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.Fitoterapia 71 2000 274277

New flavonoids from Aicennia marina

M. Sharaf, M.A. El-Ansari, N.A.M. SalehPhytochemistry and Plant Systematics Department, National Research Centre, Dokki-12311,

Cairo, Egypt

Received 21 June 1999; accepted in revised form 3 November 1999

Abstract

In addition to luteolin 7-O-methylether, chrysoeriol 7-O-glucoside and isorhamnetin3-O-rutinoside, the methanol extract of the aerial parts of Aicennia marinayielded two new

.flavonoids identified as luteolin 7-O-methylether 3-O--D-glucoside 1 and its galactosideanalogue 2. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Aicennia marina; Flavonoids; Anticancer activity

1. Introduction

. .The bark, leaves and fruits of Aicennia marina Forssk. Vierh. Avicenniaceae are used in traditional medicine to treat skin diseases 1 . Iridoid glucosides, fatty

acids, sterols and hydrocarbons were previously isolated from A. marina 2,3 . Also, in vitro antimalarial activity and cytotoxicity of A. marina were reported 4 . To the

best of our knowledge, nothing has been reported on the flavonoids of this species.The present communication deals with the isolation and structure elucidation oftwo new glycosides from the aerial parts of this plant. The anticancer activity of themajor flavonoid compounds has been preliminarily evaluated.

2. Results and discussion

The methanol extract of A. marina was fractionated on a polyamide column.

Corresponding author.

0367-326X00$ - see front matter 2000 Elsevier Science B.V. All rights reserved. .PII: S 0 3 6 7 - 3 2 6 X 9 9 0 0 1 6 9 - 0

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Purification was achieved by a combination of PPC, silica gel TLC and SephadexLH-20. Flavonoids1 and 2 were isolated.

Acid hydrolysis of 1 released glucose and luteolin 7-O-methylether. The UV

spectral data of 1 with diagnostic shift reagents indicated a flavone substituted atpositions 7 and 3, a free 4-hydroxyl group and absence of a free ortho-dihydroxyl

pattern at B-ring 5 .The 1 H-NMR spectrum suggested that 1 is a monoglucoside of luteolin 7-O-

methyl ether on the basis of the sugar C-1 proton doublet at 5.1, with a coupling . constant J 7 Hz characteristic for-linked glucose 6 .

Its 13C-NMR spectrum confirmed that 1 is a monoglucoside of luteolin 7-O-methylether. The 13 C-NMR of the aglycone part corresponded well to the shifts of

luteolin 7 , the only differences being a downfield shift of the signal assigned to .C-7 by approximately 3 ppm 164.00167.08 and upfield shift of the ortho-related

carbons, confirming the location of the OCH at C-7. The assignment of the3

aglycone and the sugar carbons are based on those given in literature 811 . The absence of the signal at 146.00, characteristic for C-3 in luteolin 7 , and thepresence of a signal at 145.20 indicated the location of the sugar moiety at C-3 12 . From the above data, compound 1 was identified as luteolin 7-O-methylether3-O--D-glucoside.

The chromatographic behaviour, UV and 1 H-NMR spectral data of 2 showedsimilarities with those of 1, the only differences being the releasing of galactoseinstead of glucose on acid hydrolysis. Therefore, compound 2 was identified asluteolin 7-O-methylether 3-O--D-galactoside.

Tested against BT-20 human carcinoma cells, responsible for breast cancer,compound 1 and its aglycone, luteolin 7-O-methylether, showed moderate cytotoxi-city with ED of 16 and 18 ml, respectively.

50

3. Experimental

3.1. Plant material

Aicennia marina aerial parts were collected in March 1998, from Abu-Ramad,

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.500 km south of Hurghada Red Sea shore and authenticated by Dr I. El-Garf. A .voucher specimen was deposited in the Herbarium of NRC CAIRC , Cairo.

3.2. Extraction and isolation

.The dried plant 1 kg was extracted with 80% MeOH. The concentrated extract .130 g was subjected to a polyamide column eluted with waterEtOH mixtures of

increasing amounts of EtOH. PPC using water, AcOHwater 3:17, BAW n-.BuOHAcOHwater 4:1:5, upper phase afforded pure samples of luteolin 7-O-

. .methylether 86 mg , chrysoeriol 7-O-glucoside 41 mg and isorhamnetin 3-O- . rutinoside 32 mg . A combination of TLC eluted with n-BuOHAcOHwater

. .3:1:1 and CHCl EtOAcacetone 5:1:4 and Sephadex LH-20, afforded1 95 mg3 .and 2 36 mg .

. .Luteolin 7-O-methylether 3-O--D-glucoside 1 . UVmax MeOH : 253, 267,346;NaOMe 261, 400;AlCl 273, 300sh, 410;AlCl HCl 260, 290sh, 374;3 3

1

.NaOAc 258, 354; NaOAcH BO 258, 362 nm; H-NMR 270 MHz, DMSO-d :3 3 6 . . . 7.70 2H, s, H-2,6 , 6.95 1H,d, J9 Hz, H-5 , 6.90 1H, s, H-3 , 6.80 1H,d, J2. . . .Hz, H-8 , 6.40 1H,d, J2 Hz, H-6 , 5.10 1H,d, J7Hz, H-1 , 3.90 3H, s, OCH ,3

. 13 3.20-3.70 m, sugar proton, hidden by hydroxyl signals ; C-NMR 270 MHz,. . . . . .DMSO-d : 64.53 C-2 , 105.23 C-3 , 181.91 C-4 , 162.10 C-5 , 99.69 C-6 , 167.086

. . . . . .C-7 , 94.78 C-8 , 156.96 C-9 , 105.23 C-10 , 120.53 C-1 , 115.81 C-2 , 145.20 . . . . . .C-3 , 151.31 C-4 , 116.13 C-5 , 121.05 C-6 , 102.39 C-1 , 74.00 C-2, 76.60 . . . .C-3 , 71.09 C-4 , 76.60 C-5 , 61.12 C-6.

. .Luteolin 7-O-methylether 3-O--D-galactoside 2 . UVmax MeOH : 251, 267,347;NaOMe 259, 406;AlCl 272, 295sh, 364;AlCl HCl 274, 295sh, 353;3 3

1 NaOAc 251, 267, 345; NaOAcH BO 251, 268, 345 nm; H-NMR 270 MHz,3 3. . . .DMSO-d : 7.60 2H, s, H-2,6 , 7.00 1H, d, J 9 Hz, H-5 , 6.95 1H, s, H-3 ,6

. . .6.90 1H, d, J 2 Hz, H-8 , 6.45 1H, d, J 2 Hz, H-6 , 5.55 1H, d, J 7Hz, H-1 , . .3.90 3H, s, OCH , 3.303.60 m, sugar proton, hidden by hydroxyl signals .3

3.3. Bioassay

.Cell line BT-20 ATCC HTB19 from human breast carcinoma , purchased from .American Type Culture Collection ATCC, Rockville, MD, USA , was used. The

.cells were maintained on minimum essential medium Eagle with non-essential .amino acids supplemented with 10% vv heat-inactivated fetal calf serum, and

incubated in a humidified atmosphere of 5% CO 95% air at 37C. This monolayer2culture was subcultured with a 0.25% trypsinEDTA solution. Cytotoxicity assay

. was performed by a microculture tetrazolium MYY Sigma method 13 .

Acknowledgements

The authors are grateful to Dr A.M. Soliman, Medicinal Chemistry Department,NRC, for measuring cytotoxic activity.

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