1. HMBC correlations confirmed the substitution sites of each residue allowing compound 3 to be identified as acacetin 7-O-(6''-p-coumaroyl)-β-D-glucopyranoside. Compounds 4-12 were identified by 1D- and 2D-NMR spectroscopy and ESI-MS analysis and by comparison of their data with those reported in the literature [9-14] as quercetin 3'-methyl ether-3-O-α- L-rhamnopyranoside (4), quercetin 3'-methyl ether-3- O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (5), apigenin 7-O-(6''-p-coumaroyl)-β-D-glucopyranoside (6), quercetin 3-methyl ether-7-O-α-Lrhamnopyranosyl-(1→6)-β-D-glucopyranoside (7), 4-hydroxyphenyl-O-α-L-rhamnopyranosyl-(1→6)-β- D-glucopyranoside (8), 4'''-methyl ether amenthoflavone (9), roseoside (10), icariside B5 (11), and ampelopsisionoside (12). Table 3: Scavenger effect on DPPH stable radical and superoxide anion of methanol fractions and compounds 1-12 isolated from C. senegalensis. Fracts or Compds DPPH Test Effect on O2 -. aIC50 (μg/ml) ± b SD A B C D E F 1 2 3 4 5 6 7 8 9 10 11 12 c Trolox d SOD 178 ± 6.7 0.61 ± 0.04 14.22 ± 1.1 2.6 ± 0.35 7.01 ± 0.6 0.37 ± 0.03 6.47 ± 1.5 0.19 ± 0.05 4.56 ± 0.8 0.36 ± 0.02 25.65 ± 3.6 0.47 ± 0.03 9.75 ± 0.9 0.085 ± 0.002 1.08 ± 0.4 0.025 ± 0.003 61.59 ± 2.5 2.5 ± 0.4 6.69 ± 0.7 0.20 ± 0.01 52 ± 0.5 0.85 ± 0.06 110 ± 24 1.35 ± 0.09 94.33 ± 0.7 0.42 ± 0.05 - - 4.31 ± 1.1 2.76 ± 0.01 527 ± 0.4 50 ± 0.4 32 ± 0.5 0.5 ± 0.01 25 ± 0.9 0.015 ± 0.03 96 ± 1.7 - - 89 ± 1.5 a concentration that inhibited radicals by 50%. b n = 6. c Trolox (50 μM) and d superoxide dismutase (SOD) (80 mU/mL) were used as standard; the results are expressed as % of inhibition. The preliminary in vitro biological analysis indicated that compounds 1-7 and 9-12 were able to quench DPPH radicals and exhibited a direct scavenging activity on superoxide anion; this radical was in fact produced by the reduction of β-mercaptoethanol, excluding the Fenton-type reaction and the xanthine/xanthine oxidase system (Table 3). Quercetin 3-O-(6''-caffeoyl)-β-D-glucopyranoside-3'- O-β-D-glucopyran-oside (1), quercetin 3'-methyl ether-3-O-α-L-rhamno-pyranoside (4), and 4'''-methyl ether amenthoflavone (9) exhibited the highest antioxidant capacity. On the other hand, the potent biological activity of quercetin is largely reported in literature [15]. Although both O2 .- and H2O2 are potentially cytotoxic, most of the oxidative damage in biological systems is caused by the . OH radical, which is generated by the reaction between O2 .- and H2O2 in the presence of transition metal ions [2]. In fact, the . OH radical can react with a number of target molecules including proteins, membrane lipids, and DNA. Table 4: Effect of methanol fractions and compounds 1-12 isolated from C. senegalensis (100 μg/mL) on DNA cleavage induced by the photolysis of H2O2 and metal chelating activity. UD of supercoiled DNA Ferrozine assay (% of native DNA) a IC50 (μg/mL) ± b SD scDNA 100 A 11 ± 2.4* - B 62.7 ± 3.7* 32 ± 4.5 C 78 ± 4.5* 47.6 ± 3.6 D 76.7 ± 2.6* 16.83 ± 2.5 E 95 ± 4.7* 19.74 ± 3.2 F 65 ± 4.6* 28.41 ± 0.9 1 36 ± 1.2* 13.65 ± 2.8 2 7 ± 1.6* 92 ± 1.9 3 9 ± 2.4* 25 ± 2.5 4 70 ± 2.7* 6.19 ± 0.19 5 10 ± 0.8* 44.64 ± 3.6 6 5 ± 0.9* - 7 37 ± 0.6* 630 ± 67 8 3.4 ± 0.4* 625 ± 50 9 73 ± 4.7* 18.31 ± 2.4 10 2.6 ± 0.6* - 11 15.3 ± 1.1* 222 ± 32 12 11.3 ± 3.1* - DMSO 75.3 ± 3.1* - c DTPA - 77.5 ± 2.3 The hydroxyl radicals generated by the photolysis of H2O2 inhibited the supercoiled DNA (SCDNA). Each value represents the mean ± SD of three experiments. *Significant vs. supercoiled DNA (p<0.001). a concentration that inhibited the ferrozine-Fe2+ formation by 50%. b n = 6. DTPA (5 μM) and DMSO (1mM) were used as standard; c the result is expressed as % of inhibition. Based on the data obtained from this study, compounds 1, 4, and 9 might also be able to modulate hydroxyl radical formation more efficiently than other compounds acting as direct scavengers and chelating iron. In fact, these natural compounds exhibited a more efficient protection against DNA strand scission induced by . OH radicals generated by UV-photolysis of H2O2 (Table 4), and showed metal

Flavonoid glycosides from Chrozophora senegalensis Natural product commnunications vol 1 02)2006 1093 chelating activity capturing ferrous ions before were purchased from GIBCO BRL Life Technologies (Grand Island.NY.USA). 18.31gm The air-dried (60 n-hexane and ex studies is due in part to the anti-oxidant action of its MeOH 9:1.and MeOH.The extracts wer active components ried residues, Experimental portion or the g)wa m wee wer sue with sodium lamp (589 nm)and a 1o cm mic were chromatog nhed over hadex H-20 Elemental analysis was obtained from a Carlo Erba column (100 cm x 5 cm)with MeoH as the eluent A 1106 elemental analyzer.UV spectra were recorded total of 115 fractions were collected (10 mL each) on a Perkin-Elme These were combined according to ILC analysi ambda 12 spectrophotometer.A spectromete the [silica 60 Fs gel-coated glass sheets with n-BuOH OH H O (60:15:25 and CHCl,-MeOH-H pac age was 4 using LC-Advan e obtain nd ne s319 spectrometer.equipped with Xcalibur software.TLC mg).4(40 mg).and 9 (30 mg ompoue.Fr A(90 mg)was purified by RP-HPLC using MeOH (Merck.Darmstadt,Germany): 10 were H2O (45:55)to give compounds 10 (6 mg. tp= 20 min).Fraction B (36 mg) detected by spraying Ce(SO4)/H2SO (Sigma min)and 12 (5 mg Aldrich, Lou was purified by RP-HPLC using MeOH- Caen-PEGPoethyleeghcol give compoun S Z in). mg. 89 mg LH-20 (Phapmy RP-HPL MeOH-H.O y ere conducted on Waters 515 and 7 (10 to= system eau ipped with a Waters 20 min).Fraction D (100 mg)was purified by 401 refractive index detector and Waters U6K iniector RP-HPLC Me0H-H20(45:55) to oive using a Cis u-Bondapak column (30 cm x 7.8 mm) compounds 1 (14.5 mg./8=10 min)and 6(6.5 mg and a mobile MeOH-H2O 20 min),while fraction E(70 mg)was purified b phase consisting of MeOH-H2O GC analyses (55:45)to yiel mixtures at a flow rate of 2 mL/mi mg.Ig 0mn). using a L p hi.Tk. MEOH-H-O1 ographed P.HPLC assay (5 mg.=28 min)and 4(6.3 mg.6 mim Plant material and rcolkcd The lea Ouercetin 3-0-(6"-caffeovl)-B-D-glucopyranoside. Bandiagara,Mali,in 1999 and identified by Prof. 3'-0-B-D-glucopyranoside(1) N'Golo Diarra of the Departement Medicine Yellow amorphous nowder Traditionelle(DMT),Bam ako,Mali where a vouche [alp:-27(co 1 MeoH) specime DNA I-dipheny- UV/Vis max (MeOH)nm (log )267 (3.99),344 (DPPH) cryl-hyd (4.32) H NMR (600 MHz,CD;OD):Table 1 idyl-5 6-bis envl-sulfonic 124-triazine (ferrozine)were obtained from Sigm ESIMMR (600 ME CDOD):Table 1. Aldrich Co(St.Louis,USA):B-nicotinamide-adenine nV- C6H602oC,54.83H4.60.Found dinucleotide(NADH)was obtained from Boehringer Mannheim GmbH(Germany).All other chemicals

Flavonoid glycosides from Chrozophora senegalensis Natural Product Communications Vol. 1 (12) 2006 1093 chelating activity capturing ferrous ions before ferrozine, with an IC50 value (concentration that inhibited the ferrozine-Fe2+ by 50%) of 13.65, 6.19 and 18.31 μg/mL, respectively (Table 4). These data also suggest that the biological effect of C. senegalensis observed from ethnopharmacological studies is due in part to the anti-oxidant action of its active components. Experimental General: Optical rotations were measured on a Perkin-Elmer 241 polarimeter equipped with a sodium lamp (589 nm) and a 10 cm microcell. Elemental analysis was obtained from a Carlo Erba 1106 elemental analyzer. UV spectra were recorded on a Perkin-Elmer-Lambda 12 spectrophotometer. A Bruker DRX-600 NMR spectrometer using the UXNMR software package was used for NMR experiments. ESIMS (negative mode) were obtained using a Finnigan LC-Q Advantage Termoquest spectrometer, equipped with Xcalibur software. TLC was performed on precoated Kieselgel 60 F254 plates (Merck, Darmstadt, Germany); compounds were detected by spraying with Ce(SO4)2/H2SO4 (SigmaAldrich, St. Louis, Mo, USA) and NTS (Naturstoffe reagent)-PEG (Polyethylene glycol 4000) solutions. Column chromatography was performed over Sephadex LH-20 (Pharmacia); reversed-phase (RP) HPLC separations were conducted on a Waters 515 pumping system equipped with a Waters R401 refractive index detector and Waters U6K injector, using a C18 μ-Bondapak column (30 cm x 7.8 mm) and a mobile phase consisting of MeOH-H2O mixtures at a flow rate of 2 mL/min. GC analyses were performed using a Dani GC 1000 instrument. A Hitachi U-2000 spectrophotometer (Hitachi, Tokyo, Japan) was used for all antioxidant assays. Plant material and chemicals: The leaves of Chrozophora senegalensis were collected in Bandiagara, Mali, in 1999 and identified by Prof. N’Golo Diarra of the Departement Medicine Traditionelle (DMT), Bamako, Mali where a voucher specimen (DMT n. 0074 ) is deposited. pBR322 plasmid DNA, 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH), diethylenetriaminepentaacetic acid (DTPA) and 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonic acid)- 1,2,4-triazine (ferrozine) were obtained from Sigma Aldrich Co (St. Louis, USA); β-nicotinamide-adenine dinucleotide (NADH) was obtained from Boehringer Mannheim GmbH (Germany). All other chemicals were purchased from GIBCO BRL Life Technologies (Grand Island, NY, USA). Extraction and isolation: The air-dried powdered leaves of C. senegalensis (600 g) were defatted with n-hexane and extracted successively by exhaustive maceration (3 x 1 L, for 48 h) with CHCl3, CHCl3- MeOH 9:1, and MeOH. The extracts were concentrated under reduced pressure to afford 13.4, 14.0, 13.8, and 62.4 g of dried residues, respectively. A portion of the MeOH extract (27.0 g) was partitioned between n-BuOH and H2O to give a n-BuOH soluble portion (9.0 g); 5.0 g of this residue were chromatographed over a Sephadex LH-20 column (100 cm x 5 cm) with MeOH as the eluent. A total of 115 fractions were collected (10 mL each). These were combined according to TLC analysis [silica 60 F254 gel-coated glass sheets with n-BuOHAcOH-H2O (60:15:25) and CHCl3-MeOH-H2O (40:9:1)] to give nine pooled fractions (A-I). Fractions G, H, and I yielded compounds 3 (19.2 mg), 4 (40 mg), and 9 (30 mg), respectively. Fraction A (90 mg) was purified by RP-HPLC using MeOHH2O (45:55) to give compounds 10 (6 mg, tR= 10 min) and 12 (5 mg, tR= 20 min). Fraction B (36 mg) was purified by RP-HPLC using MeOH-H2O (1:1) to give compounds 2 (8 mg, tR= 10 min) and 11 (12 mg, tR= 20 min). Fraction C (50.5 mg) was purified by RP-HPLC using MeOH-H2O (45:55) to give compounds 5 (28 mg, tR= 10 min) and 7 (10.8 mg, tR= 20 min). Fraction D (100 mg) was purified by RP-HPLC using MeOH-H2O (45:55) to give compounds 1 (14.5 mg, tR= 10 min) and 6 (6.5 mg, tR= 20 min), while fraction E (70 mg) was purified by RP-HPLC using MeOH-H2O (55:45) to yield compound 3 (11 mg, tR= 10 min). Finally, fraction F (85 mg) was chromatographed on a RP-HPLC using MeOH-H2O (1:1) as the eluent to afford compounds 8 (5 mg, tR= 28 min) and 4 (6.3 mg, tR= 46 min). Quercetin 3-O-(6''-caffeoyl)-β-D-glucopyranoside- 3'-O-β-D-glucopyranoside (1) Yellow amorphous powder. [α]D:-27° (c 0.1, MeOH). UV/Vis λmax (MeOH) nm (log ε): 267 (3.99), 344 (4.32) 1 H NMR (600 MHz, CD3OD): Table 1. 13C NMR (600 MHz, CD3OD): Table 1. ESIMS: m/z 787 [M - H]- . Anal. Calcd for C36H36O20: C, 54.83; H, 4.60. Found C, 54.79; H 4.62

1094 Natural Product commnications vol 1 (12)2006 Vassallo et al. Quercetin 3-methyl ether-7-0-a-L 517 min at room te (50M0,a vitamin E,was d as a m(Me0 H)nm (log:265(3.92),356 (A 05 Scavenger effect on superoxide anion:Superoxide 'H NMR (600 MHz.CD OD):Table 2. anion was generated in vitro as described by Paoletti et al.[17].The assay mixture contained in a total C NMR(600 MHz CD OD):Table 2 of ESIMS:mk769M-H可， Anal.Calcd for C37H3sOis:C,57.66:H,4.97.Found die M NADH:25 C,57.68;H5.00 mM/ nds 7-0-(6"-p-coumaroyl)-B-D-glucopyrano absorhan sured at 340 nm oxide dismutase Yellow amornhous nowde (SOD)(80 mU/mL)was used as a standard. [alp:+11 (c0.1.MeOH). UV/Vis .Me0HDnm1og269(399)321 DNA cleavage induced by hydrogen peroxide UV- (3.76) photolysis: The experiments were pertormed,as 'H NMR (600 MHz,CD;OD):Table 2. previously reported [18].in a volume of 20 ul C NMR (600 MHz,CD3OD):Table 2. containing 33 uM in bp (base pair)of pBR32. ESIMS:m591M-H可 plasmid DNA in 5 mM phosphate saline buffer(pH :C,62.84:H,4.76.Found 4).and con t.Hopr Acid of c ounds 3 A solutio of ncentration of 2.5 mM.The reaction volumes were each compound (1-3.2.0 mg each)in I N HCI held in caps of polyethylene microcentrifuge tubes mL)was stirred at 80C in a stoppered reaction vial placed directly on the surface of a transilluminator for 4 h.After cooling,the solution was evaporated (8000 uw cm)at 300 nm.The samples were -(m irradiated for 5 min at room temperature. Afte silyl)imidaz ul of a mixture,containing 0.25% and the wa red at 60 drying the 5 min.Afte bromophenol blue,0.25%xylen yanol FF and 307 partitione wer added t the ated solutic then a sing an LCp -Chirsi-Val s buffe (0.32 mm25 m).Temperatures of the injector and (45 mM Tris-borate 1 mM EDTA)Untrea ted detector were 200C for both.A temperature gradient pBR322 plasmid was included as a control in each system was used for the oven,starting at 100C for 1 run of gel electrophoresis,conducted at 1.5 V/cm for min and increasing up to 180 )°C at a rate of5C/min 15 hours.Gel was stained in ethidium bromid Peaks of the were (1 ug/mL;30 min)and photographed on Polaroid- compa son with etention time Type 667 positive land film. ne intensity of eack was by so)(mM Pyridine dens ( ed was us as a standard Antioxidant activity in cell-free systems Metal chelating activity:The chelating of ferrou Quenching of DPPH:The free radical-scavenging ions by fractions and pure compounds was estimated by the ferrozine pure compoune assay 19].Briefly natural ed by their e I, compounds were added to a solution of 0.15 mM 0 mM The reaction was initiate by the addition of mixture containe 86 DPPH ferrozine and the mixture was shaken

1094 Natural Product Communications Vol. 1 (12) 2006 Vassallo et al. Quercetin 3-methyl ether-7-O-α-Lrhamnopyranosyl-(1→6)-(2''-p-coumaroyl)-β-Dglucopyranoside (2) Yellow amorphous powder. [α]D: +18° (c 0.1, MeOH). UV/Vis λmax (MeOH) nm (log ε): 265 (3.92), 356 (4.05). 1 H NMR (600 MHz, CD3OD): Table 2. 13C NMR (600 MHz, CD3OD): Table 2. ESIMS: m/z 769 [M - H]- . Anal. Calcd for C37H38O18: C, 57.66; H, 4.97. Found C, 57.68; H 5.00. Acacetin 7-O-(6''-p-coumaroyl)-β-D-glucopyranoside (3) Yellow amorphous powder. [α]D: +11° (c 0.1, MeOH). UV/Vis λmax (MeOH) nm (log ε): 269 (3.99), 321 (3.76). 1 H NMR (600 MHz, CD3OD): Table 2. 13C NMR (600 MHz, CD3OD): Table 2. ESIMS: m/z 591 [M - H]- . Anal. Calcd for C31H28O12: C, 62.84; H, 4.76. Found C, 62.80; H 4.80. Acid hydrolysis of compounds 1-3: A solution of each compound (1-3, 2.0 mg each) in 1 N HCl (1 mL) was stirred at 80°C in a stoppered reaction vial for 4 h. After cooling, the solution was evaporated under a stream of N2. Each residue was dissolved in 1-(trimethylsilyl)imidazole and pyridine (0.2 mL), and the solution was stirred at 60°C for 5 min. After drying the solution, the residue was partitioned between water and CHCl3. The CHCl3 layer was analyzed by GC using an L-CP-Chirasil-Val column (0.32 mm x 25 m). Temperatures of the injector and detector were 200°C for both. A temperature gradient system was used for the oven, starting at 100°C for 1 min and increasing up to 180°C at a rate of 5°C/min. Peaks of the hydrolysate were detected by comparison with retention times of authentic samples of L-rhamnose and D-glucose (Sigma Aldrich) after treatment with 1-(trimethylsilyl)imidazole in pyridine. Antioxidant activity in cell-free systems Quenching of DPPH: The free radical-scavenging capacity of extracts, fractions and pure compounds was tested by their ability to bleach the stable 1,1- diphenyl-2-picrylhydrazyl radical (DPPH) [16]. The reaction mixture contained 86 μM DPPH and different concentrations of the natural compounds in 1 mL of ethanol. After 10 min at room temperature the absorbance at λ = 517 nm was recorded. Trolox (50 μM), a water-soluble derivative of vitamin E, was used as a standard. Scavenger effect on superoxide anion: Superoxide anion was generated in vitro as described by Paoletti et al. [17]. The assay mixture contained in a total volume of 1 mL, 100 mM triethanolaminediethanolamine buffer, pH 7.4, 3 mM NADH, 25 mM/12.5 mM EDTA/MnCl2, 10 mM β-mercaptoethanol; some samples contained the natural compounds at different concentrations. After 20 min incubation at 25°C, the decrease in absorbance was measured at λ = 340 nm. Superoxide dismutase (SOD) (80 mU/mL) was used as a standard. DNA cleavage induced by hydrogen peroxide UVphotolysis: The experiments were performed, as previously reported [18], in a volume of 20 μl containing 33 μM in bp (base pair) of pBR322 plasmid DNA in 5 mM phosphate saline buffer (pH 7.4), and the natural compounds at different concentrations. Immediately prior to irradiating the samples with UV light, H2O2 was added to a final concentration of 2.5 mM. The reaction volumes were held in caps of polyethylene microcentrifuge tubes, placed directly on the surface of a transilluminator (8000 μW cm-1) at 300 nm. The samples were irradiated for 5 min at room temperature. After irradiation 4.5 μl of a mixture, containing 0.25% bromophenol blue, 0.25% xylen cyanol FF, and 30% glycerol, were added to the irradiated solution. The samples were then analyzed by electrophoresis on a 1% agarose horizontal slab gel in Tris-borate buffer (45 mM Tris-borate, 1 mM EDTA). Untreated pBR322 plasmid was included as a control in each run of gel electrophoresis, conducted at 1.5 V/cm for 15 hours. Gel was stained in ethidium bromide (1 μg/mL; 30 min) and photographed on PolaroidType 667 positive land film. The intensity of each scDNA band was quantified by means of densitometry. Dimethylsulfoxide (DMSO) (1 mM) was used as a standard. Metal chelating activity: The chelating of ferrous ions by fractions and pure compounds was estimated by the ferrozine assay [19]. Briefly, natural compounds were added to a solution of 0.15 mM FeSO4. The reaction was initiated by the addition of 0.5 mM ferrozine and the mixture was shaken

Flavonoid glycosides from Chrozophora senegalensis Natural Product Communications Vol. 1 (12) 2006 1095 vigorously and left standing at room temperature for ten minutes. After the mixture had reached equilibrium, the absorbance of the solution was then measured spectrophotometrically at 562 nm. DTPA (5 μM) was used as a standard. Supplementary data: NMR spectral data for quercetin 3'-methyl ether-3-O-α-L-rhamno-pyranoside (4), quercetin 3'-methyl ether-3-O-α-Lrhamnopyranosyl-(1→6)-β-D-glucopyranoside (5), apigenin 7-O-(6''-p-coumaroyl)-β-D-glucopyranoside (6), quercetin 3-methyl ether-7-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (7), 4- hydroxyphenyl-O-α-L-rhamnopyranosyl-(1→6)-β-Dglucopyranoside (8), 4'''-methyl ether amenthoflavone (9), roseoside (10), icariside B5 (11), and ampelopsisionoside (12). Acknowledgments - This work was supported by the “Bioactive Compounds from Medicinal and Food Plants of Developing Countries” project of the Italian Ministry for University and Research (Ministero dell’Università e della Ricerca, MIUR). References [1] Devi RS, Narayan S, Mohan KV, Sabitha KE, Devi CS. (2003) Effect of a polyherbal formulation, Ambrex, on butylated hydroxy toluene (BHT) induced toxicity in rats. Indian Journal of Experimental Biology, 41, 1294-1299. [2] Halliwell B, Gutteridge JMC. (1999) Free radicals in biology and medicine. In: Studies of Generalized Light Emission (Luminescence/Fluorescence). 3rd Ed. Oxford: University Press, 387-388. [3] Kerharo J, Adam JC. (1974) Pharmacopée Sénégalaise Traditionelle: Plantes Médicinales et Toxicologiques. Vigot et Frères Ed, Paris, 1011. [4] Neuwinger HD. (2000) African Traditional Medicine. A Dictionary of Plant Use and Application. Medpharm Scientific Publisher. [5] Agrawal PK. (1989) Carbon-13 NMR of Flavonoids. Elsevier Science, Amsterdam, 294-364. [6] Wolbis M. (1989) Flavonol glycosides from Sedum album. Phytochemistry, 28, 2187-2189. [7] Markham KR, Ternai B, Stanley R, Geiger H, Mabry TJ. (1978) Carbon-13 NMR studies of flavonoids. III. Naturally occurring flavonoid glycosides and their acylated derivatives. Tetrahedron, 34, 1389-1397. [8] Itokawa H, Suto K, Takeya K. (1981) Studies on a novel p-coumaroyl glucoside of apigenin and on other flavonoids isolated from patchouli (Labiatae). Chemical & Pharmaceutical Bulletin, 29, 254-256. [9] Nawwar MAM, El-Mousallamy AMD, Barakat, HH, Buddrus J, Linscheid M. (1989) Flavonoid lactates from leaves of Marrubium vulgare. Phytochemistry, 28, 3201-3206. [10] De Tommasi N, Pizza C, Aquino R, Cumandà J, Mahmood N. (1997) Flavonol and chalcone ester glycosides from Bidens leucantha. Journal of Natural Products, 60, 270-273. [11] Markham KR, Sheppard C, Geiger H. (1987) Carbon-13 NMR of flavonoids. Part IV. Carbon-13 NMR studies of some naturally occurring amentoflavone and hinokiflavone biflavonoids. Phytochemistry, 26, 3335-3337. [12] Otsuka H, Takeda Y, Yamasaki K, Takeda Y. (1992) Structural elucidation of dendranthemosides A and B: two new β-ionone glucosides from Dendranthera shiwogiku. Planta Medica, 58, 373-375. [13] Miyase T, Ueno A, Takizawa N, Kobayashi H, Oguchi H. (1988) Studies on the glycosides of Epimedium grandiflorum Morr. var. thunbergianum (Miq.) Nakai. III. Chemical & Pharmaceutical Bulletin, 36, 2475-2484. [14] Inada A, Nakamura Y, Konishi M, Murata H, Kitamura F, Toya H, Nakanishi T. (1991) A new ionone glucoside and a new phenylpropanoid rhamnoside from stems of Ampelopsis brevipedunculata (maxim.) Trautv. Chemical & Pharmaceutical Bulletin, 39, 2437-2439. [15] Di Carlo G, Mascolo N, Izzo AA, Capasso F. (1999) Flavonoids: old and new aspects of a class of natural therapeutic drugs. Life Sciences, 65, 337-353. [16] Bonina F, Saija A, Tomaino A, Lo Cascio R, Rapisarda P, Dederen JC. (1998) In vitro antioxidant activity and in vivo photoprotective effect of a red orange extract. International Journal of Cosmetic Sciences, 20, 331-342. [17] Paoletti F, Aldinucci D, Mocalli A, Caparrini A. (1986) A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts. Analitycal Biochemistry, 154, 536-541. [18] Russo A, Cardile V, Lombardo L, Vanella L, Vanella A,Garbarino JA. (2005) Antioxidant activity and antiproliferative action of methanolic extract of Geum quellyon Sweet roots in human tumor cell lines. Journal of Ethnopharmacology, 100, 323-332. [19] Dinis TC, Madeira VM, Almeida LM. (1994) Action of phenolic derivates (acetoaminophen, salicylate and 5-aminosalycilate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Archives of Biochemistry & Biophysics, 315, 161-169