3.1. General
The 1H NMR and 13C NMR spectra were recorded at 270 and 68.5 MHz, respectively, with TMS as an internal standard using a 270-MHz JEOLJNM Ex-270/4000 NMR instrument. Optical rotations were determined on a JASCO P-1020 polarimeter using a 100-mm glass microcell. IR spectra (KBr) were recorded on a Perkin-Elmer 1650 FT-IR spectrometer. The UV spectra were recorded with a Perkin-Elmer Lambda 2UV/VIS spectrophotometer. The melting points were determined using a Digital Melting Point Apparatus (model IA 8103, Electro thermal Engineering Ltd, Soutthend-on-Sea, Essex, UK). MS were measured on a GSMS-QP-1000EX gas chromatograph-mass spectrometer SHIMADZU-Japan. For column chromatography, silica gel (Merk. 63-200 μm particle size) was used. TLC was carried out with Merk silica gel 60F254 Plates. UV light (245 and 366 mm) and spraying with vanillin-sulfuric acid reagent followed by heating (120 C) were used for detection.
3.2. Plant material
The aerial parts of the A. Majus L. were obtained from local market, Egypt, in March 2010. The plant material has been deposited at the Laboratory of Botany, Faculty of Science, and Zagazig University, Egypt.
3.3. Extraction and isolation
The air-dried plant (500 g) was powdered and extracted with hexane (1.6 l) at room temperature (25°C) for 30 min, and the hexane solution was evaporated in vacuo to give a residue (21 g). The methanol extract (32 g) was obtained by the same procedure. The hexane (20 g) was chromatographed over silica gel (200 g) using hexane with increasing amounts of ethyl acetate (5:1) to β-sitosterol (1 C29H50O). It is crystallized from methanol (20 mg; from Hexane-EtOAc 9:1, R
f = 0.22 Light petroleum: EtOAc 2:1); mp 136°C (literature mp 136-137°C) [22]. It responded to Liebermann-Burchard Reaction. IRνmax (KBr, cm-1) 3427; 1H NMR (δ, DMSO), 5.34 (1H, br, H-6), 3.51 (1H, m, H-3), 2.28-1.13 (29H, m, 11*CH2, 7*CH), 0.92 (6H, s, 2*CH3), 0.83 (3H, s, CH3), 0.80 (3H, s, CH3), 0.78 (3H, s, CH3), 0.68 (3H, s, CH3); GCMS: 414 (M+). This data confirmed compound 1 to be β-sitosterol 1 [21] using a direct comparison. The methanol extract (30 g) was chromatographed on a silica gel column using successively hexane-ethyl acetates eluent to give three coumarin compounds (2-4).
3.4. 6-Hydroxy-7-methoxy-4 methyl coumarin (2 C11H10O4)
White, amorphous solid (53 mg; from CH2Cl2-EtOAc 8:2, R
f = 0.19 Light petroleum: EtOAc 2:1); mp 204-206°C; [α]D +41.4 (CHCl3); UV 218; IR (KBr) γmax 3620 (OH), 1710 (C = O) cm-1; 1H NMR and 13C NMR, see Table 2; m/z 206 191(100), 160(17), 143(24); anal. calcd for C11H10O4 % C 64.06, % H 4.9, % O 31.3; found % C 64.03, % H 4.21, % O 31.1.
3.5. 6-Hydroxy-7-methoxy-coumarin (3 C10H8O4)
White, amorphous powder (61 mg; from CH2Cl2-EtOAc 3:1, R
f = 0.16 Light petroleum: EtOAc 2:1); mp 183-185°C; [α]D +46.6 (CHCl3); UV 220; IR (KBr) γmax 3640 (OH), 1700 (C = O); 1H NMR and 13C NMR, see Table 2; m/z 192 177(17), 161(100) 144(25); anal. calcd for C10H8O4 % C 62.04, % H 4.21, % O 33.2; found % C 61.9, % H 4.43, % O 32.8.
3.6. Xanthotoxin (4 C12H8O4)
White, amorphous powder (26 mg; from CH2Cl2-EtOAc 1:1); mp 158-160°C; [α]D +46.6 (CHCl3). The data from IR (KBr), 1H NMR and 13C NMR proposed that compound 3 is xanthotoxin [16–20]; anal. calcd for C12H8O4 % C 66.64, % H 3.71, % O 29.2; found % C 66.49, % H 3.43, % O 29.8.
3.7. Biological studies
3.7.1. Anti-inflammatory activity
The anti-inflammatory activity was evaluated by hind paw oedema method [23]. Albino rats of weighing 100-150 g, of either three compounds (2-4), using Indomethacin as a standard, were divided into five groups of six animals. The animals were maintained under normal environmental conditions. To each group, with the exception of the control group, the tested compounds (0.01 mg/100 g of body weight) were administered, injected. To one group, the standard drug Indomethacin (0.01 mg/100 g) was administered. After 1 h, carrageenan (0.1 ml, 1% w/v solution in sterile saline) was injected into the sub-plantar tissue of the left paw of all the animals. The right paw served as the reference non-inflamed paw for comparison. The initial paw volume was measured using a plethysmograph within 30 s of the injection. After 3 h, the final paw volume of each animal was measured. The percentage of reduction in the paw volume was calculated by subtracting the difference between the right and left hind paw volumes in the treated group from the difference in the control group and dividing it by the difference in the control group. The anti-inflammatory tivity of the tested compounds and the standard reference drug was determined using the formula, % anti-inflammatory activity = (1 - V
t/V
c) × 100, where V
t represented the mean increase in paw volume of rats treated with test compounds and V
c represented the mean increase in paw volume in the control group of rats.
3.7.2. Anti-viral activity
In this study, the compounds (2-4) were evaluated for their anti-viral activity. These compounds were tested against two mammalian viruses, HSV-1 and VSV. The antiviral activity were determined by means of the end titration technique that depends on the ability of plant extract dilutions to inhibit the produced cytopathogenic effect and expressed as reduction factor (R
f) of the viral titer.