The EtOH extracts of dry D. divaricata Blume rhizomes were successively partitioned which resulted in isolation of a crystalline white powder, m.p. 283–284 °C; : +3.6° (c = 0.87, CHCl₃). The LC-MS/MS analysis revealed a major peak at m/z 439.3 (M−H⁻) with very little fragmentation that confirmed the molecular formula to be C₃₀H₄₈O₂ (MW 440). Further MS/MS analysis yielded very little fragment ions, which agrees with the triterpene compound nature. The compound was therefore identified as davallic acid (Figure 1) [11] based on its ¹H- and ¹³C-NMR spectrum (Table 1) along with data comparisons with literature data.

The effect of davallic acid in inhibiting A549 cell viability is shown in Figure 2. The results indicate that davallic acid (5, 10, and 20 μM) decreased A549 cell viability within 12 and 24 h of treatment to 73% and 57% with 20 μM davallic acid, respectively. These results suggest that davallic acid exhibits potential apoptotic induction, thereby repressing cell proliferation. Moreover, we evaluated the apoptosis index of A549 cells treated with davallic acid.

The sub-G₁ phase, in which DNA fragmentation peaks, represents apoptosis generation [12,13,14]. The sub-G₁ phase was significantly increased by 34% and 42% following treatment with 10 and 20 μM davallic acid for 24 h, respectively, as compared to the control group (Figure 3).

Cells were stained with DCFH-DA to examine the production of ROS in the A549 cells, and DCF fluorescence was measured by a flow cytometer. As shown in Figure 4, both 10 and 20 μM davallic acid significantly increased ROS levels after 12 and 24 h of treatment as compared to the control group. These results revealed that davallic acid induced apoptosis and suppressed cell viability by promoting the ROS levels, activating apoptotic signals in A549 cells. Therefore, we further investigated the apoptotic pathway induced by davallic acid.

We observed that 10 and 20 μM davallic acid significantly increased cytosolic Bax levels in A549 cells treated for 24 h (Figure 5A). Furthermore, these treatments potentially suppressed a decrease in cytosolic Bcl-2 in A549 cells (Figure 5B). Moreover, we noted a significant increase in cytosolic cytochrome c in A549 cells treated with davallic acid (10 and 20 μM) for 24 h as compared to the control group (Figure 5C). These results suggest that davallic acid induced mitochondrial membrane damage and membrane potential loss, enhancing Bax expression and inhibiting Bcl-2 expression, subsequently leading to cytochrome c release.

Apoptosis is regulated by two main pathways: The TNF-α receptor pathway and the mitochondrial pathway, both of which involve the activation of caspase-3. Caspase-8 and caspase-9 are activators/initiators of the TNF-α receptor and mitochondrial pathways, respectively. The activities of caspase-3, caspase-8, and caspase-9 in A549 cells treated with davallic acid are shown in Figure 6A–C, respectively. Davallic acid (10 and 20 μM) treatment for 24 h significantly promoted the activation of caspase-3, caspase-8, and caspase-9 in the A549 cells as compared to that in the control group.

¹H- and ¹³C NMR spectra were obtained on a Bruker AM-500 spectrometer in CDC1₃ solution, using the corresponding solvent peak as the internal standard. Optical rotation was measured on a Jasco DIP-1020 digital polarimeter. Column chromatography was carried out with Sephadex LH-20 (25–100 µM, Pharmacia Fine Chemicals). TLC was conducted on silica gel plates (60 F-254, Merck) and 10% sulfuric acid solution was used as a visualizing agent following heating. Propidium iodide (PI), sodium dodecyl sulfate (SDS), Triton X-100, and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Life Technologies (Auckland, New Zealand). Dimethyl sulfoxide (DMSO) was purchased from Wako Pure Chemical Industries (Saitama, Japan). Ham’s F-12, sodium bicarbonate, hydrocortisone, penicillin, and streptomycin were purchased from HyClone Laboratories (Logan, UT, USA).

The plant, D. divaricata Blume, collected in Taipei, Taiwan, in July 2010 was kindly supplied for this study by The Forestry Bureau of Taiwan. The sliced rhizomes were used for the extraction of davallic acid. The rhizomes (25 kg) were extracted with 80% EtOH (10 L) three times at room temperature. After evaporating the solvents under vacuum at 40 °C, a residue was obtained. This residue was dissolved in H₂O and then extracted successively with hexane, ethyl acetate, and n-BuOH. The hexane extract (62 g) was fractionated on a Sephadex LH-20 column and eluted with EtOH to yield fractions A-D. The fraction C (5 g) was recrystallized from hexane/acetone. Finally, the sediment was washed with EtOH to give 1.5 g of davallic acid. The structure of the isolated compound was elucidated using Bruker Avance DRX 500 NMR and Agilent 1200/6460 LC-MS-MS for mass spectra confirmation.

A549 lung cancer cells were obtained from the Bioresource Collection and Research Center (BCRC) in Taiwan (Hsinchu, Taiwan), and cultured on F12-K medium supplemented with 10% heat-inactivated FBS and antibiotics (100 unit mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin). Cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂.

A549 cells were seeded into 48-well plates in 1 mL of F-12K medium. The cells were treated with different concentrations of samples. At the end of incubation, the cells were washed with PBS, and the supernatants were exchanged with 1 mL of medium and MTT (0.5 mg mL⁻¹) to react for 2 h at 37 °C. After washing with PBS, MTT tetrazolium was dissolved with DMSO and measured at 570 nm [10].

A549 cells (2 × 10⁶ cells/per well) were treated with various concentrations of davallic acid for different durations of time (12 h and 24 h). The collected cells were suspended in 500 µL of PBS, mixed with 10 µM 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) and incubated for 20 min at 37 °C. Afterwards, the cells were washed thrice to remove DCFH-DA. The cell pellet was then mixed with 500 µL of PBS and the ROS level was assayed by flow cytometry.

After exposure to davallic acid, the medium was aspirated and adherent cells were harvested and centrifuged at 300 × g for 5 min. Cells were washed with PBS, fixed with ice-cold ethanol at −20 °C overnight and then stained with PI at room temperature for 30 min. The cell cycle distribution was analyzed by flow cytometry using a FACScan-LSR flow cytometer equipped with CellQuest software (BD Biosciences, San Jose, CA, USA) [10].

A549 cells were lysed in ice-cold lysis buffer containing 20 mM of Tris-HCl (pH 7.4), 1% of Triton X-100, 0.1% of SDS, 2 mM of EDTA, 10 mM of NaF, 1 mM of phenylmethylsulfonyl fluoride (PMSF), 500 μM of sodium-vanadate, and 10 μg/mL of aprotinin overnight. Then, cell lysates were sonicated with ice cooling (four times each 15 s) and centrifuged (12,000 × g, 10 min) to recover the supernatant. The supernatant was taken as the cell extract. The protein concentration in the cell extract was determined using a Bio-Rad protein assay kit. The samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein spots were electrotransferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with a block buffer for 1 h, washed with PBS containing 0.05% Tween-20 (PBST) three times, and then probed with 1:5,000 diluted solution of anti-caspase-3, anti-procaspase-3, anti-caspase-8, anti-procaspase-8, anti-caspase-9, anti-procaspase-9, 1:2,000 diluted solution of anti-cytochrome, anti-Bcl-2, anti-Bax, and anti-GAPDH (1:5,000) (Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C. The membrane was washed three times each for 5 min in PBST, shaken in a solution of HRP-linked anti-rabbit IgG secondary antibody, and washed three more times each for 5 min in PBST. The expressions of proteins were detected using enhanced chemiluminescent (ECL) reagent (Millipore, Billerica, MA, USA).

Data were expressed as mean ± standard deviation (SD). Statistical significance was determined by one-way analysis of variance (ANOVA) using the general linear model procedure of SPSS Version 17.0. [31], followed by ANOVA with Duncan’s test. All comparisons were performed relative to controls, and significant differences are indicated.