LAUSR

The genus Incarvillea, composed of 16 species that occurred mainly in the Himalayas and southwest China, is notable for being a temperate and herbaceous genus of the primarily tropical and woody family Bignoniaceae [1]. Incarvillea arguta, belonging to Bignoniaceae, is distributed in the southwest area of China. As a folk herbal medicine among the Yi nationality in Yunnan Province of China (known as “Wabuyou”), I. arguta has been widely used for treating hepatitis, diarrhea, and cancer [2]. The documented effects of Incarvillea plants on inflammation have motivated a number of studies that have characterized the chemical constituents, including monoterpenoids, iridoids, sesquiterpenoids, triterpennoids, alkaloids, sterides, phenylpropanoids, flavonoids, and amides [3]. To explore the relationship of the main components with the documented effects in this plant, further investigation was conducted, which led us to the isolation of 10 compounds. The dried roots (20 kg) of I. arguta were extracted with 90% EtOH at room temperature three times. The extract was evaporated under reduced pressure to yield a dark brown residue (1.3 kg). The residue was dissolved in water to form a suspension and extracted successively with EtOAc. The EtOAc fraction (790 g) was submitted to chromatography over a silica gel column with a gradient CHCl3–MeOH (1:0 0:1) to give six fractions 1–6. Fraction 1 (401 g) was separated by silica gel column chromatography (CC, CH2Cl2–MeOH, 1:0 5:1) to yield three subfractions (Subfr. 1.1–1.3). Subfraction 1.2 was subjected to silica gel CC with chloroform–acetone (9:1) followed by crystallization (MeOH) to generate compound 1 (351 g). By the same procedure, 2 and 3 were obtained from subfractions 1.2 and 1.3, respectively. Fraction 2 (13 g) was submitted to silica gel CC eluted with petroleum ether–acetone (7.5:2.5) to yield three subfractions (2a–2c). Subfractions 2a–2b were chromatographed on silica gel eluted with chloroform–acetone (8.5:1.5) and further crystallized with acetone to yield compounds 4 (52 mg) and 5 (22 mg), respectively. Subfraction 2c (2.8 g) was separated on silica gel CC eluted with chloroform–acetone (9:1) to afford compound 6 (12 mg). Fraction 3 (27 g) was chromatographed over a silica gel column developed with petroleum–acetone (1:1) to furnish subfractions 3a–3d. Compound 7 (16 mg) was purified from subfraction 3b (1.6 g) with silica gel CC using petroleum–EtOAc (1:1) as eluent. Subfraction 3a (0.7 g) was chromatographed repeatedly over silica gel using chloroform–EtOAc (6:4) as eluent to afford compound 8 (10 mg). Subfraction 3c (3.8 g) was submited to silica gel CC eluting with chloroform–acetone (7:3) and further separated with silica gel CC eluting with chloroform–methanol (9:1) to afford compound 4 (110 mg). Subfraction 3d was chromatographed on MCI gel, eluting with a CH3OH–H2O gradient of increasing polarity, then chromatographed on silica gel columns with chloroform–acetone (6.5:3.5), and further purified using recrystallization with MeOH to afford compound 10 (22 mg). Based on the analysis of spectra including PMR and 13C NMR, compounds 1–10 were determined as -sitosterol (1), 23(Z)-9,19-cycloart-23-ene-3 ,25-diol (2), stigmast-4-en-6 -ol-3-one (3), oleanolic acid (4), ursolic acid (5), ursolic acid lactone (6), (+)-boschniakinic acid (7), daucosterol (8), ethyl caffeate (9), and luteolin (10). Compounds 2, 3, 9, and 10 were isolated from this plant for the first time. (23Z)-9,19-Cycloart-23-ene-3 ,25-diol (2), colorless needle crystals (acetone), C30H50O2. The PMR and 13C NMR spectra were published in [4]. Stigmast-4-en-6 -ol-3-one (3), colorless needle crystals (acetone), C29H48O2. The PMR and 13C NMR spectra were in accordance with those reported in the literature [5].