Diacylglycerol Acyltransferase-inhibitory Prenylated Flavonoids from Maackia amurensis Rupr

Experimental Section

General Experimental Procedures. UV spectra were taken in MeOH using a Shimadzu spectrophotometer. Nuclear magnetic resonance (NMR) spectra were obtained from a Varian Unity Inova 400 MHz spectrometer using acetone-d6 as solvents, with TMS as the internal standard. All accurate mass experiments were performed on a Micromass QTOF2 (Micromass, UK) mass spectrometer. Column chromatography was conducted using silica gel 60, Sephadex LH-20 and RP-18 For thin-layer chromatography, precoated TLC silica gel 60 F254 plates from Merck were used. HPLC runs were carried out using a Shimadzu System LC-10AD pump equipped with a model SPD-10Avp UV detector, and an Optima Pak® C18 column (10 × 250 mm, 10 m particle size, RS Tech Korea).

Bovine serum albumin and Trizma-base were obtained from Sigma Chemical Co (ST. Louis, MO, USA). [1-14C] oleoyl-CoA (250 μCi) was purchased from Amersham. For the cell culture, Dulbecco’s modified eagle medium (DMEM), ʟ-glutamine, kanamycin sulfate, and fetal bovine serum (FBS) were purchased from GIBCO-BRL (Gaithersburg, MD, USA).

Plant Material. The stem bark of M. amurensis (3.0 kg) was collected in Yanji, Jilin province, China, in May 2009. A voucher specimen of the plant (No. 2009510) was deposited at the College of Pharmacy, Beihua University, Jilin, China.

Extraction and Isolation. The stem bark (3.0 kg) of M. amurensis was extracted with MeOH at room temperature for 2 weeks and the solution was concentrated to obtain a crude extract (165.0 g, 66% inhibition at 30 μg/mL). This extract was suspended in H2O, partitioned successively with CHCl3. EtOAc and BuOH, and then the organic solvents were removed. A portion of the EtOAc-soluble fraction (10.0 g, 74% inhibition at 30 μg/mL) was chromatographed over a silica gel column using a gradient of CHCl3–MeOH (from 20:1, 19:1 to 0:1), and was separated into 10 fractions (Fr.1–Fr.10). Fr.5 (CHCl3–MeOH 10:1, 1.0 g, 77% inhibition at 30 μg/mL) was chromatographed over Silica gel, eluted with a stepwise gradient of CH2Cl2/MeOH (from 100:1, 80:1, 50:1 to 0:1) to afford 10 subfractions (Fr.5A–Fr.5J). Purification of Fr.5D (110.0 mg) by semipreparative HPLC using an gradient solvent system of 70-80% MeCN in H2O over 60 min to yield compounds 2 (4.4 mg) and 7 (6.4 mg). Fr.5F (123.0 mg) was purified by preparative HPLC using an isocratic solvent system of 50% MeCN in H2O over 30 min followed by 60% MeCN in H2O over 70 min to obtain compounds 5 (5.9 mg), 6 (5.1 mg), and 3 (3.1 mg). Fr.6 (84% inhibition at 30 μg/mL) was subjected to an RP-18 column and was eluted with MeOH-H2O (1:10, 1:2, to 10:1) to yield six fractions (F.6A-F.6F). The most active fraction, Fr.6C (171.0 mg), was further separated by a Silica gel column eluted with CHCl3-MeOH (40:1, 35:1, to 10:1) to yield 10 subfractions (Fr.6C1-Fr.6C10). Fr.6C8 was separated by HPLC, using a gradient of 20-30% MeCN in H2O as the mobile phase to produce compounds 1 (3.1 mg) and 4 (4.9 mg).

Compound 1: White amorphous powder; −28.10 (c 0.1, MeOH); UV (MeOH): λmax (log ε) 224 (4.29), 289 (4.21), 326 (3.43) nm; CD (c 0.55 MeOH): [θ]329 +3.25, [θ]301 −5.30, [θ]215 +15.74; 1H NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz, acetone-d6) spectral data see Table 1; HR-EI-MS m/z: 368.1261 [M]+ (calcd for C21H20O6, 368.1260).

Table 1.aChemical shifts in ppm relative to TMS; coupling constants (J) in Hz.

DGAT1 Assay. Preparation of microsomes from rat liver and measurement of the in vitro DGAT1 activity was measured as reported previously.12