Cytotoxic Phenylpropanoids and a New Triterpene, Turformosinic Acid, from Turpinia formosana Nakai

One new phenylpropanoid, turformosin A (1), and one new triterpene, turformosinic acid (2), together with 16 known compounds, were isolated from the stems of Turpinia formosana Nakai. All structures were elucidated on the basis of spectroscopic analysis, including 1D- and 2D-NMR techniques and MS analysis. Selected isolated compounds were evaluated for in vitro cytotoxicity against four human cancer cell lines and antioxidant scavenging effects on DPPH. (−)-(7′S,8′S)-threo-carolignan X (3) exhibited cytotoxicity against Hep2, WiDr, Daoy, and MCF-7 cell lines with ED50 values of 3.60, 4.45, 6.07, and 13.7 μg/mL, respectively. Turformosin A (1), (−)-(7′S,8′S)-threo-carolignan X (3), methoxyhydroquinone-4-β-D-glucopyranoside (5), and methoxy-hydroquinone-1-β-D-glucopyranoside (6), exhibited similar anti-oxidative activity. Hep2 cells treated with 10 μg/mL of 3 showed elevation of sub-G1 population (from 20% at 8 h to 60% at 48 h), and activation of caspase-9/caspase-3/PARP cascade. Compound 3 induced intrinsic apoptotic pathway in Hep2 cells with dose and time dependence (10 μg/mL for 8 h).


Cell Cycle Analysis and Immunobloting
To investigate how 3 affects cell viability, we performed a cell cycle analysis. Hep2 cells treated with 10 μg/mL of 3 showed an elevating subG1 population (from 20% at 8 h to 60% at 48 h), but no difference in vehicle nor 5 μg/mL of 3 ( Figure 4A). Compound 3-induced apoptosis was determined by Western blotting. The cytosolic cytochrome c increased from 8 h to 24 h in the 10 μg/mL 3-treated cells ( Figure 4B). Furthermore, levels of procaspase-9, procaspase-3, and Bcl-2 were reduced in 3-treated cells ( Figure 4C). Then, caspase-3 mediated cleavage of PARP was also found ( Figure 4C). Taken together, these results suggest that compound 3 triggers apoptosis through the intrinsic apoptosis pathway.

General
The optical rotations were measured using a JASCO DIP-370 digital polarimeter. Infrared (IR) spectra were measured on a Mattson Genesis II spectrophotometer using a KBr matrix. UV spectra were measured a Hitachi U-3200 spectrophotometer. EI-MS were measured with JEOL Finnigan MAT TSQ-46C and JEOL SX-102A mass spectrometers. High-resolution mass spectra (HR-MS) were recorded by ESI MS on JEOL JMS-700 MStation and Hewlett-Packard 5989B mass spectrometers. 1 H and 13 C NMR spectra were obtained using Bruker Avance DRX 500 MHz and Bruker Avance 400 MHz spectrometers with tetramethylsilane (TMS) as the internal standard. Two-dimensional (2D) NMR experiments (HMQC, HMBC, and ROESY) were conducted using Bruker Avance DRX-500 and Bruker Avance 400 spectrometers. Sephadex LH-20 (Lipophilic Sephadex; Amersham Biosciences, Ltd.) and silica gel (230-400 mesh; Merck & Co., Inc.) were used for column chromatography, and pre-coated silica gel (60 F-254; Merck & Co., Inc.) plates were used for TLC. The spots on TLC were detected by spraying with 5% H 2 SO 4 and then heating at 100 °C. Preparative HPLC was performed using a reverse phase column (Cosmosil 5SL-II column, 250 mm × 20 mm i.d.; Nacalai Tesque, Inc.) on a Shimadzu LC-6AD series apparatus with RID-10A refractive index and Prominence HPLC UV-Vis detectors.

Plant Material
The stems of T. formosana Nakai were collected in Ping-Tung County, Taiwan, in May, 2007. This material was identified by one of the authors (I.-S. Chen). A voucher specimen (code No. KTF200705A) has been deposited at the National Research Institute of Chinese Medicine, Taipei, Taiwan.

Cytotoxicity Assay
Cytotoxicity against Hep2 (human laryngeal carcinoma), WiDr (human colon adenocarcinoma), Daoy (human medulloblastoma), and MCF-7 (human breast adenocarcinoma) cells was measured using a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, based on reported methods [33]. Briefly, the cells were cultured in RPMI-1640 medium supplemented with serum in an atmosphere of 5% CO 2 incubated at 37 °C. Test samples and the control drug standard were prepared at concentrations of 1, 10, 20, and 40 μg/mL. After seeding 2,880 cells/well in a 96-well microplate for 4 h, 20 μL of sample or standard agent was placed in each well and incubated at 37 °C for 3 days. Twenty L of MTT were added, and incubation continued for 5 h. After removing the medium and adding DMSO (200 μL/well) into the microplate with shaking for 10 min, the formazan crystals (the product of MTT reacting with dehydrogenase existing in mitochondria) were re-dissolved, and their absorbance was measured on a model MR 7000 microtiter plate reader (Dynatech International Corporation, Edgewood, New York, NY, USA) at a wavelength of 550 nm. The ED 50 was defined as the concentration of test sample resulting in 50% reduction of the absorbance found with the untreated cells.

DPPH Radical Scavenging Activity
The stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was used for the determination of free radical-scavenging activity of the extracts and compounds. Test compounds (1-6, 9-16, and 18) (120 μL) were added to 30 μL of DPPH (0.75 mM). After 30 min at room temperature, the absorbance was recorded at 520 nm. The experiment was repeated three times. Radical-scavenging activity (%) was calculated by the following formula: {[Ab − (A − As)]/Ab} × 100, where Ab is the absorbance without sample, A is the absorbance with compound and DPPH, and As is the absorbance with compound only [34]. If the activity of the test compound was more than 70%, the ED 50 value was calculated.

Cell Cycle and DNA Content Analysis
Cells were seeded in a 6-cm dish and cultured overnight. Cells were incubated with fresh medium containing compound 3. At the indicated time points, attached cells were trypsinized and combined with those in the supernatant. Cells were fixed with 70% EtOH at −20 °C and treated with 0.1% Triton X-100 for 30 min at room temperature. Then, cells were incubated with 50 μg/mL of RNase A and 50 μg/mL of propidium iodide (Sigma, St. Louis, MO, USA) in PBS at room temperature for 15 min. The relative proportions of cells in the G 1 , S, and G 2 /M cell-cycle phases were estimated by compartment analysis of DNA fluorescence using fluorescence-activated cell sorter (FACS) flow cytometry (Becton Dickinson, San Jose, CA, USA). Data were analyzed using the CellQuest software (Verity Software House Inc., Topsham, ME, USA).

Preparation of Mitochondrial and Cytosolic Fractions
After being cultured overnight, cells were incubated with fresh medium containing compound 2. At the indicated time points, cells were trypsinized and washed twice with ice-cold PBS. Cells were resuspended in ice-cold extraction buffer [20 mM HEPES, pH 7.4, 10 mM KCl, 250 mM sucrose, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1× protease inhibitor cocktail (Merck)] and incubated for 10 min at 4 °C. Cells were homogenized with 15 strokes of a prechilled homogenizer, and the homogenates were sequentially centrifuged at 1,000 × g and 12,000 × g for 5 and 30 min, respectively, at 4 °C. The supernatant was collected as the cytosolic fraction. The pellet was re-suspended with ice-cold lysis buffer [20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM dithiothreitol, 1× protease inhibitor cocktail (Merck)] and incubated for 20 min at 4 °C. The lysates were centrifuged at 15,000 × g for 5 min, and the supernatant was collected as the mitochondrial fraction.

Immunoblotting
Samples were separated by SDS-PAGE and transferred to PVDF sheets. Membranes were blocked with 5% non-fat milk dissolved in PBST buffer [137 mM NaCl, 3 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , 0.1% (v/v) Tween-20] for 1 h and incubated with the primary antibody at 4 °C overnight. Then, membranes were washed thoroughly with PBST buffer and incubated for 1 h with the secondary antibody at room temperature. Immunoreactive protein was visualized using Immobilon western chemiluminescent HRP substrate according to the manufacturer's protocol (Millipore, Billerica, MA, USA). Antibodies against Bcl-2, Bcl-xL, and Bax were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against caspase-9 and PARP were obtained from BD Pharmingen (San Diego, CA, USA). The anti-caspase-3 antibody was purchased from Millipore (Billerica, MA, USA). Horse radish peroxidase (HRP)-conjugated anti-mouse IgG and HRP-conjugated anti-rabbit IgG were obtained from Jackson ImmunoResearch, Inc. (West Grove, PA, USA).