Leaves and stem bark of S. suaveolens were collected from Baldha Garden, Wari, Dhaka1100, Bangladesh in October 2015. The plant was authenticated in Bangladesh National Herbarium, Mirpur, Dhaka-1216 where a voucher specimen has been deposited (Accession No. DACB-43522) for future reference. After collection, leaves and stem bark of S. suaveolens were properly cleaned, air dried and ground to a coarse powder using a suitable grinding machine at the laboratory of Department of Applied Chemistry and Chemical Engineering, University of Dhaka, Dhaka-1000, Bangladesh. About 350g of powdered materials of leaves and stem bark was taken in a round bottom flask and soaked in 2.5 l of methanol for several days with occasional shaking and stirring. The mixture was filtered through a fresh cotton plug and finally with a Whatman filter paper Number 1. The filtrate was concentrated using a rotary evaporator at 40 °C under reduced pressure. The gummy concentrate (10 g) obtained was designated as crude methanolic extract of leaves of S. suaveolens. About 5 g of the crude extract was subjected to solvent-solvent partitioning according to the modified Kupchan method (VanWagenen et al. 1993) to yield nhexane, carbon tetrachloride, chloroform and aqueous soluble fractions.
NMR spectra of the isolated compounds were acquired in CDCl3 using a Bruker AMX-400 (400 MHz) instrument in Wazed Miah Science Research Centre (WMSRC), Jahangirnagar University, Savar, Bangladesh. The spectra were referenced to the residual nondeuterated solvent signal. Preparative thin layer chromatography (PTLC) was performed on pre-coated plates (TLC Silica gel 60 F254) for purification of compounds. Pure compounds on TLC and PTLC plates were visualized by spraying with vanillin-sulfuric acid followed by heating for 5 min at 110 ºC. All other chemicals, solvents and spray reagents were of analytical grade.
For separation, both n-hexane and carbon tetrachloride soluble paritionates of methanol extract of leaves and stem bark of S. suaveolens were fractionated separately by gel permeation chromatography using lipophilic Sephadex (LH-20). About 25 fractions of each 10 ml were obtained separately from both n-hexane and carbon tetrachloride soluble partitionates. Repeated preparative TLC of sub-fraction 7-9 of n-hexane soluble fraction over silica gel using dichloromethane: hexane (50:50) provided compound 1, while sub-fraction 18-25 gave compound 2 by using mixture of ethyl acetate and toluene (20:80) as mobile phase. On the other hand, purification of sub-fractions 17-19 from carbon tetrachloride soluble fraction over silica gel using mobile phase consisting of ethyl acetate and toluene (40:60) afforded compound 3. Properties of isolated compounds are:
Stigmasterol (1): White crystalline powder; 1H NMR (400 MHz, CDCl3): δ 0.71 (3H, s, Me10), 0.82 and 0.85 (each 3H, d, J = 7.2 Hz, Me2-25), 0.93 (3H, d, J = 6.4 Hz, Me-20), 0.97 (3H, t, J = 6.8 Hz, Me-28), 1.03 (3H, s, Me-13), 3.54 (1H, m, H-3), 5.03 (1H, dd, J = 15.2, 8.4, Hz, H23), 5.15 (1H, dd, J = 15.2, 8.4 Hz, H-22), 5.31 (1H, t, J = 4.8 Hz, H-6).
β-amyrin (2): (Syn. Olean-12-en-3β-ol); Yellowish white powder; 1H NMR (400 MHz, CDCl3): 0.75 (3H, s, Me-24), 0.79 (3H, s, Me-28), 0.90 (3H, s, Me-29), 0.90 (3H, s, Me-30), 0.94 (3H, s, Me-25), 0.95 (3H, s, Me-23), 1.10 (3H, s, Me-26), 1.23 (3H, s, Me-27), 3.20 (1H, dd, J = 11.5, 3.5 Hz, H3), 5.21 (1H, t, J = 3.5 Hz, H-12).
4-Methoxycinnamic acid (3): White crystal; 1H NMR (400 MHz, CDCl3): δ 3.87 (3H, s, 4- OMe), 6.34 (1H, d, J = 16.0 Hz, H-8), 6.95 (2H, d, J = 8.0 Hz, H-3, H-5), 7.53 (2H, d, J =8.0 Hz, H-2, H-6), 7.76 (1H, d, J = 16.0 Hz, H-7).