Flavonol glycosides fromMonnina sylvatica

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Phytochemistry,Vol. 30, No. 11, pp. 3781-3784,1991 Printed in Great Britain.

0031-9422/91 $3.00+0.00 © 1991PergamonPress plc

FLAVONOL GLYCOSIDES FROM M O N N I N A S YLVATICA AHMAD BASHIR, MATTHIASHAMBURGER, MAHABIRP. GUPTA,~" PABLO N. SOLIS'~and KURT HOSTETTMANN~ Institut de Pharmacognosie et Phytochimie, Ecole de Pharmacie, Universit6 de Lausanne, 2 rue Vuillermet, CH-1005 Lausanne, Switzerland; ~"FLORPAN, Facultad de Farmacia e Instituto Especializado de Analysis, Universidad de Panama, Panama, Republic of Panama

(Received 1 February 1991) Key Word Index--Monnina sylvatica;Polygalaceae; kaempferol glycosides; apiosides; 1H and 13CNMR.

Abstraet--A new kaempferol triglycoside and three known kaempferol glycosides, among them two apiosides, have been isolated from the aerial parts of Monnina sylvatica. The structures were established on the basis of acid and enzymatic hydrolysis and spectral data (UV, ~H and ~3C NMR, NOE difference measurements, D/CI and FAB-MS) of the isolates and of some derivatives. The triglycoside kaempferol 3-O-fl-D-glucosyl-(1--,2)-O-[~t-L-rhamnosyl(l~6)]-flD-galactoside is a new natural product. The configuration of the apiosyl moiety in kaempferol 3-O-fl-o-apiosyl(1-, 2)fl-D-galactoside and kaempferol 3-O-~-D-apiosyl(l~2)-O-[~t-L-rhamnosyl(l~6)]-fl-D-galactoside was established through NOE difference measurements on the peracetate.

INTRODUCTION

gas. A quasimolecular ion was observed at m/z 595 [M + H] +. The fragment ions at m/z 449 [M + H - 1 4 6 ] + Plants belonging to the family Polygalaceae are known to produce a large spectrum of structurally and biogeneti- and 287 [M + H - 308] + indicated a successive loss of a cally diverse secondary metabolites such as xanthones rhamnosyl and a galactosyl moiety. On the basis of [1], lignans [2], saponins [3, 4], flavonol glycosides [5], ~3C NMR spectral data, 1 could be identified as kaempsucrose esters [6], coumarins [7], and chromonocouma- ferol 3-O-robinobioside, a flavonoid previously reported from Strychnos variabilis [13] and Lysimachia mauritiana rins (6H,TH-[1]benzopyrano[4,3-b][1]benzopyran-6,7diones) [8]. As a part of our ongoing search for biolo- [14]. The sugar sequence of 2 was established by D/CIMS. A gically active compounds from Polygalaceae, we investigquasimolecular ion at m/z 581 [M + H] + and fragment ated Monnina sylvatiea. The geographic distribution of the genus Monnina is confined to subtropical and tropical ions at m/z 449 [M + H - 132] + and 287 [M + H - 294] + America [9], and no investigation of secondary metab- proved that apiose was the terminal sugar. Comparison of the ~3CNMR data of 2 with those reported for olites has been reported prior to our own work. kaempferol 3-O-fl-D-galactopyranoside [12, 15] revealed Fractionation of a lipophilic root extract of M. sylvatica that the signal of C-2" in 2 was shifted downfield by led to the isolation of two new biphenyls with antifungal activity [10]. We here report on the isolation and struc- 3.5ppm, whereas C-I" appeared upfield by 3.1 ppm. ture elucidation of flavonol glycosides from the leaves of Thus, the apiosyl moiety was attached at C-2", suggesting compound 2 to be identical with kaempferol 3-O-fl-Dthat species. apiofuranosyl(1-,2)-fl-D-galactopyranoside recently reported from the seeds of. Chenopodium quinoa [16]. RESULTSAND DISCUSSION However, it should be noted that the branched-chain Fractionation of a methanolic extract from the dried sugar apiose can occur in four isomeric forms, namely leaves of Monnina sylvatica (Polygalaceae) by a combina- two pairs of ~- and fl-furanosides which differ in their tion of gel chromatography on Sephadex LH-20 stereochemistries at C-1 and C-3 [17]. As has been (MeOH), low pressure liquid chromatography (LPLC) on pointed out previously [18], the magnitude of single RP-8 and semi-preparative HPLC on RP-18 afforded vicinal tH-1H couplings are insufficient to establish both four flavonoid glycosides, 1-4. Acid hydrolysis with 2 M the conformation and the relative configuration of the HC1 afforded kaempferol for all four compounds. Sugars fexible furanose system, and 13C NMR chemical shifts do identified after hydrolysis with 2 M HCI were galactose not provide a direct proof of the stereochemistry. In the and rhamnose for 1 and 3, galactose for 2, and galactose, case of 2, an anomeric carbon resonance at 6108.8 in the glucose and rhamnose for 4. In addition, mild acid ~3CNMR spectrum and a ~H-~H vicinal coupling hydrolysis of 2 and 3 with 0.05 M H2SO 4 afforded apiose. Jr",2-, of 1.4 Hz suggested a trans relationship of the UV [11] and 13CNMR [12] spectral data of 1-4 indi- hydroxyl groups and protons at C-1 and C-2, respectively [17, 19]. In furanoses with HO-1 and HO-2 eis, Ji,2 is cated that the glycosylation site is at C-3. The sugar sequence of 1 was deduced from D/CIMS always about 4 Hz [171 while a J~,2 close to 1 Hz is recorded in the positive ion mode with NH 3 as reactant normally observed wth HO-1 and HO-2 in a trans relationship [20]. However, methyl fl-D-apio-D-furanose has a JL2 of 3.5 Hz, a fact that has been explained by a :~Author to whom correspondence should be addressed. twist conformation of the furanose ring [17, 19]. A severe 3781

3782

A. BASHIRet al.

~ HO ~

_

OH

O [ ~

OH

O u

OW H OR~2~)H

overlap of the sugar protons in the I H N M R spectrum recorded at 200 MHz hindered a detailed analysis of the apiosyl moiety through NOE difference measurements, but the spectrum of the peracetate 2a allowed a full assignment of the sugar protons (see Experimental). A J1,,,.2,,, close to 0 Hz was in accord with data reported for tetra-O-acetyl-fl-D-apio-D-furanose [21], and would indicate an almost planar conformation of the furanose ring. In a series of NOE difference experiments, presaturation of the doublet at 4.77 (H-4'") resulted in a strong enhancement of the signals at 4.10 (H-5'") and 5.28 (H-2'"). This suggests that these three protons are on the same face of the ring, hence an erythro relationship of OH-2'" and OH-3"'. Strong enhancements of H-I'" and H-I" were also observed upon irradiation of H-2". Presaturation of the anomeric proton of the apiosyl moiety gave enhancement of H-2'" and H-2", confirming the position of the interglycosidic linkage. The erythro relationship was also supported by the 13C NMR data. The chemical shift difference of 3.0 ppm, observed for C-2'" and C-Y" in 2 was closer to that reported for methyl fl-O-apio-Dfuranose (A = 1.6 ppm) than that of the ct-L-threo isomer (A=0.7 ppm) [18]. Thus, the apiosyl moiety is the 3-C(hydroxymethyl)-fl-D-erythrofuranosyl isomer, and compound 2 is kaempferol 3-O-fl-D-apio-D-furanosyl(l~2)fl-D-galactoside. The negative ion FAB mass spectrum of 3 exhibited a quasimolecular ion at m/z 725 [ M - H ] - . Fragment peaks at m/z 593 [ M - H - 1 3 2 ] - , 579 [ M - H - 1 4 6 ] - , 447 [ M - - H - 2 7 8 ] - and 285 [ M - H - 4 4 0 ] indicated a branched trisaccharide moiety with apiose and rhamnose as terminal sugars. Enzymatic hydrolysis with hesperidinase afforded glycoside 2, as shown by coTLC, 1H and 13C N M R and mass spectra. Compared to the 13C NMR spectrum of 2, the signal attributable to C6" in the spectrum of 3 was shifted downfield by 5 ppm, while C-5" appeared at higher field (+2.5ppm). The rhamnosyl moiety was therefore attached to C-6", and compound 3 was identified as kaempferol fl-D-apio-Dfuranosyl (1--,2)-O-[~t-L-rhamnopyranosyl(1 ~6)]-fl-Dgalactoside. This compound has also been reported very recently from Chenopodium quinoa without explicit proof for the stereochemistry of the apiosyl moiety [16]. D/CIMS of 4 showed a quasimolecular ion at m/z 757 which is consistent with a molecular formula C33H40020 . Fragment ions at m/z 611 [ M + H - 1 4 6 ] + and 595 [M + H - 1 6 2 ] + resulted fore a simultaneous elimination of a rhamnosyl and a hexosyl unit, characteristic of a branched sugar moiety. Enzymatic hydrolysis of 4 with flglucuronidase afforded glycoside 1 (co-TLC). Three anomeric proton signals at 65.59 (d, J = 7 . 5 Hz, H-I"), 4.56 (d, J=7.3Hz, H-I'") and 4.36 (d, J=0.8Hz, H-I'"') confirmed the presence of a trisaccharide moiety. In the ~3CNMR spectrum of 4, all the signals attributable to

Rl

R2

1

H

Rha

2

Api

H

3

Api

Rha

4

Glc

Rha

HO-

0/

Api = ~ OH

OH

kaempferol, to the galactosyl and rhamnosyl moieties were found to be identical with those of I with exception of C-I" and C-2". A downfield shift of the latter signal (-8.1 ppm) in 4 and an upfield shift of the anomeric carbon signal (+ 3.8 ppm) indicated that the glucopyranosyloxy moiety was attached to C-2". Thus, the structure of 4 was established as kaempferol 3-O-/~-D-giUCOpyranosyl(1--,2)-O-[~-L-rhamnopyranosyl(1 ~6) ]-fl-Dgalactoside, a new glycoside of kaempferol. The four flavonol glycosides identified in this first investigation of polar metabolites from the genus Monnina are structurally similar to those previously reported from Securidaca [5], another genus of the family Polygalaceae confined t o subtropical and tropical regions. Whereas Securidaca diversifolia contains mainly quercctin and only few kaempferol glycosides, the flavonoids identified so far in Monnina sylvatica are exclusively glycosides of kaempferol, and acid hydrolysis of the crude MeOH extract did not afford detectable amounts of quercetin. In all the flavonol apiosides found so far in Monnina sylvatica and Securidaca diversifolia, the apiosyl moiety is attached in 2-position of the inner sugar. For the two apiosides reported here, the stereocbemistry of this branched pentose was established as 3-C(hydroxymethyl)-fl-D-erythro furanose on the basis of NOE difference experiments and a detailed analysis of tH and 13C NMR data. Apiose appears to occur frequently in glycosides of the Polygalaceae, as it has also been found in several Polygala saponins [3, 4]. EXPERIMENTAL General. Mp" uncorr. UV shift reagents were prepd according to ref. [11]. TLC was carried out on pre-coated silica gel 60 F254 aluminium sheets (Merck) and RP-8 or Diol HPTLC plates (Merck). The following solvent systems were employed: CHCI3-MeOH-H20 (13:7:1) and EtOAc-MeCOEtHOAc-H20 (5:3: l : 1) (silica gel), EtOAc-toluene (1 : 1) (Diol) and MeOH-H2 O (7: 3) (RP-8). Compounds were revealed in UV light (254 and 366 nm) and after spraying with 2-aminoethylborate/polyethyleneglycol reagent. For open CC, silica gel 40-63/am (Merck) was used. Low pressure liquid chromatography (LPLC) was carried out on pre-packed Lobar columns (LiChroprep, RP8, 40-63/am, Merck) with a flow rate of 2 ml rain- 1. Semi-prep. HPLC was performed on a LiChroprep RP-18 column (7 #m, 16 x 250 ram, i.d., Knauer) at a flow rate of 5 mi min- 1. Purity of compounds was checked by HPLC on LiChrosorb RP-8 and RP-18 columns (7/am, 4.6 x 250 mm i.d., Knauer). D/CIMS were recorded in the positive ion mode using NH 3 as reactant gas. FABMS: negative ion mode, thioglyceroi. IH and 13CNMR spectra were recorded in DMSO-d6 or CDCI3 at 200.6MHz for proton and 50.3 MHz for carbon, respectively. TMS was used as an int. standard. NOE difference measurements were carried out according to ref. [22].

Flavonol glycosides from Monnina sylvatica

Extraction and isolation. M onnina sylvatica Cham. & Schtecht was collected in August 1989 near El Valle de Anton, Cocl6 Province, Republic of Panama. A voucher specimen has been deposited at the Herbarium of the Dept. of Botany, University of Panama. Dried leaves (47 g) were successively extracted at room temp. with CH2C12 and MeOH. The MeOH extract (7,78 g) was subjected to CC on Sephadex LH-20 (MeOH). Nine fractions were collected. Compounds 3 (22 mg) and 4 (12.5 rag) were obtained from fr. 4 (400 mg) by LPLC [RP-8, MeOH-H20 (2:3). Fr. 5 (250 mg) was subjected to flash chromatography on silica gel [CHC13-MeOH-H20 (13:7:1)] to afford 3 (115 mg). Fr. 6 (130mg) was sepd by open CC on silica gel [CHC13-MeOH-H20 (75:25:4)]. Nine frs (A-I) were collected. Compound 1 (6 mg) was obtained from fr. E (15 mg) by semiprep. HPLC [RP-18, MeOH-H20 (21:29)]. Fr. G (20 mg) was subjected to CC on Sephadex LH-20 (MeOH) to afford 2 (5 mg). Fr. I (22 mg) consisted of 3. Hydrolysis of compounds 1-4 with 2 M HCI. The compounds (1 mg) were refluxed in 2 M HC1 (5 ml) for 2 hr. The aglycone was extracted with EtOAc and identified by co-TLC with an authentic sample of kaempferol [silica gel, CHC13-MeOH-H20 (75: 25: 3), Diol, EtOAc-toluene (1 : 1)]. The aq. layer was adjusted to pH 6 by addition of NaHCO 3. After lyophilization, sugars were extracted with pyridine and analysed by co-TLC with authentic samples [silica gel, EtOAc-MeOH-HzO-HOAc (13:3:3:4)]. Detection was with p-anisidine phthalate and naphthoresorcine reagents. Hydrolysis of compounds 2 and 3 with 0.05 M H2SO4. Each compound (1 rag) was refluxed in 0.05 M H2SO4 (2 ml) for 30 min. Extraction and TLC was carried out as above. Apiose was detected with naphthoresorcine reagent. Enzymatic hydrolysis with hesperidinase.Compound 3 (30 mg) was incubated with hesperidinase (5 mg, Sigma) in acetate buffer (pH 5.5) at 37° for 72 hr. The soln was extracted successively with EtOAc and n-BuOH (3 x 20 ml). The residue of BuOH layer was purified by chromatography on Sephadex LH-20 (MeOH) to afford 2 (17 rag). Hydrolysis with fl-olucuronidase. Compound 4 (1 mg) was incubated with fl-glucuronidase (1 mg, Sigma) in acetate buffer (pH 5.5) at 37° for two days. The soln was extracted successively with EtOAc and n-BuOH (3 x 5 ml). After removal of BuOH, the residue was identified as 1. Kaempferol 3-O-ct-L-rhamnopyranosyl(l ~6)-fl-D-galactopyranoside (1). Yellow powder; mp 202-204°; TLC [silica gel, EtOAc-MeCOEt-HOAc-H20, 5:3 : 1 : 1 (system A)]: R I 0.53; UV 2n,axnm (log e): 267 (4.12), 352 (4.00); (A1C13)269, 346, 391; (A1CI3+ HC1) 272, 347, 392; (NaOMe) 276, 392; (NaOAc) 274, 379; (NaOAc + H3BO3) 267, 352; D/CIMS (NH3, pos. ion mode) m/z 595 [ M + H ] +, 449 [ M + H - 1 4 6 ] +, 287 [ M + H - 3 0 8 ] + ; 1H and 13CNMR corresponded with [13, 14].

Kaempferol 3-O.fl-D-apio-D-furanosyl(l ~ 2)-fl-D-oalactopyranoside (2). Yellow powder; mp 166-169°; TLC (system A): R I 0.49; [ct]o- 52° (MeOH; c 0.1); UV 2m~ nm (log e): 267 (4.09), 352 (3.97); (AICI3) 275, 303sh, 347, 392; (A1CI3 + HC1) 274, 303sh, 345, 392; (NaOMe) 275, 323, 392; (NaOAc) 274, 304sh, 382; (NaOAc +H3BO3) 267, 309sh, 348; D/CIMS (NH3, pos. ion mode) m/z 581 [ M + H ] +, 449 [ M + H - 1 3 2 ] +, 287 [ M + H - 2 9 4 ] + ; aHNMR (200.6MHz, DMSO-dr): 63.00-3.90 (unresolved sugar protons), 5.31 (1H, d, J = l . 4 Hz, H-I'"), 5.6 (ill, d, J = 7.5 Hz, H-I"), 6.12 (1H, d, J = 1.8 Hz, H-6), 6.35 (1H, d, J = 1.8 Hz, H-8), 6.85 (2H, d, J = 8.9 Hz, H-3' and H-5'), 8.10 (2H, d, J=8.9 Hz, H-2' and H-6'); 13CNMR (50.3 MHz, DMSO-dr): 6 59.9 (C-6"), 64.2 (C-4'"), 68.2 (C-4"), 73.6 (C-3"), 73.8 (C-5'"), 74.9 (C-2"), 75.8 (C-5"), 76.06 (C-2'"), 79.1 (C-3'"), 93.6 (C-8), 98.2 (C1"), 98.8 (C-6), 103.2 (C-10), 108.8 (C-I"'), 114.9 (C-3' and C-5'),

3783

120.9 (C-I'), 130.6 (C-2' and C-6'), 132.7 (C-3), 155.1 (C-9), 156.2 (C-2), 159.8 (C-4'), 161.1 (C-5), 177.1 (C-4). Acetylation of compound 2. A soln of 2 (16mg) in Ac20-pyridine (1 : 1) (2 ml) and dimethylaminopyridine (few mg) was kept at room temp. for 16 hr. After the addition of ice H20 , the aq. layer was extracted with Et20. The solvent was evapd and the residue purified by CC on silica gel (toluene-EtOAc, 1 : 1) to afford nonaacetate 2a (8 mg). Amorphous powder; mp 88-90.5°; D/CIMS (pos. ion mode) m/z 959 [M + HI +; XHNMR (200.6 MHz, CDCI3): 61.89, 1.98, 2.01, 2.04, 2.05, 2.08, 2.32, 2.33, 2.39 (3H, each, s, OAc), 3.78 (3H, m, H-5" and H-6"), 4.00 (1H, dd, J = 10.3, 7.6 Hz, H-2"), 4.10 (1H, d, J = 10.5 Hz, Ha-5'"), 4.29 (1H, d, J = 10.5 Hz, Hb-5'"), 4.77 (1H, d, J = 12.5 Hz, Ha-4'"), 4.92 (1H, d, J = 12.5 Hz, Hb-4'"), 5.00 (1H, dd, J = 10.3, 3 Hz, H-3"), 5.21 (1H, s, H- 1'"), 5.28 (1H, s, H-2'"), 5.29 (1H, d, J = 3 Hz, H-4"), 5.52 (1H, d, J=7.6 Hz, H-I"), 6.81 (1H, d, J=2,2 Hz, H-8), 7.2 (2H, d, J = 9 Hz, H-3' and H-5'), 7.29 (1H, d, J=2.2 Hz, H-6), 8.1 (2H, d, J = 9 Hz, H-2' and H-6').

Kaempferol 3-O-[O-~-D-apio-D-furanosyl(l~2)-O-[ct-Lrhamnopyranosyl(1 ~6)]-fl-D-galactopyranoside (3). Yellow amorphous powder; TLC (system A): R r 0.33; [~t]t)-67 ° (MeOH; c 0.1); UV 2ma, nm (log e): 267 (4.09), 304 (3.49), 352 (4.00); (A1C13)274, 305sh, 347, 392; (AICla + HC1) 275, 305sh, 347, 390; (NaOMe) 274, 323, 392; (NaOAc) 274, 304, 373; (NaOAc +HsBO3) 267, 348; FAB-MS (neg. ion mode) m/z 725 [M - H l -, 593 [ M - H - 132]-, 579 [ M - H - 146]-, 447 [ M - H - 2 7 8 ] - , 285 [ M - H - 4 4 0 ] - ; 1HNMR (200.6 MHz, DMSOd6): 61.07 (3H, d, J = 6.1 Hz, H-6""), 3.10-3.90 (unresolved sugar protons), 4.38 (1H, br s, H-I'"'), 5.34 (1H, d, J = 1.2 Hz, H-I'"), 5.54 (1H, d, J=7.8 Hz, H-I"), 6.21 (1H, d, J = 2 Hz, H-6), 6.44 (1H, d, J = 2 Hz, H-8), 6.88 (2H, d, J = 9 Hz, H-3' and H-5'), 8.1 (2H, d, J = 9 Hz, H-2' and H-6'); 13C NMR (50.3 MHz, DMSOdr): 617.7 (C°6'"'), 64.4 (C-4'"), 65.1 (C-6"), 68.2 (C-5'"'), 68.3 (C4"), 70.3a (C-2'"'), 70.6a (C-3'"'), 71.9 (C-4'"'), 73.3b (C-5"), 73.4b (C3"), 73.9 (C-5'"), 74.8 (C-2"), 76.2 (C-2'"), 79.2 (C-3'"), 93.6 (C-8), 98.7 (C-6), 99.1 (C-I"), 100.0 (C-I'"'), 103.8 (C-10), 108.8 (C-I"), 115.1 (C-3' and C-5'), 120.9 (C-I'), 130.8 (C-2' and C-6'), 132.8 (C3), 155.8 (C-9), 156.3 (C-2), 159.9 (C-4'), 161.2 (C-5), 164.2 (C-7), 177.3 (C-4). a'bAssignments interchangeable.

Kaempferol 3-O-fl-rJ-glucopyranosyl(1--*2)-O-[ct-L-rhamnopyranosyl(1 ~6)]-fl-D-galactopyranoside (4). Light yellow powder: mp 196-198°; TLC (system A): Ry 0.25; [~]o--38 ° (MeOH; c0.l); 2maxnm (log e): 267 (4.17), 300sh, (3.93), 351.6 (4.11); (A1Cls) 274, 303sh, 348, 392; (A1C13+ HC1) 274, 304sh, 345, 392; (NaOMe) 274, 325, 392; (NaOAc) 274, 372; (NaOAc + HaBO3) 267, 352; D/CIMS (NH3, pos. ion mode) m/z 757 [M + H I +, 611 [M + H - - 146] +, 595 [M + H - 162] +, 287 [ M + H -4701+; 1HNMR (200.6MHz, DMSO-dr): 61.05 (3H, d, J =6.1 Hz, H-6"'), 3.00-3.90 (unresolved sugar protons), 4.36 (1H, d, J=0.8 Hz, H-I'"), 4.56 (1H, d, J=7.2 Hz, H-I'"), 5.59 (1H, d, J = 7.5 Hz, H-I"), 6.17 (1H, d, J = 1.9 Hz, H-6), 6.39 (1H, d, J = 1.9 Hz, H-8), 6.88 (2H, d, J = 8.8 Hz, H-3' and H-5'), 8.05 (2H, d, J =8.8 Hz, H-2' and H-6'); 13CNMR (50.3 MHz, DMSO-dr): 617.7 (C-6"'), 60.8 (C-6"'), 64.8 (C-6"), 67.6 (C-4"), 68.1 (C-5"'), 69.7 (C-4'"), 70.3a (C-2'"'), 70.5~ (C-3'"'), 71.8 (C-4'"'), 72.9b (C-5"), 73.5 b (C-3"), 74.1 (C-2'"), 76.5c (C-5'"), 76.8c (C-3'"), 79.1 (C-2"), 93.5 (C-8), 98.5 (C-I"), 98.7 (C-6), 100.0 (C-I'"'), 103.7 (C-l'"), 103.9 (C-10), 115.1 (C-3' and C-5'), 120.8 (C-I'), 130.8 (C-2' and C6'), 132.7 (C-3), 155.8 (C-9), 156.2 (C-2), 159.8 (C-4'), 161.1 (C-5), 164.2 (C-7), 177.2 (C-4). ~-CAssignments interchangeable.

Acknowledgements--Financial support has been provided by the Swiss National Science Foundation, the Sandoz Foundation, Basel and the Herbette Foundation of the University of Lausanne. A studentship was awarded to A.B. by the Swiss

3784

A. BASHIRet al.

Commission F6d6rale des Bourses pour Etudiants Etrangers. The Organization of American States is acknowledged for the support of the Project FLORPAN in Panama through its Regional Scientific and Technological Development Program. Thanks are due to Professor M. D. Correa (Curator of the Herbarium of the University of Panama) for the identification of the plant material. REFERENCES 1. Ghosal, S., Basumatari, P. C. and Banerjee, S. (1981) Phytochemistry 20, 489. 2. Hokanson, G. C. (1979) J. Nat. Prod. 42, 378. 3. Sakuma, S. and Shoji, J. (1981) Chem. Pharm. Bull. 30, 810. 4. Sakuma, S. and Shoji, J. (1981) Chem. Pharm. Bull. 30, 2431. 5. Hamburger, M., Gupta, M. and Hostettmann, K. (1985) Phytochemistry 24, 2689. 6. Hamburger, M. and Hostettmann, K. (1985) Phytochemistry 24, 1739. 7. Hamburger, M., Gupta, M. and Hostettmann, K. (1985) Planta Med. 50, 215. 8. Di Palo, E. R., Hamburger, M., Stoeckli-Evans, H., Rogers, C. and Hostettmann, K. (1989) Helv. Chim. Acta 72, 1455. 9. Taylor, C., M. (1985) Rhodora 87, 159.

10. Bashir, A., Hamburger, M., Rahalison, L., Monod, M., Gupta, M. P., Soliz, P. and Hostettmann, K. (1991) Planta Med. 57, 192. 11. Markham, K. R. (1982) Techniques of Flavonoid Identification. Academic Press, London. 12. Markham, K. R., Ternai, B., Stanley, R., Geiger, H. and Mabry, T. J. (1978) Tetrahedron 34, 1389. 13. Brasseur, T. and Angenot, L. (1986) Phytochemistry 25, 563. 14. Yasukawa, K. and Takido, M. (1987) Phytochemistry 26, 1224. 15. Lin, C. N., Arisawa, M., Shimizu, M. and Morita, N. (1982) Phytochemistry 21, 1466. 16. Simone, F. D., Dini, A., Pizza, C., Saturnino, P. and Schettino, O. (1990) Phytochemistry 29, 3690. 17. Angyal, S. J., Bodkin, C. L., Mills, J. A. and Pojer, P. M. (1977) Aust. J. Chem. 30, 1259. 18. Snyder, J. R. and Serianni, A. S. (1987) Carbohydrate Res. 166, 85. 19. Ritchie, R. G. S., Cyr, N., Korsch, B., Koch, H. J. and Perlin, A. S. (1975) Can. J. Chem. 53, 1424. 20. Angyal, S. J. and Pickles, V. A. (1972) Aust. J. Chem. 25, 1695. 21. Tronchet, J. M. J. and Tronchet, J. (1974) Carbohydrate Res. 34, 263. 22. Kinns, M. and Sanders, J. K. M. (1984) Maon. Reson. 56, 518.

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