File 2517 (14).qxd Bangladesh J. Sci. Ind. Res. 41(1-2), 93-96, 2006 Introduction Cassia Linn. includes a large tropical genus of 580 species of herbs, shrubs and trees of the leguminosae family, including Cassia alata and many of them are found in Indo- Pak-Bangladesh subcontinent.1,2 Cassia alata Linn. is a small tree generally grown in the gardens and not far away from human dwellings.3 Its use as medicine, especially in the eradication of Herpescircinatus is confirmed by Mekenna et al.4 Earlier investigations on Cassia alata reported the presence of only hydroxymethy- lanthraquinones (unidentified) and chryso- phanic acid as isolated compounds.5 Hauptmann and co-workers6 isolated rhein (an antibiotic) and its reduced form with other anthraquinone derivatives which they could not identify. Though some anthraquinones were isolated and character- ized by some scientists,7,8,9 none reported on the isolation of flavonoids. The present work had, therefore, been taken up to make a complete chemical investigation on the plant Cassia alata. A flavone was isolated from the plant as a new source. The structure of the flavone was elucidated by spectroscopic techniques. Materials and Methods A Reichart micro melting point apparatus was used for recording the melting point. UV spectra (MeOH) was recorded on a Shimadzu UV-240 spectrophotometer, IR Spectra (KBr) on a Shimadzu IR-460 instrument, 1H- NMR spectra(CD3OD) on a Bruker AM-500 FT NMR spectrometer (500 MHz) using TMS as internal standard, 13C-NMR spectra A Flavone from the Leaves of Cassia Alata M. S. Rahaman,a AJM Moynul Hasan,a M. Y. Alib and M. U. Alib aBCSIR Laboratories, Binodpur Bazar, Rajshahi-6206 and bDepartment of Chemistry, Rajshahi University, Bangladesh Abstract Studies were carried out on the leaves of Cassia alata. A flavone 3,5,7,4�-tetrahy- droxy flavone was isolated as a new source from the leaves of Cassia alata with the help of column and thin layer chromatography using a gradient of organic solvents with increasing polarity. The compound was characterized on the basis of UV, IR, 1H- NMR, 13C-NMR and Mass spectrometry. (CD3OD) on a Bruker AM-500 FT NMR spectrometers (100 MHz) and Mass spectra on a Varian-MAT 112S spectrometer. Electron Impact (EI), Peak Matching experi- ments were performed on a MAT-312A mass spectrometer. Fresh leaves were collected from the plants grown in the adjoining areas of BCSIR Laboratories, Rajshahi campus during August-September period. The leaves were washed with water to remove extraneous materials and then dried in shade. Care was taken to avoid exposure to sunlight. The dried material was crushed to powder. The air-dried Cassia alata leaf powder (6.6 Kg) was soaked in 80 % ethanol for a week. The ethanolic extract was then filtered and the solvent was removed under reduced pres- sure to obtain a viscous residue (494 g). The crude residue was then defatted with n-henane. The defatted mass was dried under reduced pressure to give a residue (162g). The defatted extract was then treated with water, shaken well to resolve into water soluble and water insoluble parts. The water soluble part was extracted with ethyl acetate. The ethyl acetate soluble part was chro- matographed over a silica gel (70-230 mash) column and successively eluted using n-hexane and ethyl acetate. Elution of the column with n-hexane : ethyl acetate (40:60 v/v) afforded a compound designated as compound-1 along with minor impurities. Purification of the compound by preparative thin layer chromatography (PTLC) The compound 1 with some impurities was applied to a PTLC card of silica gel 60GF254 (thickness 0.1mm) and eluted with n-hexane : ethyl acetate (4:1 v/v). A distinct single band (Rf 0.49) was observed on the PTLC card. The band was collected and washed out with ethyl acetate to obtain a yellow solid (compound 1, 12.5mg, m.p 276-278O C, Rf = 0.49). Spectroscopic analysis of compound 1 UVλmax (MeOH)nm : 366, 323, 266, 203, 195 IRυmax (KBr)cm-1 : 3490(O - H), 2900 (C - H), 1670(C = O), 1570 -1480(C = C) EIMS m/z (rel. int%) : 286(100), 257(9), 241(1), 229(8), 193(0.85), 184(1), 143(10), 121(20), 105(3), 93(5), 69(8). Peak matching m/z (formula) : 286.41360 (C15H10O6). 1H-NMR (500MHz) δδTMS : (CD3OD) : δ6.28 [H-6, 1H, d, J(H-6, H-8) 2.1 Hz] δ6.54 [H-4, 1H, d, J(H-8, H-6) 2.1Hz] δ8.09 [H-2 / , H-6 / , 2H, dd, J(H-2/, H-3/) 9.0Hz, J(H-2/, H-6/) 2.1 Hz] δ7.01 [H-3 / , H-5 / , 2H, dd, J(H-5/, H-6/) 9.0Hz, J(H-3/, H-5/) 2.1 Hz] 94 A Flavone from the leaves 41(1-2) 2006 13C-NMR (CD3OD, 100MHz) : 147.9, 137.0, 177.2, 160.4, 99.2, 162.4, 94.4, 158.1,104.5, 123.7, 130.6, 115.2, 165.4, 116.2, 130.6 (Table I). Results and Discussion The ethyl acetate triturate of the ethanolic extract of Cassia alata leaves aforded com- pound 1 after purification by preparative TLC. Compound 1 was suggested to be a flavonoid as it exhibited light yellow appear- ance on silica gel card and deep yellow colour when sprayed with ceric sulphate reagent. The absorption at 3490 and 1670 cm-1 in the IR spectrum (KBr) of the compound were indicative of hydroxyl and carboxyl functions respectively. The IR spectrum also Rahaman, Hasan, Ali and Ali 95 Table I. 13C-NMR (CD3OD, 100 MHz) chemical shifts of 3,5,7,4 /-tetrahydroxy flavone. C. No. Multiplicity (DEPT) 13C-NMR (δ) 1H-NMR (δ) 1JHH(Hz) C-2 C 147.9 C-3 C 137.0 C-4 C 177.2 C-5 C 160.4 C-6 CH 99.2 6.28 d, J = 2.1 C-7 C 162.4 C-8 CH 94.4 6.54 d, J = 2.1 C-9 C 158.1 C-10 C 104.5 C-1 / C 123.7 C-2 / CH 130.6 8.09 dd, J = 9.0, 2.1 C-3 / CH 116.2 7.01 dd, J = 9.0, 2.1 C-4 / C 165.4 C-5 / CH 116.2 7.01 dd, J = 9.0, 2.1 C-6 / CH 130.6 8.09 dd, J = 9.0, 2.1 showed absorption at 2900 and 1570- 1480cm-1 due to CH and C = C functions. The EI mass spectrum showed the molecular ion as well as base peak at m/z 286. The moleular formula was established with the help of 13C-NMR, 1H-NMR and peak matching experiments as C15H10O6 corre- sponding to the mass m/z 286.41360. 15 sig- nals appeared in the broad-band spectrum of copound-1, which were resolved with the help of DEPT experiment into six methine and nine quaternary carbons. The 1H-NMR spectrum of compound 1 displayed a doublet at δ6.28 with coupling constant 2.1 Hz for H-6 proton and another doublet resonated at δ6.54 having coupling constant 2.1 Hz for H-8 proton. Two protons at 2 / and 6 / positions gave a double doublet at δ8.09 having coupling constant 9.0 and 2.1 Hz respectively. Two protons at 3 / and 5 / positions gave another double doublet at δ7.01 having coupling constants 9.0 and 2.1 Hz. From the spectral informations, it became apparent that the compound 1 belonged to the flavone series and was characterized as 3,5,7,4 / tetrahydroxy flavone. Acknowledgement The authors are grateful to Professor M. Iqbal Choudhary, H. E. J Research Institute of Chemistry, University of Karachi, Pakistan for giving excellent facilities to perform all spectroscopic analysis there. References 1. The Wealth of India. A Dictionary of Indian Raw Materials and Industrial products, Vol. II, C.S.I.R, New Delhi, India, (1950) 93. 2. R. N. Chopra, R. L. Badhwar and S. Ghose. Poisonous Plants of India, Vol. I, Academic Publishers, Jaipur, India, (1984) 364. 3. G. Watt : A Dictionary of the Economic products of India, Vol. II, New Delhi, (1972) 211. 4. Mekenna : Madras Med. Jour., Vol. I, 431; Arthur : Indian Ann. of Med. Science, Vol. III (1956) 632. 5. R. N. Chopra, S. L. Nayar and I. C. Chopra : Glossary of Indian Medicinal Plants. Council of Scientific and Indian Research, New Delhi, India, (1980). 6. L. H. Hauptmann and L. L. Nazario : J. Am. Chem. Soc., 72 (1950) 1492. 7. Hemlata and Suruj B. Kalidhar. Phitochemistry, 32 (1993) 1616-1617. 8. K. Sahyender Yadav and Suraj B. Kalidhar, Planta Med., 60 (1994) 601. 9. K. N. Rai and S. N. Prasad. J. Indian Chem. Soc., 71 (1994) 653-654. 96 A Flavone from the leaves 41(1-2) 2006