Lupane and Ursane-Type Triterpenoids from Pergularia tomentosa

Pergularia tomentosa L. (Apocynaceae) grows naturally as a perennial twining herb with much milky latex in semi-arid zones of the Sahel and Arabian Peninsula where preparations from its root and shoot are used to treat skin diseases and other ailments [1]. It is a member of the subfamily Asclepiadoideae [2] and is often classified as a toxic plant that causes spasm and gastroenteritis when eaten [3]. P. tomentosa showed antitumor [4], molluscicidal [5], and hypoglycemic [6] properties. Cardenolides [4, 7] and taraxasterol-type triterpenes [8] have been isolated from the root and leaf. The cardenolides and alkaloids pergularinine and tylophorinidine are responsible for the cytotoxic, antitumor, and antimicrobial properties [7, 9] of the Asclepiadaceous plants.

In this paper we report lupane 1, ursane triterpenoids 2 and 3, and a triacylglycerol (4) as new analogues from this plant, along with stigmast-5-en-3-O-β-glucoside (5). The stem of P. tomentosa was collected from the plant growing in Al-Rusayl Muscat near the armed forces hospital in September 2015. The plant was identified by comparison with a voucher specimen previously deposited at the herbarium of Biological Sciences Department at Sultan Qaboos University.

The air-dried powdered stem of P. tomentosa (1 kg) was extracted sequentially by maceration with petroleum ether (7.5 L × 2), EtOAc (7.5 L × 2), and EtOH (7.5 L × 2), each for one week, to afford 33.5 g residue of petroleum ether, 64.0 g of EtOAc, and 4.2 g of EtOH extract, respectively. The EtOAc extract (15.8 g) was purified on silica gel (120 g) and eluted using gradients of Me₂CO in hexane as mobile phase to give fractions 1–30. Fraction 10 (10.6 g) eluted with petroleum ether–Me₂CO (9:1) had a distinctive ester smell. It was further purified on silica gel (120 g), eluting with gradients of CHCl₃ in petroleum to give fractions 10-1–10-44. Fraction 10-22 gave 20.9 mg of 3β-O-acetyl lupeol (1) [10], with R_f 0.85 (CHCl₃–hexane, 1:3), and fraction 10-15 gave 11.27 mg of 3β-O-acetyl-α-amyrin (2), R_f 0.47 (CHCl₃–hexane, 1:3). A portion of fraction 12–14 (63.0 mg, yellowish brown semisolid) gave 6.0 mg of triacylglycerol (4) [11] after purification by prep TLC. The MALDI-TOF mass spectral data revealed the molecular formula C₅₆H₉₆O₆ for compound 4. The ion peak at m/z 886.690 amu [M + Na]⁺ confirmed the presence of a C₃ glyceryl substructure and two C₁₈ and one C₁₇ fatty acyl groups in compound 4. The peaks at m/z 638.451 and 378.416 resulted from loss of the C₁₇ fatty acyl group at β (2) from [M + Na]⁺ and C₁₈ fatty acyl group at α (1 or 3) from the resulting daughter ion. The EtOH extract (4.2 g) was loaded on silica gel (20 g) and eluted with hexane, CHCl₃, EtOAc, and EtOAc–EtOH (9:1) in that order. The fraction eluted with EtOAc afforded a white solid (6.6 mg) of stigmast-5-en-3-O-β-glucoside (5) [12] after trituration with Me₂CO, CHCl₃ and EtOAc.

The petroleum ether fraction (11.9 g) was also loaded on silica gel (120 g) and eluted with gradients of Me₂CO in hexane to give fractions PT-1–PT-24. Fraction PT-10 (3.2 g) was purified on silica gel (40 g) using gradients of CHCl₃ in hexane as mobile phase to afford fractions PT-10-1–PT-10-56. Fractions PT-10-41–PT-10-44 gave 11.2 mg of compound 3 [10], R_f 0.07 (hexane–CHCl₃, 3:2).

Compounds 1–3 are low-level oxidation triterpenes and are reported for the first time from P. tomentosa. In contrast to the leaf and root, the stem of the plant is rich in triterpenoids.

The EtOAc extract inhibited Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli at 250 μg/mL but none of compounds 1–3 showed strong antimicrobial activity [13] (inhibitory concentration > 125 μg mL^–1), significant DPPH free radical scavenging activity [14] (percentage inhibition (%IP) < than 10%), and lethality to brine shrimp larvae [15] (BST LC₅₀ > 1000 μg mL^–1).

3 β - O -Acetyl lupeol (1), mp 199.8–201.4°C. IR (neat, ν, cm^–1): 2931, 1731, 1636, 1450, 1363, 1241, 1023, 977, 874. For ¹H and ¹³C NMR, see Table 1. MALDI-TOF-MS m/z 491.2380 [M + Na]⁺ (calcd for C₃₂H₅₂O₂Na, 491.3865).

Table 1. ¹H (700 MHz) and ¹³C (176 MHz) NMR Data for Compounds 1–3 (CDCl₃, δ, ppm, J/Hz)

3 β - O -Acetyl- α -amyrin (2), mp 232.9–234.8°C. IR (neat, ν, cm^–1): 2927, 1733, 1610, 1456, 1366, 1243, 1025, 985, 903. For ¹H and ¹³C NMR, see Table 1. MALDI-TOF-MS m/z 491.1930 [M + Na]⁺ (calcd for C₃₂H₅₂O₂Na, 491.3865).

α -Amyrin (3), mp 166.8–169.4°C. IR (neat, ν, cm^–1): 3294, 2849, 1464, 1387, 1189, 1036, 995. ¹H and ¹³C NMR, see Table 1.

Triacylglycerol (4). IR (neat, ν, cm^–1): 2910, 2850, 1457, 1365, 1057, 1097, 721. ¹³C NMR (176 MHz, CDCl₃, δ ppm): Ln1: 173.5 (C-1), 34.2 (C-2), 25.0 (C-3), 29.2 (C-4), 29.3 (C-5), 29.3 (C-6), 29.7 (C-7), 27.3 (C-8), 130.2 (C-9), 128.0 (C-10), 25.8 (C-11), 127.9 (C-12), 130.8 (C-13), 27.3 (C-14), 29.4 (C-15), 32.1 (C-16), 22.8 (C-17), 14.3 (C-18); 62.2 (C-1′), 68.3 (C-2′), 62.2 (C-3′); Ln3: 173.4 (C-1), 34.2 (C-2), 24.9 (C-3), 29.2 (C-4), 29.3 (C-5), 29.3 (C-6), 29.6 (C-7), 27.3 (C-8), 130.2 (C-9), 128.0 (C-10), 25.8 (C-11), 127.9 (C-12), 130.7 (C-13), 27.3 (C-14), 29.3 (C-15), 32.1 (C-16), 22.8 (C-17), 14.3 (C-18); Ln2: 173.0 (C-1), 34.3 (C-2), 24.6 (C-3), 29.2 (C-4), 29.3 (C-5), 29.3 (C-6), 29.5 (C-7), 27.3 (C-8), 130.2 (C-9), 128.0 (C-10), 25.8 (C-11), 127.9 (C-12), 130.4 (C-13), 32.0 (C-14), 31.7 (C-15), 22.7 (C-16), 14.2 (C-17). MALDI-TOF-MS m/z 887.6900 [M +Na]⁺ (calcd for C₅₆H₉₆O₆Na, 887.7105).

Stigmast-5-en-3- O - β -glucoside (5), mp 208.9°C. IR (neat, ν, cm^–1): 3418, 2960, 1662, 1421, 1348, 1071, 1017, 672. MALDI-TOF-MS m/z 599.5290 [M + Na]⁺ (calcd for C₃₅H₆₀O₆Na, 599.4288).