A New Ursane-Type Nor-Triterpenoid from the Leaves of Eucommia ulmoides Oliv.

A new ursane-type nortriterpenoid, (11S,12S)-4-methyl-11,12-epoxy-2-hydroxy-3-oxoursa-1,4-dine-28-oic acid γ-lactone (1), named ulmoidol A, together with ten known compounds: ulmoidol (2), corosolic acid (3), 2α,3α-dihydroxy-24-nor-4(23),12-oleanadien-28-oic acid (4), oleanolic acid (5), ursolic acid (6), cycloart-3β, 25-diol (7), foliasalacioside B1 (8), (6R,7E,9R)-9-hydroxy-4,7-megastigmadien-3-one-9-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside (9), (6R,7E,9R)-9-hydroxy-4,7-megastigma-dien-3-one-9-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside (10), and quercetin 3-O-sambubioside (11) were isolated from the leaves of Eucommia ulmoides Oliv. The structure of compound 1 was determined by extensive spectroscopic analysis, and its absolute configuration was determined by CD experiments and a computational method. Compounds 3, 4, 7–10 were isolated from this plant for the first time. Compounds 3 and 4 showed inhibition to PTPIB activities, with IC50 values of 0.69 and 3.98 μM, respectively.


Introduction
Eucommia ulmoides Oliv. mainly grows along the Yangtze River and in southern China. Its bark has traditionally been applied in China as an antihypertensive, diuretic, sedative, tonic and nourishing agent [1]. The pharmacological effects of E. ulmoides are recorded as strengthening the internal organs, bones and muscles, and preventing senescence [2]. The extract of its leaves also showed activities against hypertension and bacteria [2,3]. Previous chemical investigations on this plant have resulted in the isolation of a series of lignanoids [4][5][6][7], phenylpropanolds [8][9][10], iridoids [9,11], flavones [11,12], guttapercha [2], polysaccharides [2], and terpenes [13,14]. As part of a program to study the chemical diversity of traditional Chinese medicines and their biological effects, an ethanol extract of E. ulmoides has been investigated. We describe herein the isolation, structure elucidation of a new ursane-type nortriterpenoid, ulmoidol A (1), and ten known compounds 2-11 from the EtOAc-soluble portion of the ethanol extract of E. ulmoides (Figure 1). On the basis of theoretical calculations of its electronic circular dichroism (ECD), the absolute configuration of compound 1 was also established. Compounds 3 and 4 showed inhibition to PTPIB activities with IC 50 values of 0.69 and 3.98 μM, respectively.

General Procedures
Optical rotations were measured on a P2000 automatic digital polarimeter. UV spectra were taken with a Hitachi UV-240 spectrophotometer. CD spectra were measured on a JASCO J-815 spectro-polarimeter. IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer. NMR measurements were performed on INOVA-500 and Bruker AV500-III spectrometers. HRESIMS were obtained using an Agilent 1100 series LC/MSD Trap SL mass spectrometer. Preparative HPLC was carried out on a Shimadazu LC-6AD instrument with a SPD-20A detector, using a YMC-Pack ODS-A column (250 × 20 mm, 5 µm). Column chromatography (CC) was performed with silica gel (200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China) and ODS (50 µm, YMC, Tokyo, Japan). TLC was carried out with glass precoated silica gel GF 254 plates.

Plant Material
The

Extraction and Isolation
The dried leaves of E. ulmoides (2.0 kg) were powdered and extracted with 95% ethanol (30 L × 3) under reflux. The filtrate was evaporated under reduced pressure to yield a dark brown residue (320 g). The residue was suspended in water (2,000 mL) and then successively partitioned with EtOAc (3 × 1,000 mL) and n-BuOH (3 × 1,000 mL). After removing the solvent, the EtOAc-soluble portion (120 g) was fractionated via silica gel CC eluting with a CHCl 3 -MeOH gradient (100:0-3:1) to afford ten fractions A 1 -A 10 on the basis of TLC analysis. Fraction A 2 (16.301 g) was chromatographed over silica gel (200-300 mesh) eluted with a CHCl 3 -MeOH gradient (100:1, 50:1) to give 2 (25 mg), 3 (8 mg), 4 (8 mg), 5 (450 mg), 6 (360 mg), and 7 (9 mg) and a mixture (20 mg [25] Recombinant human GST-PTP1B protein was overexpressed by hGST-PTP1B-BL21 E. coli and purified by GST affinity chromatography. The reagent pNPP was used as substrate for the measurement of PTP1B activity. Compounds were pre-incubated with the enzyme at room temperature for 5 min. Assay was performed in final volume of 100 μL in the active system containing 50 Mm HEPES, 5 mM DTT, 150 mM NaCl, 2 mM EDTA, and 2 mM pNPP (pH 7.0), incubated at 30 °C for 10 min, stopped by addition of 50 μl 3 M NaOH. Then, the absorbance was determined at 405 nm wavelength. The similar system without GST-PTP1B protein was used as blank. The effects of compounds 1-6, 8-10 on PTP1B activity were measured, and the IC50 value was calculated by nonlinear regression.

Conclusions
A new ursane-type nortriterpenoid, ulmoidol A (1), together with ten known compounds were isolated from the leaves of Eucommia ulmoides Oliv. The structure of compound 1 was determined by extensive spectroscopic analysis, and the absolute configuration was determined by CD experiments and computational methods. Compounds 3, 4, 7-10 were isolated from this plant for the first time.
Compounds 1-6, and 8-10 were tested for inhibition of PTP1B activities, and compounds 3 and 4 showed inhibit activities with IC 50 values of 0.69 and 3.98 μM, respectively.