《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_P21-P25

1086 Natural Product Communications Vol.1(12)2006 Speranzaetal isolated from the enzymatic hydrolysis of 1 copyd the CD sp of pep-br mixture of the tetra-esters was found to be coinciden with that of the corresponding mixture of ester prepared from an authentic sample of D-glucose and in agreement with data reported for a-and B-D- glucopyranosides tetra-p-bromobenzoates [9,10]. (a-L-themmno Treatment of the diglycoside 2 with a-rhamnosidase om --ylpyares and CD Tha is involyed in ar the 8o-nosition of the r 2 stems from NOE correlations and coupling 5mnA0cha15304 constants of the anomeric proton of this hexopyranose (Table 1).Finally,the crude established monoglycoside isolated from the rhamnosidase catalyzed on the basis of the NOE association between H-4 and hydrolysis separation was neate HO Ar-CH2-O-protons and of the correlation observed in on and the HMBC This identified as D-xylose in agreement with NMR data (C2) of 1 and 2 (Table 1),indicating the presence of a B-xylopyranoside residue [12].Therefore. the structure of kenvaloside was concluded to be 1-(B-D otad that th glucopyranosyloxy)-8-(a-L-rhamnopyranosyloxy)-3- the o of the kemvaloside (B-D- kylopyranosyl-oxmethyl)naphthalene(1). biogenetic considerations suggesting the cyclization Experimental of a decarboxylated polyketide (heptaketide)chain as the key step in the formation of the naphthalen nucleus [7]. mbiguous tion or h their CD spectra 1-500 ent.and IR spectra on a Perkin-Elmer FT-IR 1725X spectrometer.NMR spectra were recorded on and hydrolysis experiments,as follows. a Bruker AVANCE 400 Spectrometer using a XWIN- NMR software package;chemical shifts()are given When 1 was submitted to B-glucosidase-catalyzed in ppm and were referenced to the CDOD signals hydrolysis,a diglycoside(2)was obtained showing in (u 3.30.c 49.0).ESI-HRMS spectra were acquired its NMR spectrum the loss of the B-glucopyranosyl residue the 1-position the and spectra on (H7)nd ofth F TLC W ed anta methyl upfi alun the remain unchanged.The exp ected but not axiomatic eluents: 4.EtOAc-EtOH-H2O.100:20:13:B. [8].D-configuration of glucose was proven by BuOH-AcOEt-H2O,7:2:1;components were detected application of the exciton chirality method developed under an UV lamp and by spraying with either 0.5% by Nakanishi to characterize methyl glycosides at the Fast Blue B salt (phenols)or with 4%ceric sulfate nano-gram level [9,10].The monosaccharide ammonium molybdate solution (sugars).followed by

1086 Natural Product Communications Vol. 1 (12) 2006 Speranza et al. The existence of an Ar-CO-CH3 (δH 1.95, δC 30.84, 206.70) and of an Ar-glycosyloxymethyl group in 2- and 3-positions, respectively, could be established on the basis of the NOE association between H-4 and Ar-CH2-O- protons and of the correlation observed in the HMBC spectrum between the glycosyloxymethyl protons and the aromatic carbon linked to the acetyl group (C-2). Such an assumption was in agreement with the strong similarity in the UV and IR spectra of 1 with those of dianellidin dimethyl ether (3) [6]. In addition, it can be noted that the carbon skeleton and the oxygenation pattern of the kenyaloside aglycone (1, where R1 = R2 = R3 = H) are both consistent with biogenetic considerations suggesting the cyclization of a decarboxylated polyketide (heptaketide) chain as the key step in the formation of the naphthalene nucleus [7]. The unambiguous identification of the three monosaccharides, including their absolute configuration, the orientation of the glycosidic bond, and the location of each of them on the aglycone moiety, resulted from combined NMR spectral data and hydrolysis experiments, as follows. When 1 was submitted to β-glucosidase-catalyzed hydrolysis, a diglycoside (2) was obtained showing in its NMR spectrum the loss of the β-glucopyranosyl residue from the 1-position of the naphthalene nucleus (Table 1). In fact, both chemical shifts and NOE correlations of the upfield aromatic proton (H-7) and of the methylene protons at 3-position remain unchanged. The expected, but not axiomatic [8], D-configuration of glucose was proven by application of the exciton chirality method developed by Nakanishi to characterize methyl glycosides at the nano-gram level [9, 10]. The monosaccharide isolated from the enzymatic hydrolysis of 1 was converted into the mixture of methyl α- and β- glucopyranosides, which were per-p-bromobenzoylated: the CD spectrum of the resulting mixture of the tetra-esters was found to be coincident with that of the corresponding mixture of esters prepared from an authentic sample of D-glucose and in agreement with data reported for α- and β-Dglucopyranosides tetra-p-bromobenzoates [9, 10]. Treatment of the diglycoside 2 with α-rhamnosidase from Fusarium oxysporum [11] gave L-rhamnose, as demonstrated by derivatization of the sugar and CD spectra comparison, as described above. [9, 10]. That L-rhamnose is involved in an α−glycosidic linkage at the 8-O-position of the naphthalene nucleus in 1 and 2 stems from NOE correlations and coupling constants of the anomeric proton of this hexopyranose (Table 1). Finally, the crude monoglycoside isolated from the rhamnosidasecatalyzed hydrolysis of 2, after separation of L-rhamnose, was heated in HCl solution and the released sugar processed and analyzed according to Nakanishi’s method [9, 10]. This pentose was identified as D-xylose in agreement with NMR data of 1 and 2 (Table 1), indicating the presence of a β-xylopyranoside residue [12]. Therefore, the structure of kenyaloside was concluded to be 1-(β-Dglucopyranosyloxy)-8-(α-L-rhamnopyranosyloxy)-3- (β-D-xylopyranosyl-oxmethyl)naphthalene (1). Experimental General experimental techniques: Optical rotations were measured on a Jasco P-1030 polarimeter, UV spectra on a Hewlett Packard 8452A Diode Array Spectrophotometer, CD spectra on a Jasco J-500 instrument, and IR spectra on a Perkin-Elmer FT-IR 1725 X spectrometer. NMR spectra were recorded on a Bruker AVANCE 400 Spectrometer using a XWINNMR software package; chemical shifts (δ) are given in ppm and were referenced to the CD3OD signals (δH 3.30, δC 49.0). ESI-HRMS spectra were acquired on a Bruker Daltonics FT-ICR APEX-II mass spectrometer and ESI MS spectra on a ThermoFinnigan LCQ Advantage instrument. Analytical TLC was performed on silica gel 60 F254 aluminum sheets (Merck) using the following eluents: A, EtOAc-EtOH-H2O, 100:20:13; B, nBuOH-AcOEt-H2O, 7:2:1; components were detected under an UV lamp and by spraying with either 0.5% Fast Blue B salt (phenols) or with 4% ceric sulfate/ ammonium molybdate solution (sugars), followed by OR1 R2 O CH2-OR3 CH3 O 8 1 5 4 1 : R1 = O H HO H HO H H H OH OH (β-D-glucopyranosyl) O H HO H HO H H H OH H HO H H3C H HO O H OH H R2 = R3 = (α-L-rhamnopyranosyl) (β-D-xylopyranosyl) 2 : R1 = H R2 , R3 as in 1 3 : R1 = R2 = CH3 OR3 = H dimethyldianellidin 1' 1" 1

Naphthalene triglycoside from Kenyan Aloe species Natural Product Communications Vol. 1 (12) 2006 1087 Table 1: NMR data of compounds 1 and 2 in CD3OD at 400 MHz (1 H) and 100 MHz (13C).a, b kenyaloside (1) Compound 2 C/H position δΗ (J, Hz) δC Selected 1 H-1 H NOEs δΗ (J, Hz) δC Selected 1 H-1 H NOEs 1 152.69 152.56 2 123.06 122.96 3 134.10 134.12 4 7.48 s 119.77 7.52; 4.73, 4.94; 4.31 7.47 s 119.73 7.51; 4.71, 4.93 4a 137.03 137.05 5 7.52 d (8.0) 122.94 7.48; 7.45 7.51 dd (8.3, 1.1) 122.83 7.47; 7.45 6 7.45 dd (7.6, 8.0) 127.97 7.52; 7.33 7.45 dd (7.8, 8.3) 127.97 7.51; 7.32 7 7.33 d (7.6) 109.92 7.45; 5.77 7.32 dd (7.8, 1.1) 109.91 7.45; 5.75 8 153.68 153.66 8a 114.74 114.71 COCH3 206.70 206.59 COCH3 1.95 s 30.84 1.89 31.62 CH2O 4.73 d (12.4) 4.94 d (12.4) 68.80 4.31, 7.48 4.71 d (12.4) 4.93 d (12.4) 68.78 4.27, 7.47 1’ 4.39 d (8.0) 102.46 2’ 3.23 dd (8.0, 8.8) 73.67 3’ 3.33 me 76.85c 4’ 3.33 me 70.67d 5’ 3.33 me 77.10c 6’ 3.67 dd (5.2, 12.0) 3.89 dd (1.6, 12.0) 61.64 1” 5.77 d (1.8) 100.85 7.33 5.75 d (1.9) 100.83 7.32 2” 4.21 dd (1.8, 3.4) 70.56 4.20 dd (1.9, 3.5) 70.66 3” 3.85 dd (3.4, 9.2) 71.58 3.84 dd (3.5, 9.3) 71.55 4” 3.57 t (9.2) 72.42 3.56 t (9.3) 72.40 5” 3.71 m 70.72d 3.70 m 70.71 CH3(5”) 1.31 d (6.4) 17.02 1.29 d (6.1) 17.02 1”’ 4.31 d (7.2) 103.09 4.73, 4.94, 7.48 4.27 d (7.3) 103.30 4.71, 4.93 2”’ 3.29 dd (7.2, 8.8) 73.82 3.23 dd (7.3, 9.0) 73.97 3”’ 3.50 t (8.8) 75.11 3.35 t (9.0) 76.85 4”’ 3.71 m 77.65 3.50 m 70.23 5”’ 3.33 me 4.06 dd (5.2, 12.0) 63.58 3.19 dd (10.1, 11.5) 3.88 dd (5.4, 11.5) 65.94 a Spectra recorded at 40°C; b all assignments were based on extensive 1D and 2D NMR measurements (COSY, TOCSY, NOESY, APT, HMQC and HMBC); c,d signals with the same superscript are interchangeable; e covered by the CH3OH signal. heating at 150°C. Silica gel 60, 63-200 μm and 40-63 μm (Merck) was used for column and flash chromatography, respectively. Plant material: The commercial exudate of Kenyan Aloe species used in this investigation was purchased from Sessa Carlo spa (Sesto S. Giovanni, Italy). A voucher specimen is kept at the Dipartimento di Chimica Organica e Industriale, Università di Milano Extraction and isolation: The dried exudate of Kenyan Aloe species (250 g) was finely powdered and extracted with water (750 mL) with vigorous mechanical stirring for 24 h at room temperature. After filtration of the insoluble material, the aqueous solution was partitioned with ethyl acetate (2 x 1 L) and lyophilized to give a brown residue (120 g). Of this residue, 40 g was adsorbed onto sea sand and fractioned by flash chromatography (silica gel, 1.5 Kg) eluting with EtOAc containing increasing amounts of MeOH. Separation was monitored by TLC (eluent A) and fractions containing 1 (Rf 0.38) were combined, concentrated (3.5 g) and further purified by flash chromatography (silica gel, 500 g) eluting with EtOAc-EtOH-H2O, 100:20:10. Fractions were combined on the basis of TLC analysis (eluent A) and evaporated to dryness. The residue (ca. 400 mg) was chromatographed over a Sephadex LH-20 column eluted with MeOH-H2O (1:1) to give kenyaloside (1) (200 mg, 0.08% overall yield) as an amorphous powder, pure by TLC (eluent A). Kenyaloside [1-(β-D-glucopyranosyloxy)-8-(α-Lrhamnopyranosyloxy)-3-(β-D-xylopyranosyloxymethyl)naphthalene (1)] [α]D:- 84.4º (c 0.25, MeOH). Rf : 0.38 (AcOEt-EtOH-H2O, 100:20:13). IR (KBr): 1695, 1652,1615 cm-1. UV/Vis λmax (MeOH) nm (log ε): 226 (4.72), 260 (4.36), 290sh (4.30), 338 (3.94) [for dimethyl dianellidin (3) [6]: 223 (4.68), 253 (4.04), 331 (3.61)]. 1 H NMR (400 MHz, CD3OD): Table 1. 13C NMR (100 MHz, CD3OD): Table 1. ESI-HRMS: m/z [M + Na+ ] calcd for C30H40NaO17 695.21577, found 695.21326. ESI MS: m/z 695 [M + Na+ ], 549 [M-146+Na+ ]. Enzymatic hydrolyses: β-Glucosidase (almond emulsin, Sigma, 30 mg) was added to a solution of

1088 Natural Product Communications Vol. 1 (12) 2006 Speranza et al. kenyaloside (1, 50 mg) in H2O (25 mL), and the mixture was incubated at 37° for 3 h under nitrogen. After adding MeOH, the solution was filtered and concentrated under reduced pressure. Column chromatography of the aqueous residue (eluent: EtOAc-EtOH-H2O, 100:20:13) gave two fractions. The less polar fraction, after further purification by column chromatography eluting with AcOEt-EtOH (from 5:1 to 1:1), furnished the diglycoside 2 (30 mg, 79%) as a pale yellow powder. The more polar fraction was submitted to column chromatography (eluent AcOEt-EtOH, from 3:1 to 1:1) to give glucose (9 mg, 67%), identified by TLC comparison with an authentic sample (eluent B, Rf 0.31). Compound 2 (20 mg), dissolved in 50 mM phosphate buffer pH 6 (5 mL), was incubated with α-rhamnosidase from Fusarium oxyporum CCF 906 (0.2 U) [11] at 35°C for 24 h. After concentration, MeOH was added, the precipitate removed by filtration and the solvent evaporated under reduced pressure. Repeated column chromatographic purification (eluent AcOEt-EtOH, from 5:1 to 1:1 and from 3:1 to 1:1) furnished rhamnose (5 mg, Rf 0.62, eluent B, co-TLC with an authentic sample) and a yellow product (10 mg, Rf 0.73, eluent A). This was hydrolyzed without further purification (0.1 N HCl, dioxane-H2O, 1:1, 5 mL; 70°C, 5 h, under nitrogen) to give xylose (Rf 0.50, eluent B, co-TLC with an authentic sample), which was purified (1.5 mg) by column chromatography (eluent AcOEtEtOH, from 3:1 to 1:1). 8-(α-L-Rhamnopyranosyloxy)-3-(β-Dxylopyranosyloxymethyl)naphthalen-ol (2) [α]D:- 36.2º (c 0.07, MeOH). Rf : 0.54(AcOEt-EtOH-H2O, 100:20:13). IR (KBr): 1635 cm-1. UV/Vis λmax (MeOH) nm (log ε): 225 (4.49), 258sh (4.09), 297 (3.99), 334 (3.80). 1 H NMR (400 MHz, CD3OD): Table 1. 13C NMR (100 MHz, CD3OD): Table 1. ESI MS: m/z 533 [M + Na+ ], 387 [M-146+Na+ ]. Determination of the absolute configuration of the isolated sugars: The isolated monosaccharides were converted into methyl glycopyranosides followed by treatment with excess p-bromobenzoyl chloride, as in ref. 9. Comparison of the CD spectra of the resulting per-p-bromobenzoates with those of the analogous derivatives prepared from authentic samples allowed the D-configuration for glucose and xylose and the Lconfiguration for rhamnose to be established. Acknowledgments – Thanks are due to Dr Lavinia Durì for technical assistance and to MIUR for financial support. References [1] Speranza G, Morelli CF, Tubaro A, Altinier G, Durì L, Manitto P. (2005) Aloeresin I, an anti-inflammatory 5-methylchromone from Cape aloe. Planta Medica, 71, 79-81. [2] Trease GE, Evans WC. (1983) Pharmacognosy, Baillière Tindall, London, 404-408. [3] Durì L, Morelli CF, Crippa S, Speranza G. (2004) 6-Phenylpyrones and 5-methylchromones from Kenya aloe. Fitoterapia, 75, 520-522. [4] Reynolds T. (2004) Aloe chemistry. In Aloes. The genus Aloe. Reynolds T (Ed). CRC Press, Boca Raton, USA. 39-74. [5] Dagne E, Bisrat D, Viljoen A, Van Wyk B-E. (2000) Chemistry of Aloe species. Current Organic Chemistry, 4, 1055-1078. [6] Batterham T, Cooke RG, Duewell H, Sparrow LG. (1961) Colouring matters of Australian plants. VIII. Naphthalene derivatives from Dianella species. Australian Journal of Chemistry, 14, 637-642. [7] Speranza G, Corti S, Manitto P. (1994) Isolation and chemical characterization of a new constituent of Cape aloe having the 1,1-diphenylethane skeleton. Journal of Agricultural and Food Chemistry, 42, 2002-2006. [8] (a) Buckingham J. (2005) Dictionary of Natural Products on CD-ROM. Chapman & Hall/CRC, England; (b) Editorial (1997) Planta Medica, 63, 195. [9] Golik J, Liu H-W, Dinovi M, Furukawa J, Nakanishi K. (1983) Characterization of methyl glycosides at the pico- to nano-gram level. Carbohydrate Research, 118, 135-146. [10] Nakanishi K, Kuroyanagi M, Nambu H, Oltz EM, Takeda R, Verdine GL, Zask A. (1984) Recent application of circular dichroism to structural problems, especially oligosaccharide structures. Pure and Applied Chemistry, 56, 1031-1048. [11] Monti D, Pišvejcová A, Křen V, Lama M, Riva S. (2004) Generation of an α-L-rhamnosidase library and its application for the selective derhamnosylation of natural products. Biotechnology and Bioengineering, 87, 763-771. [12] Pham TN, Hinchley SL, Rankin DWH, Liptaj T, Uhrínpp D. (2004) Determination of sugar structures in solution from residual dipolar coupling constants: methodology and application to methyl β-D-xylopyranoside. Journal of the American Chemical Society, 126, 13100-13110

New Flavonoid Glycosides from Chrozophora senegalensis and Their Antioxidant Activity Antonio Vassalloa , Giuseppina Cioffia , Francesco De Simonea , Alessandra Bracab , Rokia Sanogoc , Angelo Vanellad , Alessandra Russod and Nunziatina De Tommasia* a Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy b Dipartimento di Chimica Bioorganica e Biofarmacia, Università di Pisa, Via Bonanno 33, 56126 Pisa, Italy c Departement Medicine Traditionelle (DMT), INRSP, B.P. 1746, Bamako, Mali d Dipartimento di Chimica Biologica, Chimica Medica e Biologia Molecolare, Università di Catania, v.le A. Doria 6, 95125 Catania, Italy detommasi@unisa.it Received: June 27th, 2006; Accepted: September 27th, 2006 Dedicated to the memory of Professor Ivano Morelli. Bioassay-directed fractionation of an antioxidant methanol extract of the leaves of Chrozophora senegalensis using DPPH assay led to the isolation of three new flavonoid glycosides, quercetin 3-O-(6''-caffeoyl)-β-D-glucopyranoside-3'-O-β-Dglucopyranoside (1), quercetin 3-methyl ether-7-O-α-L-rhamnopyranosyl-(1→6)-(2''-p-coumaroyl)-β-D-glucopyranoside (2), acacetin 7-O-(6''-p-coumaroyl)-β-D-glucopyranoside (3), along with five known flavonoids, one phenolic derivative, and three megastigmane glycosides. Their structures were established on the basis of detailed spectral analysis. All isolated compounds were tested for their antioxidant activity on DPPH stable radical, superoxide anion, metal chelating activity, and DNA cleavage induced by the photolysis of H2O2. Quercetin 3-O-(6''-caffeoyl)-β-D-glucopyranoside-3'-O-β-D-glucopyranoside (1), quercetin 3'-methyl ether-3-O-α-L-rhamnopyranoside (4), and 4'''-methyl ether amenthoflavone (9) exhibited the highest antioxidant capacity being also able to modulate hydroxyl radical formation more efficiently than other compounds acting as direct hydroxyl radical scavengers and chelating iron. Keywords: Chrozophora senegalensis, Euphorbiaceae, flavonoids, antioxidant activity. In recent years, a global trend toward the use of natural phytochemicals present in herbs and functional foods as antioxidants was further increased after that it had been reported that some commonly used synthetic antioxidant compounds, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole, have long-term toxicological effects, including carcinogenicity [1]. Of particular interest as possible sources of natural antioxidants are medicinal plants traditionally used to treat conditions related to oxidative stress, such as rheumatism and inflammation. In this regard, many phytochemicals with diversified biological properties have shown promise for the prevention and/or treatment of all diseases in which oxidative stress plays a key role [2]. Chrozophora senegalensis (Lam) A Juss. ex Spreng, syn. Croton senegalensis (Euphorbiaceae family) is a small tree widely distributed in Mali where it grows wild and is used in folk medicine for the treatment of diarrhea, rheumatism, teniasis, stomachache, rachitis, and venereal diseases. The leaf and root decoctions are also drunk for hairloss [3, 4]. To confirm the use of C. senegalensis in Malian traditional medicine, the extracts of the leaves were evaluated for in vitro antioxidant activity. A bioassay-guided fractionation procedure showed that NPC Natural Product Communications 2006 Vol. 1 No. 12 1089 - 1095

1090 Natural Product Communications Vol.1(12)2006 Vassallo et al. extract was the active one.while all the Table 1:'Hand"CNMR dataofco d 1(CD.OD.600 MHzr res u were nactive ow) methanol extract led to the isolation and structural characterization of three new flavonoids (1-3). together with some known compounds,including five 6- 6.16d(15) flavonoids (4-7 and 9).one phenolic derivative (8). 6.33d15 and three megastigmane glycosides (10-12) 901 06% 7.67d(2.5) C-DEPT NMR and elemental analysis ans of ESI-MS ([M-H]peak at m/ -3456 Analysis of 600 MHz NMR spectra suggested a flavonoid skeleton for compound 1. TheH-NMR 3-0-Gls 358 patter for ring A (two me .0 6.16 and 6.33 395dd(5. 12.00 3-0.G Hz 2.5Hz I51 TheH-NM 9.0 trans-safl 4.32dd(5.0120 and a caffeoyl residue (Table 1).Two anomeric protons arising from the sugar moieties appeared at 7.00d1.5 6 5.26 and 4.88 each (IH,d,J=7.5 Hz),which 15 correlated respectively with signals at 8 103.4 and 681d(8.8 104.7 ppm in the HSQC spectrum.All the 'H-and 456 signals of were assignec ing 6206 ID-TOCS 7.39d(160 HSQC and HMB experime ts ncn飞 of pron accomplished by DOF-COSY and 1D-TOCSY experiments and allowed the identification of the BC.DEPT NMR analyses and was st sugars as two terminal B-D-glucopyranosvl units.The configurations of the sugar nits were assigned after hydrolysis of 1 with 1 N HCI.The hydrolysate was ether derivative [5]. displayed signal for and GC retention times compared tha with the The lower field shifts of H2-6(4.32 experiments, 0 the fication of e B-D-g one glucosy ti information could be ed by 2D-NMR was deter orted fo nd was the HMBC exneriment indicated 1.The presence of one p-coumarovl moietv between85.26(H-1"and135.6(C-3).δ4.88(H-1") shown in the H-NMR spectrum by the signals at and149.0(C-3).84.32and4.23(H2-6")and170.0 7.45 and 6.73 each (2H.d.J=8.5 Hz)and 57.41 and (COO).Thus.the structure of 1 was determined as 6.38 each (IH,d,J=16.0 Hz).The HSQC spectrum quercetin 3-0-(6"-caffeoyl)-B-D-glucopyranoside-3'- showed glycosidation shifts for C-6"(8 67.5)and O-B-D-glucopyranoside. acylation shift for H-2"(8 4.74)and C-2"(74.5)of the B-D-glucopyranosyl unit.An unambiguous The molecular formula CHOs for compound 2 determination of the sequence and linkage sites was was determined by ESI-MS ([M-HT at m769).C. n c

1090 Natural Product Communications Vol. 1 (12) 2006 Vassallo et al. the methanol extract was the active one, while all the other residues were inactive (data not shown). Subsequent fractionation and analysis of the methanol extract led to the isolation and structural characterization of three new flavonoids (1-3), together with some known compounds, including five flavonoids (4-7 and 9), one phenolic derivative (8), and three megastigmane glycosides (10-12). Compound 1 was isolated as a yellow amorphous powder. Its molecular formula was established as C36H36O20 by means of ESI-MS ([M-H]- peak at m/z 787), 13C, 13C-DEPT NMR, and elemental analysis. Analysis of 600 MHz NMR spectra suggested a flavonoid skeleton for compound 1. The 1 H-NMR spectrum (Table 1) indicated a 5,7-dihydroxylated pattern for ring A (two meta-coupled doublets at δ 6.16 and 6.33, J = 1.5 Hz) and a 3’,4’- dihydroxylation pattern for ring B (ABX system signals at δ 6.80, d, J = 8.5 Hz; 7.58, dd, J = 8.5, 2.5 Hz; 7.67, d, J = 2.5 Hz), allowing the aglycon to be recognized as quercetin [5]. The 1 H-NMR spectrum of 1 also showed signals ascribable to sugar moieties and a caffeoyl residue (Table 1). Two anomeric protons arising from the sugar moieties appeared at δ 5.26 and 4.88 each (1H, d, J = 7.5 Hz), which correlated respectively with signals at δ 103.4 and 104.7 ppm in the HSQC spectrum. All the 1 H- and 13C-NMR signals of 1 were assigned using 1D-TOCSY, DQF-COSY, HSQC, and HMBC experiments. Complete assignments of proton and carbon chemical shifts of the sugar portion were accomplished by DQF-COSY and 1D-TOCSY experiments and allowed the identification of the sugars as two terminal β-D-glucopyranosyl units. The configurations of the sugar units were assigned after hydrolysis of 1 with 1 N HCl. The hydrolysate was trimethylsilylated, and GC retention times compared with those of authentic sugar samples prepared in the same manner. The lower field shifts of H2-6''' (δ 4.32 and 4.23) of one glucosyl unit suggested the substitution site of the caffeoyl moiety. Unequivocal information could be obtained by 2D-NMR spectra; the HMBC experiment indicated correlations between δ 5.26 (H-1''') and 135.6 (C-3), δ 4.88 (H-1'') and 149.0 (C-3'), δ 4.32 and 4.23 (H2-6''') and 170.0 (COO). Thus, the structure of 1 was determined as quercetin 3-O-(6''-caffeoyl)-β-D-glucopyranoside-3'- O-β-D-glucopyranoside. The molecular formula C37H38O18 for compound 2 was determined by ESI-MS ([M-H]- at m/z 769), 13C, Table 1: 1 H and 13C NMR data of compound 1 (CD3OD, 600 MHz)a . position δH δC 2 159.0 3 135.6 4 179.0 5 163.5 6 6.16 d (1.5) 100.0 7 166.3 8 6.33 d (1.5) 94.2 9 159.0 10 105.8 1' 123.1 2' 7.67 d (2.5) 117.2 3' 149.0 4' 146.4 5' 6.80 d (8.5) 116.0 6' 7.58 dd (2.5, 8.5) 123.5 3'-O-Glc 1'' 4.88 d (7.5) 104.7 2'' 3.58 dd (7.5, 9.0) 74.8 3'' 3.52 t (9.0) 77.3 4'' 3.42 t (9.0) 71.2 5'' 3.53 m 78.4 6''a 3.95 dd (5.0, 12.0) 62.4 3-O-Glc1''' 5.26 d (7.5) 103.4 2''' 3.56 dd (7.5, 9.0) 73.6 3''' 3.49 t (9.0) 77.7 4''' 3.40 t (9.0) 71.8 5''' 3.59 m 75.6 6'''a 4.32 dd (5.0, 12.0) 64.2 trans-caffeoyl 1 128.4 2 7.00 d (1.5) 115.4 3 147.5 4 150.1 5 6.81 d (8.8) 116.2 6 6.82 dd (1.5, 8.8) 123.1 α 6.07 d (16.0) 114.6 β 7.39 d (16.0) 147.4 COO 170.0 a Coupling pattern and coupling constants (J in Hertz) are in parentheses. 13C-DEPT NMR analyses and was supported also by elemental analysis. Its 1 H- and 13C-NMR spectra (see Table 2) indicated that it was a quercetin 3-methyl ether derivative [5]. Its 1 H-NMR spectrum further displayed signals for two sugar residues that were easily clarified with the help of 1D-TOCSY and DQF-COSY experiments, leading to the identification of one β-D-glucopyranosyl and one α- L-rhamnopyranosyl residue. The configuration of sugar units was determined as reported for compound 1. The presence of one p-coumaroyl moiety was shown in the 1 H-NMR spectrum by the signals at δ 7.45 and 6.73 each (2H, d, J = 8.5 Hz) and δ 7.41 and 6.38 each (1H, d, J = 16.0 Hz). The HSQC spectrum showed glycosidation shifts for C-6'' (δ 67.5) and acylation shift for H-2'' (δ 4.74) and C-2'' (δ 74.5) of the β-D-glucopyranosyl unit. An unambiguous determination of the sequence and linkage sites was obtained from an HMBC experiment, showing cross peak correlations between δ 5.06 (H-1'') and 164.5