An acylated flavonol glycoside from Lasiobema japonica

Phytochemistry, Vol. 29, No. 3, pp. 1013-1014, Printed in Great Britain,

AN ACYLATED TETSUO

IWAGAWA,

1990. 0

FLAVONOL

JUN-ICHI

KAWASAKI,

GLYCOSIDE

TSUNAO

HASE,

and Department

Civilculture,

SHIZUO

MUJO

FROM LASIOBEMA SAKo,t

TSUTOMU

~KUBO,$

0031-9422/90 $3.00+0.00 1990 Pergamon Press plc

JAPONICA MUNETAKA

ISHIDA~

KIM$

of Chemistry, Faculty of Science, Kagoshima University, 1-21-35 Korimoto, Kagoshima 890, Japan; TLaboratory of Faculty of Agriculture, Kagoshima University, l-21-24 Korimoto. Kagoshima 890, Japan; $Taiyo Kagaku Co. Ltd., l-3 Takaramachi Yokkaichi 530, Japan (Received 21 June 1989)

Key Word Index-Lasiobemajaponica;

Leguminosae; acylated flavonol glycoside; antibacterial

activity; flavonoids.

Abstract-A new acylated flavonol glycoside quercetin 3-cc-arabinopyranoside-2”-gallate, having antibacterial activity, has been isolated from the leaves of Lasiobema japonica. Quercetin, hyperin and guaijavarin were also identified.

INTRODUCTION

Lasiobema japonica (Bauhiniajaponica) (Leguminosae) is the only member of the genus Lasiobema found in Japan. The methanolic extract of the leaves was found to have antibacterial activity against Escherichia coli B. This finding attracted our attention to the chemical constituents. This paper describes the isolation and characterization of a new antibacterial acylated flavonol glycoside (1). RESULTS AND DISCUSSION

The molecular formula of 1 was established as C2,H,,0,, by the MS and 13CNMR spectra. The UV spectrum exhibited absorption maxima at 267 (E 31000) and 355 nm (E 22 000) which is typical for a flavonoid [ 11. In the IR spectrum signals of an ester carbonyl at 1700 cm-‘, besides those of a hydroxyl group at 3300 cm-‘, a conjugated carbonyl at 1655 cm-’ and a phenyl group at 1605 and 1500 cm-i appeared. The ‘HNMR spectrum showed that 1 was a quercetin derivative. Two doublets with meta-coupling at 66.18 (lH, J=2.0 Hz) and 6.39 (lH, d, J =2.0 Hz) were due to H-6 and H-8, respectively. Signals at 66.85 (lH, d, J =8.4Hz),7.49(1H,d,.l=2.2Hz)and7.70(1H,dd,.I=2.2 and 8.4 Hz) were characteristic for a 3,4-disubstituted B ring. A singlet at 6 7.03 (2H, s) was due to a galloyl group. Upon hydrolysis with 2 M hydrochloric acid, 1 afforded quercetin, gallic acid and L-arabinose. Acetylation of 1 with acetic anhydride and pyridine gave a nona-acetate (2), whose ‘HNMR spectrum showed two sugar acetyl signals at 6 2.01 and 2.13 (3H each, s) and seven aromatic acetyl signals at 62.30-2.40 (3H x 7, s). The placement of the sugar at C-3 in 1 was assumed from the UV shift. After addition of NaOAc and AlCl, + HCl, absorption maxima at 267 and 355 nm shifted to 273 and 363, and 267, 364 and 410 nm, respectively [l]. Furthermore, the chemical shifts at C-2 (6 156.12), C-3 (6 132.93) and C-4 (6 177.0) in the 13C NMR spectrum supported the above assignment [Z]. Attachment of the galloyl group through an ester linkage at C-2 in arabinose was determined by the

downfield shift of H-2” (65.33) in the ‘H NMR spectrum. The acylated position was also confirmed by comparison of the 13CNMR spectrum with that of quercetin 3-a-Larabinopyranoside. Thus, a downfield shift of C-2” (0.56 ppm) and upfield shifts of C-l” and C-3” (2.72 and 0.95 ppm, respectively) were observed. The r-configuration of the arabinose moiety was evident from the coupling constant (J=6.2 Hz) at 65.59 due to an anomeric proton. Based on the above data, 1 is quercetin 3-a-~arabinopyranoside-2”-gallate. Its antibacterial activity was tested with paper disc (8 mm dia. and 1.5 mm thick) method. It appeared to have a potent antibacterial activity in nutritional agar medium at 100 ppm concentration against E. coli B. EXPERIMENTAL Mps: uncorr.

‘H NMR

spectra

were measured

at 400 MHz

using TMS as int. standard.

Extraction and isolation. Lasiobema japonica was collected on the campus of Kagoshima University and deposited (Herbarium sample No. 9). The fresh leaves (2 kg) were extracted twice with MeOH. After concn of the combined MeOH solns, H,O was added and successively extracted with Et,O, EtOAc and nBuOH. Part (6 g) of the EtOAc extract (11 g) was chromatographed on silica gel with Et,O-MeOH with increasing proportions of MeOH. The fractions eluted with Et,O-MeOH (4: 1) were further applied to a column of polyamide with MeOH-H,O (1 : 1) to give a flavonoid mixture (1.3 g). The mixture was submitted to a column of LH-20 with MeOH to afford methyl gallate (69 mg), quercetin (47 mg), hyperin (211 mg), guaijaverin (64 mg) and 1 (85 mg) in order of eluting. Compound 1. Yellow prisms from MeOH-H,O, mp 192-193”, [aIn - 125” (MeOH; c 0.33); ‘%NMR (d,-DMSO): 664.93 (C5”). 66.84 (C-4”), 69.66 (C-3”), 72.11 (C-2”), 93.38 (C-8), 98.61 (C-6 and C-l”), 103.77 (C-lo), 108.78 (C-2”’ and C-6”‘), 115.24 (C-2’ or C-S’), 115.46 (C-5’ or C-2’), 119.39 (C-l”‘), 120.64 (C-l’), 122.24 (C-6’), 132.93 (C-3). 138.32 (C-4”‘), 144.89 (C-3’), 145.35 (C-3”’ and C-5”‘). 148.55 (C-4) 156.12 (C-2 and C-9). 161.07 (C-5), 164.04 (C-7”‘), 164.92 (C-7), 177.0 (C-4); FABMS: m/z 587 [M + 11’. The nona-acetate 2 of 1 was obtained as an amorphous

1013

1014

Short Reports

powder, mp 130-131’; ‘HNMR (CDCI,): 62.01 and 2.13 (3H each, s, OAc), 2.30 (3H x 3, s, OAc), 2.33 (3H x 2, s, OAc), 2.35 and 2.40 (3H each, s, OAc), 3.60,3.88 (IH each, br d, J = 13.0 Hz, H”-5 x2),5.23-5.30(2H,m,H-3”and4”),5.58(1H,dd.J=7.3and8.8, H-2”), 5.68 (lH, dd. J=7.3 Hz, H-l”), 6.81 (lH, d, 5=2.2 Hz, H6),7.28(1H,d,J=2.2Hz,H-8),7.33(1H,d,J=8.4Hz,H-5’),7.83 (ZH, s, H-2”’ and H-6”‘). 7.97 (lH, d, J=1.8 Hz, H-2’), 7.98 (lH, dd, J = 1.8 and 8.4 Hz, H-6’); FABMS: m/z 965 [M + 11’. Hydrolvsis of 1. A soln of 1 (5.5 mg) in MeOH (1 ml) was refluxed with 2 M HCl (1 ml) for 6.75 hr. The resulting ppt. was recrystallized to give quercetin (1.4 mg), mp > 300 The IR and ‘HNMR spectra of quercetin were identical with those of an authentic sample 137, The presence of gallic acid in the aq. soln was confirmed by co-TLC [solvent svstem: nBuOH-HOAc--H,O, 3: 1: 11.The aq. soln was neutralized with Amberlite IRA-45 (2 g). The sugar from the aq. soln was identified as arabinose by co-PC [solvent system: n-BuOHHOACEH,O, 4: I : 5. upper phase]. Hyperin. Yellow prisms from MeOH, mp 232-233”. The spectral data were identical with those of an authentic sample 141. Hydrolysis gave quercetin and galactose. Guaijaverin. Yellow prisms from MeOH, mp 232%236”, ‘%I NMR (d,-DMSO): d64.16 (C-5”), 65.96 (C-4”), 70.61 (C-3”),

71.55 (C-2”), 93.44(C-8), 98.62 (C-6), 101.33 (C-l”), 103.73 (C-lo), 115.26 (C-2’ or C-S’), 115.64 (C-5’ or C-2’), 120.79 (C-l’), 121.93 (C-6’), 133.63 (C-3). 144.87 (C-3’), 148.51 (C-4’), 156.12 (C-2 or C-9), 156.21 (C-9 or C-2), 161.12 (C-5), 164.31 (C-7), 177.38 (C-4) [2]. The spectral data were identical with those of an authentic sample [S]. Hydrolysis gave quercetin and arabinose. Acknowledgements--- We thank Dr Y. Umeno. Taiho Co. Ltd. for the FABMS spectra measurements.

REFERE’VCES

1. Mabry, T. J., Markham, K. R. and Thomas, B. M. (1970) The Systematic Ident$ication of Flaronoids. Springer, New York. 2. Markham, K. R., Ternai, B., Stanley. R., Geiger. H. and Mabry, T. J. (1978) Tetruhedron, 34. 1389. 3. lwagawa, T. and Hase. T. (1979) Rep. Fat. Sci. Kagoshima Univ. (Math. Ph!-s. Chem.) 12, X5. 4. Iwagawa, T. and Hase. T. 11988) Rep. Fat. Sci. Kugoshima C’nir. (Mark. Phys. Chum.) 21, 81 Y. S. (1958) J. Chent. Sot. 5. Khadem, H, El and Mohammed, 3320.

Phytochemistry, Vol. 29, No. 3, pp. 1014 1016. 1990. Printed in Great Britain.

0031.9422190 %3.00+0.00 Pergamon Press plc

TWO ISOFLAVONES P. K. KACHROO, Phytochemistry

Section,

T. K. RAZDAN,

C.O.R.D.,

University

Yakugyo,

FROM

M. A. QURISHI,

IRIS KASHMZRZANA

M. A. KHUROO,

of Kashmir, Naseembagh, Canal Road, Jammu-

S. KOUL*

Srinagar-190006, 002, India

and K. L. DHAR*

India; *Division

of Chemistry,

R.R.L.,

(Received 27 June 1989) Key Work Index-Iris

kashmiriana;

Iridaceae;

rhizomes;

iriskashmirianin;

isoiriskashmirianin;

Abstract-3’-Hydroxyand 4’-hydroxy-5,5’-dimethoxy-6,7-methylene-dioxyisoflavones omes of Iris kashmiriuna.

Iris kashmiriana Baker is a widely growing plant species endemic to Kashmir and Pakistan [l]. Like other species of the genus, this plant elaborates isoflavonoids [2,3]. On chemical re-investigation, the rhizomes of the plant afforded two new isomeric isoflavones, iriskashmirianin 1 and isoiriskashmirianin 2, in addition to irilone 3 [4] irisolone 4 [S] and irisolone methylether 5 [6]. The structural elucidation of the two new isoflavones is reported here. The compounds 1 and 2 showed identical mass, [M]’ at m/z 342.3105, C,sH,,O,, and were found to be isomerit isoflavones by their similar behaviour towards ferric chloride and Na-Hg/HCI [7] tests, characteristic UV absorptions [S], i. f$‘” nm 268,330 (sh) and 260,330 (infl), IR bands at v ff: cm- ’ 1660 (C=O), 1610 (C=C) and

isoflavones.

were identified

in the rhiz-

the ‘HNMR signal at 67.88 (lH, s), due to H-2. Their ‘H NMR spectra contained signals for two methoxyls. Two of the oxygens, in both compounds, were found to be present as methylenedioxy groups; I’:!$ j 940cm-‘, 65.82 (2H, s), by the positive Labat’s test [9]. The presence of a phenolic group; Ykt:. , cm _ 1 3220 and 3240, 65.36 and 4.67 (1H each, hr s, exch. D,O), respectively, was evident from the formation of their respective monoacetates 6 and 7. S2.45 and 2.43 (3H, s). In the mass spectrum. which showed similar fragmentation patterns, the compounds exhibited the molecular ion peak as the base peak. The densely populated fragment ion peaks, arising from the RDA fragmentation, at m,/~ 194, from ring A. and 148, from B-ring, placed the