New Phytoconstituents from the Aerial Parts of Uraria lagopoides.

Abstract

Four new compounds, namely 7′-carboxylic-n.-heptacosanyl-glutarate, n.-nonacosan-6-ol-1,13-dioic acid, 4,12-dimethyl-n.-tetradeca-6,8,10-triene-1-ol-4-oic acid, and 8,14-13,17-diseco-stigmast-5,22-diene-3-α-ol, along with 2-hydroxytricontane and β.-sitosterol glycoside, have been isolated from the aerial parts of Uraria lagopoides. D.C. (Papilionaceae) and characterized by combination of chemical and spectral data analysis.

Keywords:

• Anti-inflammatory, 7′-carboxylic-n.-heptacosanyl-glutarate
• 4,12-dimethyl-n.-tetradeca-6,8,10-triene-1-ol-4-oic acid, 8,14-13,17-diseco-stigmast-5,22-diene-3-α-ol
• n.-nonacosan-6-ol-1,13-dioic acid, Uraria lagopoides

Introduction

Uraria lagopoides. D.C. (Papilionaceae), a perennial herb locally known as “prisniparni,” finds use as a remedy for several ailments in the indigenous system of medicine (Kirtikar & Basu, 1994; Chopra et al., 1956; Dey, 1994; Narian, 1999; Nadkarni, 1976). The alcohol and aqueous extracts of the aerial parts of the plant were found to have significant anti-inflammatory and analgesic activities (Hamid et al., 2004). A literature survey indicated that flavonoids, which exhibit anti–snake venom activity (Xiafen et al., 1986), and fatty acids (Grampurohit et al., 1993), are only phytochemicals reported so from this plant. In continuation of our earlier work on this plant, (Hamid et al., 2004), we herein report the isolation and characterization of four new compounds, 7′-carboxylic-n.-heptacosanyl-glutarate (2), n.-nonacosan-6-ol-1,13-dioic acid (3), 4,12-dimethyl-n.-tetradeca-6,8,10-triene-1-ol-4-oic acid (4), and 8,14–13,17-diseco-stigmast-5,22-diene-3-α-ol (5), along with two known compounds, namely, 2-hydroxytricontane (1) and β.-sitosterol glycoside (6), from the chloroform extract of U. lagopoides. (Fig. 1).

Figure 1 Constituents of Uraria lagopoides..

Materials and Methods

General experimental methods

Melting points were determined on a Perfit apparatus and are uncorrected. IR spectra were recorded in KBr pellet on Win IR FTS 135 instrument (Biorad, USA). ¹H (300 MHz), ¹³C (75 MHz), and 2D NMR spectra were recorded by Bruker spectrospin NMR instrument in CDCl₃, using TMS as internal standard. EIMS were scanned at 70 eV on a Jeol D-300 instrument (Jeol, USA). Column chromatography was carried out using silica gel (Merck, 60-120 mesh) and thin-layer chromatography on silica gel G coated TLC plates (Merck). The spots were visualized by spraying with 10% alc. H₂SO₄ as well as 5% cerric ammonium sulfate followed by heating at 105°C for 5 min.

Plant material

The aerial parts of Uraria lagopoides. D.C. were procured from a Khari Baoli, New Delhi, and authenticated by Dr. M.P. Sharma, Botany Department, Jamia Hamdard (Hamdard University). A voucher specimen (KB/ND/PL/UL/01) has been deposited in the Phytochemical Research Laboratory of this university.

Extraction and isolation

The dried and coarsely powdered plant material (2 kg) was extracted exhaustively with chloroform in a Soxhlet apparatus. Solvent was removed under reduced pressure in a Büchi rotavapor to give a yellowish mass. The extract was adsorbed on silica gel to form slurry and loaded on a silica gel column packed in petroleum ether. Elution was carried out with petroleum ether, petroleum ether–chloroform mixtures in varying proportions and chloroform to isolate the compounds. Elution of the column with petroleum ether afforded a sticky mass, which on purification afforded colorless amorphous powder of compound 1 (25 mg, 0.0013% yield). Petroleum ether–chloroform [(9:1), (4:1), (7:3), (5:5)] furnished compound 2 (45 mg, 0.00223% yield), compound 3 (30 mg, 0.0015% yield), compound 4 (30 mg, 0.0015% yield), and compound 5 (35 mg, 0.0018% yield), respectively. Elution of the column with chloroform yielded compound 6 (36 mg, 0.0018% yield). The physiochemical and spectral data of the isolated compounds are reported below.

2-Hydroxytricontane (1): Colorless amorphous powder, m.p. 92–95°C; IR data ν._max (KBr) cm⁻¹: 3400, 2920, 2850, 1464, 1380, 1270, 1060, 750, 723 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 3.59 (1H, m, CHOH), 1.52 (3H, d, CH₃-CHOH, J. = 6.5 Hz), 1.39 (1H, s, OH, D₂ O exchangeable), 1.25 (54H, brs, 27 × CH₂), 0.83 (3H, t, CH_3, J. = 6.5 Hz); ¹³C NMR (75 MHz, CDCl₃): δ 67.74 (C-2, CHOH), 23.86 (C-1, CH₃), 14.13 (C-30, CH₃), 39.72 (C-3), 32.04 (C-28, CH₂), 29.76 (C-26, CH₂), 29.70 (C-6, 7, CH₂), 29.54 (20 × CH₂), 29.41 (C-27, CH₂), 22.59 (C-29, CH₂); EIMS m/z. (rel. int. %): 438 [M]⁺ (2.1) (C₃₀H₆₂O), 423 (2.3), 395 (3.0), 393 (6.8), 365 (4.5), 337 (6.1), 309 (5.2), 98 (34.2), 97 (45.0), 85 (53.0), 83 (54.0), 71 (67.3), 69 (63.5), 57 (94.0), 55 (86.4), 45 (64.3), 43 (100), 41 (89.8).

7′-Carboxylic-n.-heptacosanyl-glutarate (2): Crystallized from petroleum ether–ethyl acetate, m.p. 76–77°C; IR data ν._max (KBr) cm⁻¹: 3350, 3260, 2919, 2850, 1737, 1680, 1473, 1376, 1172, 957, 920, 721 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): 2.26 (2H, t, J. = 7.44 Hz, H-2), 1.44 (1H, brs, H-3), 1.60 (2H, brs, H-4), 4.07 (2H, m, H-1′), 1.34 (2H, brs, H-2′), 1.30 (4H, brs, H-6′, 8′), 1.25 (38H, brs, 19 × CH₂), 1.17 (2H, brs, H-3′), 1.55 (1H, m, H-7′), 1.04 (2H, brs, H-13′), 0.87 (3H, t, J. = 6.0 Hz); ¹³C NMR (75 MHz, CDCl₃): δ 173.17 (C-1), 34.43 (C-2), 28.67 (C-3, C-4), 183.12 (C-5), 64.39 (C-1′), 31.92 (C-2′), 25.94 (C-3′), 25.04 (C-4′, C-5′), 29.69 (C-6′, C-8′), 39.81 (C-7′), 22.68 (C-9′-26′), 14.11 (C-27′), 182.36 (C-28′); EIMS m/z. (rel. int. %): 554 [M]⁺C₃₃H₆₂O₆(2.8),516 (8.1), 497 (6.8), 480 (16.3), 467 (10.4), 440 (4.9), 439 (8.4), 423 (5.3), 416 (15.6), 385 (36.7), 357 (29.8), 341 (1.5), 329 (31.1), 298 (33.6), 281 (6.3), 273 (78.9), 225 (1.3), 215 (1.2), 197 (1.3), 194 (30.7), 175 (8.6), 169 (1.5), 147 (33.1), 135 (17.1), 131 (16.1), 115 (11.3), 101 (41.2), 87 (41.4), 85 (60.5), 74 (61.3), 59 (99.8), 57 (50.1), 43 (100).

n.-Nonacosan-6-ol-1,13-dioic acid (3): Crystallized from petroleum ether–ethyl acetate; m.p. 120–122°C; IR: ν._max (KBr) 3435, 3350, 2921, 2850, 1680, 1465, 1380, 1064, 725 cm⁻¹; ¹H NMR (300 MHz, CDCl₃): δ 1.60 (1H, brs, H-13), 1.56 (2H, brs, H-2), 3.65 (1H, brm, w_1/2 = 16.2), 1.25 (28H, brs, 14 × CH₂), 1.27 (14H, brs, 7 × CH₂), 0.89 (3H, t, J. = 6.90 Hz, Me-29); ¹³C NMR (75 MHz, CDCl₃),: δ 183.11 (COOH), 32.42 (C-2), 62.62 (C-6), 25.30 (C-12), 39.00 (C-13), 22.17 (C-14), 29.19 (20 × CH₂), 31.44 (2 × CH), 12.68 (Me-29), 177.81 (C-30); EIMS m/z. (rel. int. %): 498 [M]⁺ C₃₀H₅₈O₅ (7.4), 469 (6.2), 439 (3.2), 425 (3.5), 410 (3.10), 397 (3.5), 380 (3.5), 367 (1.7), 352 (6.2), 287 (4.1), 283 (9.1), 273 (9.1), 247 (15.5), 225 (13.5), 215 (3.9), 211 (2.3), 201 (3.5), 189 (5.5), 174 (5.7), 145 (13.0), 131 (21.2), 115 (42.8), 101 (78.0), 87 (47.9), 73 (47.3), 59 (100), 57 (64.3), 45 (61.2).

4,12-Dimethyl-n.-tetradeca-6,8,10-triene-1-ol-4-oic acid (4): Colorless crystals from petroleum ether–chloroform, m.p. 152–153°C; UV data λ_max (CHCl₃) nm (log ε): 241 (4.2): 282 (4.7); IR data ν_max (KBr) cm⁻¹: 3410 3360, 2958, 2919, 1706, 1680, 1625, 1434, 1310, 1287, 1217, 1164, 1142, 1121, 1023, 983, 930; ¹H NMR data (300 MHz, CDCl₃): 4.23 (2H, t, J. = 5.81 Hz, H₂-1), 1.60 (2H, m, H₂-2), 2.66 (2H, brm, H₂-3), 2.90 (2H, d, J. = 7.21 Hz, H₂-5), 7.00 (4H, brm, H-6, 7, 10,11), 7.52 (1H, dd, J. = 3.33, 3.36 Hz, H-8), 7.71 (1H, dd, J. = 3.30, 3.33 Hz, H-9), 2.10 (1H, brm, H-12), 1.34 (2H, m, H-13), 0.87 (3H, dd, J. = 7.50, 7.50 Hz, H₃-14), 1.42 (3H, brs, H₃-15),1.21 (3H, d, J. = 6.50 Hz, H₃-17); ¹³C NMR data (75 MHZ, CDCl₃): 68.14 (C-1), 29.65 (C-2), 28.90 (C-3), 34.26 (C-4), 37.40 (C-5), 124.76 (C-6), 131.24 (C-7), 135.87 (C-8), 132.07 (C-9), 130.85 (C-10), 128.78 (C-11), 38.71 (C-12), 31.88 (C-13), 10.93 (C-14), 30.28 (C-15), 170.58 (C-16), 14.01 (C-17); EIMS, m/z. (rel. int.): 280 [M]⁺ (C₁₇H₂₈O₃) (22.3), 264 (100), 223 (4.2), 221 (11.0), 149 (10.1), 144 (5.6), 131 (4.2), 59 (5.6), 57 (36.3).

8,14-13,17-Diseco-stigmast-5,22-diene-3-α.-ol (5): Colorless crystals from petroleum ether–chloroform, m.p. 121–122°C; UV data λ_max (MeOH) nm (log ε): 240 (4.1); IR data ν_max (KBr) cm⁻¹: 3475, 2954, 2852, 1635, 1465, 1383, 1324, 1243, 1193, 1110, 1060; ¹H NMR data (300 MHz, CDCl₃): 1.25 (1H, dd, J. = 9.32, 3.40, 2.45, H-1_α.), 2.26 (1H, m, H-1_β.), 1.85 (1H, m, H-2_α.), 1.82 (1H, m, H-2_β.), 3.51 (1H, brm, W_1/2 = 10.71, H-3_β.), 2.02 (1H, m, H-4_α.), 1.94 (1H, m, H-4_β.), 5.35 (1H, brs, H-6), 2.18 (1H, brs, H-7_α.), 2.14 (1H, brs, H-7_β.), 2.28 (1H, brs, H-8_α.), 2.27 (1H, brs, H-8_β.), 1.44 (2H, brs, H₂-9), 1.37 (1H, brs, H-11_α.), 1.28 (1H, brs, H-11_β.), 1.14 (1H, brs, H-12_α.), 1.80 (1H, brs, H-12_β.), 1.54 (1H, m, H-13), 1.52 (1H, m, 13_α.), 1.51 (1H, m, 13_β.), 1.08 (2H, m, H₂-14), 1.65 (1H, m, 16_α.), 1.53 (1H, m, 16_β.), 2.23 (2H, brs, H₂-17), 0.67 (3H, d, J. = 5.40 Hz, H₃-18), 1.01 (3H, brs, H₃-19), 1.99 (1H, m, H-20), 0.93 (3H, d, J. = 6.30 Hz, H₃-21), 5.14 (1H, dd, J. = 8.46, 8.43 Hz, H-22), 5.02 (1H, dd, J. = 8.43, 8.40 Hz, H-23), 1.69 (1H, m, H-24), 1.48 (1H, m, H-25), 0.84 (3H, d, J. = 6.90 Hz, H-26), 0.82 (3H, d, J. = 6.27 Hz, H-27), 1.48 (2H, m, H₂-28), 0.91 (3H, d, J. = 6.03 Hz, H₃-29); ¹³C NMR data (75 MHZ, CDCl₃): 37.24 (C-1), 31.87 (C-2), 71.73 (C-3), 42.25 (C-4), 140.73 (C-5), 121.65 -(C-6), 29.68 (C-7), 33.92 (C-8), 50.12 (C-9), 36.12 (C-10), 21.06 (C-11), 39.75 (C-12), 45.81 (C-13), 28.22 (C-14), 24.29 (C-15), 25.37 (C-16), 26.07 (C-17), 11.84 (C-18), 19.79 (C-19), 56.04 (C-20), 18.76 (C-21), 138.28 (C-22), 129.25, (C-23) 56.74 (C-24), 29.14, (C-25), 19.36 (C-26), 19.01 (C-27), 23.05 (C-28), 11.97 (C-29): EIMS, m/z. (rel. int.): 416 [M]⁺ C₂₉H₅₂O (11.3), 401 (99.7), 398 (6.7), 383 (6.8), 331 (7.3), 305 (7.6), 277 (5.8), 263 (17.0), 259 (10.2), 227 (5.8), 214 (8.1), 193 (1.2), 175 (1.5), 167 (1.2), 160 (8.7), 144 (29.7), 139 (8.7), 130 (26.8), 111 (22.6), 101 (29.8), 85 (8.7), 83 (28.3), 72 (48.1), 54 (71.3), 43 (100).

β.-Sitosterol-glycoside (6): Colorless amorphous mass from chloroform, m.p. 315–320°C; UV data λ_max (MeOH) nm (log ε): 207 (4.15); IR data ν_max (KBr) cm⁻¹: 3440, 3300, 2951, 2930, 1620, 1459, 1375, 1121, 1055, 955, 850, 785; ¹H NMR data (300 MHz, DMSO-d.₆): δ 5.32 (1H, d, J. = 4.64 Hz, H-6), 4.80 (1H, brs, H-1′), 4.86 (1H, brs, w_1/2 18.0 Hz, H-3_α.), 4.25 (1H, d, J. = 5.02 Hz, H-5′), 3.69 (1H, m, H-2′), 3.65 (2H, m, H-3′ and H-4′), 3.09 (2H, m, H₂-6′), 1.24 (3H, brs, Me-19), 0.96 (3H, d, J. = 6.50 Hz, Me-21), 0.89 (3H, d, J. = 6.01 Hz, Me-26), 0.83 (3H, d, J. = 6.5 Hz, Me-27), 0.91 (3H, t, J. = 6.03 Hz, Me-29), 0.66 (3H, brs, Me-18); ¹³C NMR data (75 MHz, DMSO-d.₆): δ 36.80 (C-1), 30.54 (C-2), 73.37 (C-3), 40.32 (C-4), 140.30 (C-5), 121.09 (C-6), 29.23 (C-7), 31.37 (C-8), 49.58 (C-9), 35.45 (C-10), 20.54 (C-11), 40.45 (C-12), 45.14 (C-13), 56.14 (C-14), 23.81(C-15), 28.98 (C-16), 55.38 (C-17), 11.57 (C-18), 19.59 (C-19), 36.17 (C-20), 18.82 (C-21), 33.34 (C-22), 27.73 (C-23), 45.14 (C-24), 28.65 (C-25), 19.59 (C-26), 19.02 (C-27), 23.81 (C-28), 11.60 (C-29), 100.79 (C-1′), 76.55 (C-2′), 78.32 (C-3′), 78.76 (C-4′), 79.20 (C-5′), 61.09 (C-6′); Mass spectral data, EIMS, m/z. (rel. int.): 577 [M]⁺C₃₅H₆₁O₆ (4.2), 414 (3.9), 400 (3.1), 399 (15.3), 396 (11.5), 381 (6.7), 367 (3.6), 273 (3.0), 255 (32.5), 240 (3.8), 231 (8.1), 213 (23.2), 198 (11.6), 173 (14.3), 163 (15.9), 161 (24.4), 159 (32.6), 145 (53.3), 133 (41.6), 121 (32.3), 119 (32.5), 107 (53.1), 105 (50.3), 95 (49.2), 93 (39.8), 83 (5.6), 81 (70.1), 71 (31.2), 69 (7.3), 67 (52.5), 55 (85.3), 43 (100).

Results and Discussion

Compound 1 in its IR spectrum demonstrated bands at 3400, 2850, 1464, 1380, 1270, 1064, and 723 cm⁻¹ indicating the presence of hydroxyl group and the aliphatic nature of the compound. On acetylation, compound 1 gave a monoacetate (IR 1735 cm⁻¹), suggesting the presence of one hydroxyl group. The ¹H NMR spectrum of 1 showed a triplet at δ 0.83 (J. = 6.5 Hz) for a terminal methyl group and a doublet at δ 1.52 (J. = 6.5 HZ) assigned to the methyl group attached to carbinol carbon. The single methine proton appeared at δ 3.59 as a multiplet. The remaining methylenes were observed as a broad band appearing at δ 1.28 and integrating for 54 protons.

Appearance of a number of ion peaks at a regular interval of 14 mass units in the mass spectrum of 1 confirmed that the compound was an aliphatic alcohol. The appearance of fragment ions at m/z. 393 (M-C₂H₅) and m/z. 423 (M-CH₃) indicated the position of hydroxyl group at C-1. The ¹³C NMR spectrum further supported the above assignments by exhibiting signals at δ 14.13 for C-29 terminal methyl group and at δ 23.86 for methyl group attached to carbinol carbon. The carbinol carbon resonated at δ 67.74, with the rest of the methylene groups appearing at δ 39.72 (C-2), 32.04 (C-27), 29.76 (C-25), 29.70 (C-5, 6), 29.41 (C-26), 22.59 (C-28), and 29.54 (20 CH₂). On the basis of this evidence, 1 was found to be 2-hydroxytricontane (Mehta et al., 1999).

Compound 2 was assigned a molecular formula C₃₃H₆₂O₆ as indicated by a weak molecular ion peak at m/z. 554 in EIMS and the ¹³C NMR spectrum. It did not respond positively to tetranitromethane and bromine water suggesting the saturated nature of the molecule. It showed effervescence with sodium bicarbonate solution indicating the presence of carboxylic functionality. The presence of an ester carbonyl, carboxylic group, and the aliphatic nature of the molecule was revealed by absorption bands at 1737, 3350, 1680 and 2919, 2850, 1473, 1376, 1172, and 721 cm⁻¹, respectively, in its IR spectrum.

The ¹H NMR spectrum of 2 exhibited a three-proton triplet at δ 0.87 (J. = 6.0 Hz) for one terminal methyl group suggesting the presence of carboxylic group at the other terminal position. A broad singlet at δ 1.25 appeared for 46 methylene protons present in an identical environment. A triplet at δ 2.26 accounted for CH₂ adjacent to a carbonyl group, and a two-proton multiplet at δ 4.07 was assigned to an oxymethylene group. The spectrum exhibited a multiplet at δ1.55 for the C-7′ methine proton while three broad signals at δ1.60, 1.34, and 1.30 were assigned correspondingly to C-4, C-2′, C-6′, and C-8′ methylene protons. All these data suggested a long-chain aliphatic ester with two carboxylic groups attributable to C-7′ and C-5 positions. The position of the ester group was established by its mass fragmentation pattern. In the mass spectrum, the separation of most of the peaks by 14 mass units and appearance of C_nH_2n+1, C_nH_2n,, C_nH_2n-1 ion series confirmed its long-chain aliphatic nature (Dyer et al., 1984). Characteristic peaks of an ester group involving β.-fission of McLafferty rearrangement were observed at m/z. 74 and 480, and appearance of fragments at m/z. 87, 467, 115, 439, 131, 423 were observed due to α. and CO-O cleavage, which attested to the ester functionality at C-1 position (McLafferty, 1971). Further, the position of two carboxylic groups at C-5 and C-7′ was confirmed by significant fragment ions at m/z. 87, 74, 115, 215, 273 in its mass spectrum. The ¹³C NMR spectrum was in agreement with the ¹H NMR and MS assignments. It exhibited signals at δ183.12, 182.36, 173.17, 64.39, 39.81 for two carboxylic carbons, C-1 carbonyl carbon, C-1′ oxygenated carbon, and C-7′ methine carbon, respectively. Moreover, hydrolysis of compound 2 yielded an acid moiety, which was identified as glutaric acid on comparison with the authentic sample. Based on these cumulative findings, the structure of this new compound has been formulated as 7′-carboxylic-n.-heptacosanyl-glutarate.

Compound 3 was assigned a molecular formula C₃₀H₅₈O₅ as suggested by the molecular ion peak at m/z. 498 in EIMS and its ¹³C NMR spectrum. In its IR spectrum, it showed bands at 3435, 3350, 1680, 2921, 2850, 1465, 1380, 1064, and 725 cm⁻¹ indicating the presence of hydroxyl, carboxylic, and aliphatic chain. The positive reaction with sodium bicarbonate solution and acetylation of compound 3 to yield a monoacetate supported the presence of carboxylic group and a hydroxyl group in 3. The ¹H NMR spectrum of 3 exhibited only one signal as a three-proton triplet at δ0.89 (J. = 6.5 Hz) for one terminal methyl group, which ruled out the presence of second terminal methyl group in 3 and suggested the presence of one terminal carboxylic group. A broad signal at δ1.56 was assignable to methylene protons adjacent to carboxylic group. A one-proton multiplet was observed at δ 1.60, which was ascribed to one methine proton. A broad multiplet at δ3.65 was assigned to carbinol proton and its half bandwidth of 16.20 Hz, suggested its α.-orientation. The methylene protons resonated at δ1.27 and 1.25 as broad signals. These observations suggested 3 to be a long-chain secondary alcohol with two carboxylic groups. Appearance of a number of ions with a uniform difference of 14 mass units and the absence of fragment ion [M-CH₃]⁺ confirmed the straight chain of the molecule. The position of carboxylic groups and a secondary alcoholic group was determined by its mass fragmentation pattern. In its EI mass spectrum, the fragment ions at m/z. 87, 101, 215, 225, 273, 283 suggested the attachment of carboxylic groups at C-1 and C-13. The fragment ions at m/z. 101, 367, 131, 397 showed the position of secondary hydroxyl group at C-6. The ¹³C NMR spectrum supported the presence of two carboxylic groups at δ183.11 and 177.81 and a carbinol carbon at δ62.62. These cumulative observations led us to formulate the structure of 3 as n.-nonacosan-6-ol-1,13-dioic acid.

Compound 4 in its IR spectrum exhibited absorption bands for hydroxyl (3410 cm⁻¹), carboxylic group (3350, 1680 cm⁻¹), and unsaturation (1625 cm⁻¹). Its positive reaction with bromine water and sodium bicarbonate solution confirmed the existence of unsaturation and carboxylic group, respectively, in the molecule. The molecular ion peak at m/z. 280 in EIMS and ¹³C NMR spectrum were consistent with the molecular formula C₁₇H₂₈O₃. The loss of methyl group from M⁺ resulted in a fragment ion at m/z. 256. The other important fragments appearing at m/z. 221 and 59, 131 and 149, 223 and 57 due to C₃/C₄, C₄/C₅, and C₁₁/C₁₂ fission, respectively, suggested that 4 was a hydroxy acid with substituents at C-1 (OH), C-4 (COOH), C-4 (CH₃), C-6, C-8, C-10 (double bonds), and C-12 (CH₃). The ¹H NMR spectrum of 2 exhibited two one-proton double doublets at δ7.71 (J. = 3.30, 3.33 Hz) and 7.52 (J. = 3.33, 3.36 Hz) assigned correspondingly to C-9 and C-8 vinylic protons. Theother vinylic protons resonated as a four-proton broad signal at δ7.00. A two-proton triplet at δ4.23 (J. = 5.81 Hz) was due to C-1 oxymethylene protons. Two doublets at δ2.90 (J. = 7.21 Hz) and 1.21 (J. = 6.50 Hz) were ascribed correspondingly to C-5 methylene protons (attached to C-6 olefinic carbon) and C-17 secondary methyl protons, respectively. Three multiplets at δ2.66, 1.34, and 2.10 were assignable to C-3, C-13 (methylene), and C-12 (methine) protons, respectively. A three-proton double doublet at δ0.87 (J. = 7.50, 7.50 Hz) and a broad signal integrating for three protons at δ1.42 were associated with C-14 primary methyl and C-15 tertiary methyl protons, respectively. Thus, the possible structure of 4 could be 4,12-dimethyl-n.-tetradeca-6,8,10-trien-1-ol-4-oic acid. The ¹³C NMR spectrum supported the proposed structure. The signals at δ124.76, 131.24, 135.87, 132.07, 130.85, and 128.78 were assigned to C-6, C-7, C-8, C-9, C-10, and C-11 olefinic carbons. The acid carbonyl carbon and carbinol carbon appeared at δ170.58 and 68.14, respectively. This constitutes the first report of the occurrence of such compound in an Uraria. species.

Compound 5 responded positively to the Liebermann-Burchard reagent. Its molecular weight was established as 416 [M]⁺ relating to the molecular formula C₂₉H₅₀O on the basis of mass and ¹³C NMR data. Its IR spectrum showed the presence of hydroxyl group (3475 cm⁻¹) and unsaturation (1635 cm⁻¹). The mass spectrum exhibited the prominent fragment ions at m/z. 401 [M-CH₃]⁺, 398 [M-H₂O]⁺, 277 [M-side chain]⁺, and 259 [277-H₂O]⁺. These fragments suggested that it was a C₂₉ sterol possessing one double bond and a C₁₀ unsaturated side chain. The ion fragments at m/z. 54 [C_{1, 10}-C_{4, 5}-H₂O]⁺, 83 [C_1,2-C_{5, 10}-C_{6, 7}]⁺ indicated that the hydroxyl group was located in the ring A, which was placed at C-3 on biogenetic ground, and that the double bond was situated at C-5 position. The fragment ion at m/z. 85 [C₂₃-C₂₄]⁺ suggested the presence of a second double bond at C-22. The mass spectrum indicated the presence of an ethyl group in the side chain, which was placed at C-24 on the basis of biogenetic analogy, as well as similarities in chemical shifts of the protons and carbons of the side chain with the related compounds (Ansaria Ali, 2001). The fragment ion at m/z. 277 [C_17-C₂₀]⁺ and not m/z. 273 as in β.-sitosterol, a difference of 4 mass units, suggested two open rings in 3. The ion fragments at 193 [C₁₂-C₁₃]⁺; 263 and 167 [C₁₇-C₁₈] suggested that rings C and D were open. Therefore, 3 has an identical carbon framework as that of β.-sitosterol with C and D rings open (Gupta et al., 1992). The ¹H NMR spectrum exhibited a one-proton doublet at δ5.35 (J. = 4.65 Hz) assigned to H-6 vinylic proton. Two one-proton double doublets at δ5.14 (J. = 8.46, 8.43 Hz) and 5.02 (J. = 8.43, 8.40 Hz) were ascribed to C-22 and C-23 vinylic protons, respectively. A broad multiplet at 3.51 with W_1/2 10.71 Hz demonstrated the presence of 3 β.-methine proton (equatorial) interacting with C-2 (axial), C-2 (equatorial), and C-4 (axial) protons. Three doublets, integrating for three protons at δ0.93 (J. = 6.30 Hz), 0.84 (J. = 6.90 Hz), and 0.82 (J. = 6.27 Hz) were due to C-21, C-26, and C-27 secondary methyl groups, respectively, and a three-proton triplet at δ0.91 (J. = 6.03 Hz) was ascribed to C-29 primary methyl protons. Another three-proton doublet at δ0.67 (J. = 5.40 Hz) was assigned to C-18 secondary methyl protons. Hence, mass fragmentation pattern and ¹³C DEPT (Distortionless enhancement by polarization transfer) data confirm that ring D was open. C-19 tertiary methyl protons appeared as a broad signal at δ1.01. The presence of all the methyl protons in the region δ0.67–1.01 suggested that these functionalities were attached to saturated carbons. The ¹³C NMR and DEPT experiments suggested the presence of six methyl, twelve methylene, nine methine and two quaternary carbons, of which C-3 carbinol carbon, C-22 and C-23 olefinic carbons were observed at δ71.73, 138.28, and 129.25, respectively. In the ¹³C-¹H COSY (correlation spectroscopy)-90 spectrum, the correlation of C-5 with H₂-4, C-6 with H₂-7, C-19 with H₂-1 and H-9, C-18 with H₂-12, H₂-14, and H₂-17, and C-21 with H-20 and H-22 was observed, thus allowing complete correlation of the protonated carbon resonance with ¹H NMR spectrum and confirming the proposed structure. Treatment of 5 with Jones reagent yielded ketonic steroid. The ketone responded positively to a Zimmermann test for 3-oxo steroids, which in turn did not respond to NaBH₄ reduction to regenerate the parent alcohol, confirming the axial orientation of the hydroxyl group in 5 (Ali, 2001). On the basis of this evidence, the structure of 5 was elucidated as 8–14,13–17-diseco-stigmast-5,22-diene-3α.-ol.

Compound 6 was obtained as a colorless amorphous powder from chloroform eluents. Its structure was established, on the basis of spectral data (Ansari & Ali, 2001) and comparison with the authentic sample (m.p., m.m.p., TLC, Co-TLC) as stigmasta-5-en-3-β.-D-glucopyranoside (β.-sitosterol-β.-D-glycoside).