Phytochemical studies on Inula hookeri

Abstract

The genus Inula, a variable perennial herb distributed in Asia, Europe, Africa, and, predominantly, in the Mediterranean, comprises ca. 100 species of the Compositae family belonging to the tribe Inuleae [1]. I. hookeri C. B. Clarke is a traditional medicinal plant used to treat myoneuralgia in folk herbal medicine of Tibet, China. In order to study the chemical constituents of the plants, 27 compounds were isolated and identified. All the compounds were isolated from this plant for the first time. The whole plants of I. hookeri were collected from Weixi country, Yunnan Province, P. R. China, in August 2010. The botanical identification was made by Prof. Zhang Hanmin, Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai, P. R. China. A voucher specimen (No. CX 20100801) was deposited at School of Pharmacy, Shanghai Jiao Tong University, Shanghai, P. R. China. The air-dried whole parts of I. hookeri (10.0 kg) were powdered and percolated with 95% EtOH at room temperature (30 L 5 h 3). The combined filtrates were concentrated under reduced pressure to give a crude extract (0.8 kg), which was further suspended in H2O and then extracted with petroleum ether (PE, 60–90 C), EtOAc, and n-BuOH successively. The EtOAc extract (130.5 g) was subjected to silica gel (100–200 mesh, 1.3 kg) column chromatography (CC, 10 100 cm) eluting with a CH2Cl2–MeOH gradient system (100:0, 100:1, 50:1, 20:1, 10:1, 5:1, and 0:1, v/v) to give fractions A–H according to TLC analysis. Fractions A–F (12.1 g, 6.1 g, 12.6 g, 19.0 g, 4.1 g, and 9.3 g) were applied to MCI gel CC (4 40 cm) eluting with 80% MeOH to afford subfractions A1–F1 (10.1 g, 4.3 g, 9.5 g, 10.0 g, 3.0 g, and 7.2 g), respectively. Subfraction A1 was further purified over Sephadex LH-20 CC (3 100 cm) eluting with CH2Cl2–MeOH (1:1), yielding compounds 5 (9.3 mg), 7 (6.0 mg), 26 (10.0 mg), and 27 (12.2 mg). Subfraction B1 was further purified over Sephadex LH-20 CC (3 100 cm) eluting with CH2Cl2–MeOH (1:1), followed by preparative HPLC (65% MeOH), to yield compounds 4 (5.0 mg), 8 (3.2 mg), 9 (4.1 mg), and 16 (4.5 mg). Subfraction C1 was further purified over Sephadex LH-20 CC (3 100 cm) eluting with CH2Cl2–MeOH (1:1), followed by preparative HPLC (50% MeOH), to yield compounds 2 (6.4 mg), 3 (7.3 mg), and 13 (5.2 mg). Subfraction D1 was further purified over Sephadex LH-20 CC (3 100 cm) eluting with CH2Cl2–MeOH (1:1), followed by preparative HPLC (45% MeOH), to yield compounds 6 (4.6 mg), 14 (5.3 mg), 17 (4.2 mg), 20 (4.6 mg), and 25 (7.3 mg). Subfraction E1 was further purified over Sephadex LH-20 CC (3 100 cm) eluting with MeOH, followed by preparative HPLC (40% MeOH), to yield compounds 1 (6.6 mg), 10 (3.3 mg), 11 (7.3 mg), 12 (5.2 mg), 15 (8.6 mg), 18 (9.5 mg), 23 (7.5 mg), and 24 (5.8 mg). Subfraction F1 was further purified over Sephadex LH-20 CC (3 100 cm) eluting with MeOH, followed by preparative HPLC (35% MeOH), to yield compounds 19 (5.6 mg), 21 (8.3 mg), and 22 (7.2 mg). Compound 1. Colorless oil, C14H20O5. ESI-MS (positive) m/z 291 [M + Na]+; ESI-MS (negative) m/z 267 [M – H]–. The above data were consistent with those reported in [2], and 1 was identified as 8,10-dihydroxy-9-isobutyryloxythymol. Compound 2. Colorless oil, C18H26O6. ESI-MS (positive) m/z 361 [M + Na] +; ESI-MS (negative) m/z 337 [M –H]–. The MS and NMR data were in agreement with those reported in [2], and 2 was identified as 8-hydroxy-9,10diisobutyryloxythymol. Compound 3. Colorless oil, C19H28O6. ESI-MS (positive) m/z 375 [M + Na]+; ESI-MS (negative) m/z 351 [M – H]–. The above data were consistent with those reported in [3], and 3 was identified as 8-hydroxy-9-[(isobutyryl)oxy]-10-(2methylbutanoyl)thymol.