Novel Oleanane-Type Triterpene Glycosides from the Saponaria officinalis L. Seeds and Apoptosis-Inducing Activity via Mitochondria

Saponaria officinalis L., commonly known as “Soapwort”, is a rich source of triterpene glycosides; however, the chemical constituents of S. officinalis seeds have not been fully identified. In this study, we conducted a systematic phytochemical investigation of the seeds of S. officinalis and obtained 17 oleanane-type triterpene glycosides (1–17), including seven new glycosides (1–7). The structures of 1–7 were determined based on a detailed analysis of NMR spectroscopic data and chromatographic and spectroscopic analyses following specific chemical transformation. The cytotoxicities of the isolated compounds were evaluated against HL-60 human promyelocytic leukemia cells, A549 human adenocarcinoma lung cancer cells, and SBC-3 human small-cell lung cancer cells. The cytotoxicities of 1, 4, and 10 toward HL-60 cells and SBC-3 cells were nearly as potent as that of cisplatin. Compound 1, a bisdesmosidic triterpene glycoside obtained in good yield, arrested the cell cycle of SBC-3 cells at the G2/M phase, and induced apoptosis through an intrinsic pathway, accompanied by ROS generation. As a result of the mitochondrial dysfunction induced by 1, mitochondria selective autophagy, termed mitophagy, occurred in SBC-3 cells.

In the statistical data for the ten-year relative survival of Japanese cancer patients between 2002 and 2006, the survival rates of lung cancer and leukemia patients were 18.1% and 20.5% for males (aged 15 to 99), and 31.2% and 20.5% for females (aged 15 to 99), respectively [17]. Although lung cancer and leukemia patients successfully go into remission, they are not satisfied with current long-term medical treatment owing to relatively high-frequency recurrence. Small-cell lung cancer is generally treated with
Compound 6 (C65H102O33) was shown to be essentially analogous to 16, including the sugar moieties attached to C-3 and C-28 of the aglycone, based on the 1 H-and 13 C-NMR Compound 1 was obtained as an amorphous solid, and its molecular formula was assigned as C 76 H 120 O 41 based on the accurate sodium adduct ion at m/z 1711.7161 [M + Na] + (calculated for C 76 H 120 NaO 41 : 1711.7203) using high-resolution electrospray ionization time-of-flight mass spectroscopy (HR-ESI-TOF-MS) and 13 C-NMR spectral data. In the 1 H-and 13 C-NMR spectra of 1, the following signals were observed: six tertiary methyl groups The monosaccharides were identified based on HPLC analysis using a combi-nation of optical rotation and refractive index detectors. As the sugar moieties of 1 were composed of eight monosaccharides, their numerous proton signals overlapped severely, precluding their assignment using conventional NMR analysis. To address this issue, onedimensional total correlation spectroscopy (1D-TOCSY) and heteronuclear single quantum coherence (HSQC)-TOCSY spectroscopy in addition to 1 H-1 H correlation spectroscopy (COSY) and HSQC spectroscopy were applied. The 1 H-NMR subspectra of individual glycosyl units were obtained by selective irradiation of each anomeric proton signal and other non-overlapping proton signals. Subsequent analysis of the 1 H-1 H COSY spectrum allowed for the sequential assignment of the proton resonances for the pentaglycosyl moiety attached to C-28 of the aglycone, enabling us to identify their multiplet patterns and coupling constants. The HSQC and HSQC-TOCSY spectra correlated the proton signals with the corresponding one-bond-coupled carbon shifts. The assigned 1 H-and 13 C-NMR signals indicated that the C-28 pentaglycosyl moiety consisted of a 2,4-disubstituted β-D-  [16]. In the heteronuclear multiple bond correlation (HMBC) spectrum of 1, 3 J C,H correlations were observed between H-1""" of Glc and C-3""' of Rha, H-1"""' of Xyl (II) and C-4""' of Rha, H-1""' of Rha and C-2"" of Fuc, H-1"""" of Qui and C-4"" of Fuc, and between H-1"" of Fuc and C-28 of the aglycone.  [24], this is the first time it has been isolated from a natural source and its structure determined based on extensive NMR spectroscopic data.

Apotosis-Inducing Activity of 1
Compound 1 exhibited potent cytotoxic activity against SBC-3 cells and was obtained in a good yield. Thus, the apoptosis-inducing activity of 1 in SBC-3 cells was evaluated. Prior to assessing the apoptosis-inducing activity, SBC-3 cells were exposed to either 1 or cisplatin for 24 h to obtain IC 50 values. The IC 50 values of 1 and cisplatin were calculated to be 7.3 and 8.6 µM, respectively, based on the dose-response curves ( Figure 4). Thus, the apoptosis-inducing activity of 1 was evaluated at 10 µM.  (1) Data are represented as the mean value ± S.E.M. of the three experiments performed in triplicate.

Apoptosis Induced by 1
After SBC-3 cells were treated with 1 for 24 h, the cells were stained with Annexin V and propidium iodide (PI), and the apoptotic cell ratio was analyzed using flow cytometry. The percentage of early (Q4 area) and late (Q2 area) apoptotic cell populations increased significantly to 10 ± 0.32% and 33 ± 1.5% for 1 compared to 2.0 ± 0.058% and 2.2 ± 0.12%, respectively, for the vehicle control ( Figure 5).

Cell Cycle Arrest at the G 2 /M Phase by 1
To determine the cell cycle distribution of SBC-3 cells treated with 1, flow cytometry analysis was performed using PI staining. After 12 h of treatment with 1, the cell population in the G 2 /M phase (P5 area) increased to 35 ± 0.45% from 23 ± 0.48%, which was the value obtained after treatment with the vehicle control ( Figure 6A,B). Furthermore, the sub-G 1 phase (P2 area) population of SBC-3 cells treated with 1 for 24 h was 28 ± 0.23%, while that after treatment with the vehicle control was 4.1 ± 0.15% ( Figure 6C,D). These data indicated that 1 arrested SBC-3 cell proliferation in the G 2 /M phase and induced apoptotic cell death.

Caspase Activation and PARP Cleavage by 1
Caspases, which are cysteine proteases that cleave after aspartic acid residues in a substrate, play an important role in apoptosis and are subclassified into initiator caspases (caspase-8 and -9) and executioner caspases (caspase-3, -6, -7) [25]. During apoptosis, PARP cleavage is a useful hallmark of this type of cell death [26]. To confirm the contribution of caspases to the induction of apoptosis by 1, Western blotting analysis was performed. After SBC-3 cells were treated with 1 for 24 h, proteins were extracted and subjected to Western blotting analysis. As a result, the activation of caspase-8, -9, and -3, and PARP cleavage were observed (Figure 7).

Apotosis-Inducing Activity of 1
Compound 1 exhibited potent cytotoxic activity against SBC-3 cells and was obtained in a good yield. Thus, the apoptosis-inducing activity of 1 in SBC-3 cells was evaluated. Prior to assessing the apoptosis-inducing activity, SBC-3 cells were exposed to either 1 or cisplatin for 24 h to obtain IC50 values. The IC50 values of 1 and cisplatin were calculated to be 7.3 and 8.6 μM, respectively, based on the dose-response curves (Figure 4). Thus, the apoptosis-inducing activity of 1 was evaluated at 10 μM.

Apoptosis Induced by 1
After SBC-3 cells were treated with 1 for 24 h, the cells were stained with Annexin V and propidium iodide (PI), and the apoptotic cell ratio was analyzed using flow cytometry. The percentage of early (Q4 area) and late (Q2 area) apoptotic cell populations increased significantly to 10 ± 0.32% and 33 ± 1.5% for 1 compared to 2.0 ± 0.058% and 2.2 ± 0.12%, respectively, for the vehicle control ( Figure 5).

Apoptosis Induced by 1
After SBC-3 cells were treated with 1 for 24 h, the cells were stained with Annexin V and propidium iodide (PI), and the apoptotic cell ratio was analyzed using flow cytometry. The percentage of early (Q4 area) and late (Q2 area) apoptotic cell populations increased significantly to 10 ± 0.32% and 33 ± 1.5% for 1 compared to 2.0 ± 0.058% and 2.2 ± 0.12%, respectively, for the vehicle control ( Figure 5).  analysis was performed using PI staining. After 12 h of treatment with 1, the cell population in the G2/M phase (P5 area) increased to 35 ± 0.45% from 23 ± 0.48%, which was the value obtained after treatment with the vehicle control ( Figure 6A,B). Furthermore, the sub-G1 phase (P2 area) population of SBC-3 cells treated with 1 for 24 h was 28 ± 0.23%, while that after treatment with the vehicle control was 4.1 ± 0.15% ( Figure 6C,D). These data indicated that 1 arrested SBC-3 cell proliferation in the G2/M phase and induced apoptotic cell death.

Caspase Activation and PARP Cleavage by 1
Caspases, which are cysteine proteases that cleave after aspartic acid residues in a substrate, play an important role in apoptosis and are subclassified into initiator caspases (caspase-8 and -9) and executioner caspases (caspase-3, -6, -7) [25]. During apoptosis, PARP cleavage is a useful hallmark of this type of cell death [26]. To confirm the contribution of caspases to the induction of apoptosis by 1, Western blotting analysis was performed. After SBC-3 cells were treated with 1 for 24 h, proteins were extracted and subjected to Western blotting analysis. As a result, the activation of caspase-8, -9, and -3, and substrate, play an important role in apoptosis and are subclassified into initiator caspases (caspase-8 and -9) and executioner caspases (caspase-3, -6, -7) [25]. During apoptosis, PARP cleavage is a useful hallmark of this type of cell death [26]. To confirm the contribution of caspases to the induction of apoptosis by 1, Western blotting analysis was performed. After SBC-3 cells were treated with 1 for 24 h, proteins were extracted and subjected to Western blotting analysis. As a result, the activation of caspase-8, -9, and -3, and PARP cleavage were observed (Figure 7).

Mitochondrial Dysfunction Induced by 1
There are two major apoptosis-inducing pathways: intrinsic and extrinsic. The intrinsic pathway is also known as the mitochondrial pathway and participates in the activation of caspase-9 [27]. Because the activation of caspase-9 in SBC-3 cells treated with 1 was confirmed, the mitochondrial membrane potential was evaluated employing the JC-1 assay. When cells are stained with JC-1 dye, at low mitochondrial membrane potentials, the concentration of JC-1 is low and it exists predominantly as a monomer, exhibiting green

Mitochondrial Dysfunction Induced by 1
There are two major apoptosis-inducing pathways: intrinsic and extrinsic. The intrinsic pathway is also known as the mitochondrial pathway and participates in the activation of caspase-9 [27]. Because the activation of caspase-9 in SBC-3 cells treated with 1 was confirmed, the mitochondrial membrane potential was evaluated employing the JC-1 assay. When cells are stained with JC-1 dye, at low mitochondrial membrane potentials, the concentration of JC-1 is low and it exists predominantly as a monomer, exhibiting green fluorescence with emission; at high mitochondrial membrane potentials, the dye accumulates in the mitochondria and the dye aggregates yield a red to red-colored emission. SBC-3 cells were treated with 1 for 24 h, and then analyzed using a flow cytometer. As shown in Figure 8, the population of the mitochondrial membrane potential depolarized cells significantly increased compared to that observed with the vehicle control. Additionally, the expression of Bcl-2 and Bax was evaluated employing Western blot analysis. Bcl-2 and Bax belong to the Bcl-2 family, which regulates the intrinsic apoptotic pathway. Bcl-2 is an anti-apoptotic protein, whereas Bax acts as a pro-apoptotic effector [28]. In SBC-3 cells treated with 1, the expression level of Blc-2 was remarkably diminished, and the ratio of Bcl-2/Bax was lower than that of the vehicle control ( Figure 9). These data suggest that 1 causes mitochondrial dysfunction in SBC-3 cells.

ROS Generation by 1
Reactive oxygen species (ROS) exhibit beneficial or harmful effects on cells and tissues. ROS have been reported to be associated with intrinsic and extrinsic apoptotic pathways [29,30]. SBC-3 cells were incubated with either 2.5 mM of N-acetylcysteine (NAC; negative control), 100 µM of tert-butyl hydroperoxide (TBHP; positive control), or 1 for 24 h, and then analyzed using a flow cytometer. As depicted in Figure 10, the cell fluorescence intensities of the control-and NAC-treated groups were weak, while the peaks of the cell populations of the TBHP-or 1-treated groups moved to the right side. Based on these results it can be concluded that ROS generation occurred in SBC-3 cells treated with 1.

Mitophagy Occurrence
Mitophagy is defined as mitochondria-selective autophagy and plays a role in the elimination of defective mitochondria [31]. Several reports have suggested that depolarization of the mitochondrial membrane potential and ROS production are involved in mitophagy [32][33][34]. As 1 induced depolarization of the mitochondrial membrane potential and ROS generation, we investigated whether mitophagy occurred in SBC-3 cells. SBC-3 cells were treated with 1 or 7.5 µM of carbonyl cyanide m-chlorophenylhydrazone (CCCP; positive control) for 24 h, and then stained with Mtphagy Dye and Lyso Dye. Mtphagy Dye is a fluorescent dye that binds mitochondria and emits red fluorescence, the intensity of which increases under acidic conditions when the mitochondria are fused with lysosomes. Lyso Dye stains lysosomes and emits green fluorescence. SBC-3 cells incubated with 1 emitted intense red fluorescence and exhibited co-localization with lysosomes, while those treated with the control did not (Figure 11). These findings suggest that mitophagy had occurred in SBC-3 cells.
Bcl-2/Bax was lower than that of the vehicle control ( Figure 9). These data suggest that 1 causes mitochondrial dysfunction in SBC-3 cells.

ROS Generation by 1
Reactive oxygen species (ROS) exhibit beneficial or harmful effects on cells and tissues. ROS have been reported to be associated with intrinsic and extrinsic apoptotic pathways [29,30]. SBC-3 cells were incubated with either 2.5 mM of N-acetylcysteine (NAC; negative control), 100 μM of tert-butyl hydroperoxide (TBHP; positive control), or 1 for 24 h, and then analyzed using a flow cytometer. As depicted in Figure 10, the cell fluorescence intensities of the control-and NAC-treated groups were weak, while the peaks of the cell populations of the TBHP-or 1-treated groups moved to the right side. Based on these results it can be concluded that ROS generation occurred in SBC-3 cells treated with 1.   positive control) for 24 h, and then stained with Mtphagy Dye and Lyso Dye. Mtphagy Dye is a fluorescent dye that binds mitochondria and emits red fluorescence, the intensity of which increases under acidic conditions when the mitochondria are fused with lysosomes. Lyso Dye stains lysosomes and emits green fluorescence. SBC-3 cells incubated with 1 emitted intense red fluorescence and exhibited co-localization with lysosomes, while those treated with the control did not (Figure 11). These findings suggest that mitophagy had occurred in SBC-3 cells.

General Experimental Procedures
Optical rotations were measured using a P-1030 automatic digital polarimeter (JASCO, Tokyo, Japan). IR spectral data were obtained on a Fourier-transform infrared (FT-IR) 620 spectrometer (JASCO). NMR spectral data were collected using a Bruker AVIIIHD-600 (