Abstract
A new bioactive flavone glycoside was isolated from the methanol soluble fraction of the flowers of Butea monosperma O. Kuntze, which was identified as 5,7-dihydroxy-3,6,4′-trimethoxyflavone-7-O-α-L-xylopyranosyl-(1 → 3)-O-α-L-arabinopyranosyl-(1 → 4)-O-β-D-galactopyranoside (1) by several colour reactions, chemical degradations and spectral analysis. The compound 1 shows antimicrobial activity against various fungal species.
Introduction
Butea monosperma O. Kuntze belongs to the family Leguminosae sub-family mimosae and is commonly known as ‘Palash’ in Hindi being distributed throughout the greater part of India Citation1-2. Its bark is used to cure night blindness and elephantiasis, its seeds are used as an anthelmintic whereas the flowers are astringent, depurative, diuretic and aphrodisic. Various compounds have been reported by earlier workers [Citation3] from this plant. The present paper deals with the isolation and structural elucidation of a new flavone glycoside 1 from the flowers of B. monosperma which shows antifungal activity.
Materials and methods
Plant material
The flowers of Butea monosperma O. Kuntze, were collected around the Sagar region and taxonomically authenticated by the Department of Botany, Dr H.S. Gour University, Sagar, India. The voucher specimen (No. XII) is deposited in the Natural Products Laboratory, Department of Chemistry, Dr H.S. Gour University, Sagar (M.P.) India.
General
Melting points are uncorrected. The IR spectra were recorded in KBr discs. 1H-NMR spectra were run at 300 MHz using TMS as an internal standard and CDCl3 as solvent. 13C-NMR spectra were run at 90 MHz using DMSO-d6 as solvent.
Extraction and isolation
The powdered air-dried flowers (3 kg) of this plant were extracted with 95% MeOH by a Soxhlet apparatus for 6-7 days. The total methanolic extract was concentrated under reduced pressure to give a light brown amorphous mass, 4.75 g, which was successively partitioned with various solvents of increasing polarity such as pet-ether (60–80°C), C6H6, CHCl3, EtOAc, Me2CO and MeOH. The petroleum ether, benzene, chloroform, ethyl acetate and acetone soluble parts after concentration gave very small amounts of residue and therefore these fractions were rejected. The methanol fraction was concentrated under reduced pressure to a light brownish syrupy mass, 2.85 g, at room temperature. Thin layer chromatography showed three spots, indicating it to be a mixture of three compounds 1, 1a and 1b which were separated by TLC and purified by colomn chromatography over a SiO2-gel column using chloroform and methanol as solvents in various proportions (8:2, 8:4, 8:6). The eluents (8:4) and (8:2) obtained from the various fractions on the removal of the solvent gave compounds 1a and 1b which were found in very small quantity so that it was not possible to further examine these two compounds. The eluents (8:6) obtained from the various fractions on evaporation of the solvent gave Compound 1 which was further purified by colomn chromatography and found to be homogeneous on thin layer chromatorgraphy.
Compound 1 was crystallized from acetone solution to yield 1.75 g. It had C34H42O20, [M]+770 (FABMS), m.p. 260–262°C, (found: C, 54.53; H, 5.46%. calcd.: C, 54.55; H, 5.45%). IR cm− 1 3452, 2990, 2918 (–CH-stretching), 2870 (–OMe), 1630 (C = O), 1625 (aromatic ring system) and 1530, 1495, 1245, 1136, 1062, 874, 830 cm− 1. Compound 1 was also characterized by 1H-NMR and 13C-NMR spectrum (See Tables and ).
Table I. 1H-NMR (300 MHz, CDCl3) spectrum of compound 1.
Table II. 13c-nmr (90 Mhz, Dmso-d6) Spectrum Of Compound 1.
Acid hydrolysis of compound 1
Compound 1 (900 mg) was refluxed with 15 ml of 10% H2SO4 on a water bath for 7–8 hr. On cooling a crystalline compound 2 deposited which was separated by filtration. Compound 2 (615 mg) was crystallized from methanol to give light yellow needles m.p. 214–216°C, C18H16O7, [M]+ 344 (FABMS) (found: C, 62.81; H, 4.62. calcd. C, 62.79; H, 4.65%). It was identified as 5,7 dihydroxy-3,6,4′-trimethoxyflavone as aglycone by comparison of its spectral data with reported literature values.
The aqueous hydrolysate was neutralized with BaCO3, and BaSO4 was filtered off. The filtrate was concentrated under reduced pressure and examined by paper chromatography examination on Whatman filter paper No.1 (30 × 6 cm) using n-Butanol:Acetic acid: Water (B:A:W, 4:1:5) as solvent mixture and aniline hydrogen phathalate as spraying agent. After one hour, the sugars were identified as D-galactose (Rf 0.16), L-arabinose (Rf 0.21) and L-xylose (Rf 0.27) (Co-PC).
Alkaline degradation of compound 2
Compound 2 (50 mg) was treated with 50 ml of 35% KOH in 15 mL of MeOH. The contents were cooled, acidified with HCl and extracted with Et2O. The ethereal layer was washed with water and dried over Na2CO3, to yield a compound with C7H8O4, m.p. 217–220°C, [M]+156 and identified as monomethoxy phloroglucinol. The aqueous phase was acidified with HCl, extracted with Et2O and washed with water to give a compound with C8H8O3, mp 182–185°C, [M]+152 and identified as ρ-methoxy benzoic acid.
Permethylation of 1 followed by acid hydrolysis
Compound 1 (35 mg) was treated with 10 ml of MeI and 25 mg of Ag2O in 12 mL of DMF for two days and then filtered. The filtrate was concentrated under reduced pressure to yield a syrupy mass which was hydrolysed with 12% methanolic H2SO4 for 8 h to give a methylated aglycone and methylated sugars and filtered. The methylated aglycone was identified as 7-hydroxy-5,3,6,4′, tetramethoxyflavone. The aqueous hydrolysate was neutralized with BaCO3 and the BaSO4 filtered off. The filtrate was concentrated under reduced pressure and subjected to paper chromatography using n-Butanol: Acetic acid: Water (4:1:5) as solvent and aniline hydrogen phthalate as spraying reagent. The sugars were identified as 2, 3, 6-tri-O-methyl-D-galactose, 2, 4, di-O-methyl-L-arabinose and 2,3,4-tri-O-methyl-L-xylose (Co-PC).
Enzymatic hydrolysis of compound 1
Compound 1 (25 mg) was treated with 5 ml of an aqueous solution of takadiastase at 40° for 38 hr to yield L-xylose (Rf 0.27) first, followed by L-arabinose (Rf 0.21) and 5, 7 dihydroxy-3, 6, 4′-trimethoxy flavone–7-O-β-D-galactopyranoside as proaglycone. The proaglycone was hydrolysed with almond emulsion, which liberated D-galactose (Rf 0.16) and the aglycone.
Antifungal activity of compound 1
The antifungal activity of the compound 1 was examined by the Filter Paper Disc Method [Citation9]. The compound (400 mg) was tested at different concentrations using methanol as a solvent. The compound was prepared in methanol and water in different concentrations. The sterile filter paper discs (5 mm diam.) were soaked with various test samples dried at 50°C. Saboraud's broth[Citation10] media with 4% agar was used for the preparation of plates and inoculated with the spore and mycelium suspension (2 × 106 spore/ml) of fungi obtained from a 10 days old culture. The diameters of the zone of inhibition were recorded at 27 ± 1°C after 48 hr, and the results are reported in (See in Results and discussion).
Table III. Antifungal activity of compound 1.
Results and discussion
Compound 1 C34H42O20, [M]+m/z 770 (FABMS) gave a positive response to Shinoda [Citation4] and Molisch test indicating it to be a flavonoid glycoside. Acid hydrolysis of 1 with 12% methanolic sulphuric acid gave the aglycone 2 identified as 5, 7 dihydroxy-3, 6, 4′ trimethoxy flavone by comparison of its spectral data with reported literature values [Citation5]. The structure of the aglycone was further confirmed by alkaline degradation of 2 which afforded two products; one with C7H8O4, mp 217-220°C, [M]+156 identified as monomethoxy phloroglucinol and ρ-methoxy benzoic acid mp 182–185°C, C8H8O3, [M]+152, which further confirmed the position of the hydroxyl and methoxyl groups in compound 1.
The 1H-NMR spectrum of compound 1 (see ) showed a doublet at δ 6.71 for H-8, δ 7.21 for H-3′, H-5′, doublet at δ 7.78 for H-2′, H-6′, three singlets at δ 3.85, δ 3.93, δ 3.75 for 3-OMe, 6-OMe and 4′-OCH3, respectively. The anomeric protons of the sugars showed doublets at δ 5.48, δ 4.98, δ 5.63 which were assigned to H-1″, H-1″′ and H-1″″ of D-galactose, L-arabinose and L-xylose, respectively.
The position of the sugar moiety in compound 1 was established by permethylation [Citation6] of 1 followed by acid hydrolysis. The permethylated sugars, which were identified as 2, 3, 6-tri-O-methyl-D-galactose, 2, 4-di-O-methyl-L-arabinose and 2, 3, 4-tri-O-methyl-L-xylose Citation7-8, showed that the C-1″″ of L-xylose was linked with C-3″′ of L-arabinose, C-1″′ of L-arabinose was linked to C-4″ of D-galactose and C-1″ of D-galactose was linked to C-7 of the aglycone. The linkage (1 → 3) and (1 → 4) between both the sugars were further confirmed by 13C-NMR spectrum [see .
Enzymatic hydrolysis of compound 1 with takadiastase enzyme liberated L-xylose first followed by L-arabinose and 5, 7-dihydroxy-3, 6, 4′-trimethoxyflavone 7-O-β-D galactopyranoside as proaglycone, which confirmed the presence of the α-linkage between L-xylose and L-arabinose as well as between L-arabinose and the pro-aglycone. The proaglycone on further hydrolysis with almond emulsin enzyme confirmed the presence of the β-linkage between the aglycone and D-galactose.
On the basis of the above evidence, the structure of compound 1 was identified as 5, 7-dihydroxy-3, 6, 4′-trimethoxyflavone-7-O-α-L-xylopyranosyl-(1 → 3)-O-α-L-arabinopyranosyl-(1 → 4)-O-β-D-galactopyranoside (1).
The results given in show that the antifungal activity of the methanol soluble fraction of compound 1, was strong (high) against Fusarium oxysporum and Penicillium digitatum and less active against Aspergillus niger and Tricoderma viride even at very dilute concentrations. The investigation thus reveals that compound 1 may be potentially useful for diseases caused by these microorganisms.
| Abbreviations | ||
| TMS: | = | Tetramethylsilane |
| DMSO: | = | Dimethyl sulphoxide |
| DMF: | = | Dimethyl formamide |
| Co-PC: | = | Paper chromatography with authentic sample |
Acknowledgements
The authors are grateful to the Director, Central Drug Research Institute, Lucknow for providing the spectral analysis, Prof. V.K. Saxena, Head, Department of Chemistry, Dr H.S. Gour University, Sagar, M.P. India for providing laboratory facilities and Dr Archana Mehta, Department of Botany, Dr H.S. Gour University, Sagar (M.P.) for providing facilities for microbial activity.
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