Structural Characterization of Cholestane Rhamnosides from Ornithogalum saundersiae Bulbs and Their Cytotoxic Activity against Cultured Tumor Cells

Previous phytochemical studies of the bulbs of Ornithogalum saundersiae, an ornamental perennial plant native to South Africa, resulted in the isolation of 29 new cholestane glycosides, some of which were structurally unique and showed potent cytotoxic activity against cultured tumor cell lines. Therefore, we aimed to perform further phytochemical examinations of methanolic extracts obtained from Ornithogalum saundersiae bulbs, isolating 12 new cholestane rhamnosides (1–12) and seven known compounds (13–19). The structures of the new compounds (1–12) were identified via NMR-based structural characterization methods, and through a sequence of chemical transformations followed by spectroscopic and chromatographic analysis. The cytotoxic activity of the isolated compounds (1–19) and the derivatives (1a and 6a) against HL-60 human promyelocytic leukemia cells and A549 human lung adenocarcinoma cells was evaluated. Compounds 10–12, 16, and 17 showed cytotoxicity against both HL-60 and A549 cells. Compound 11 showed potent cytotoxicity with an IC50 value of 0.16 µM against HL-60 cells and induced apoptotic cell death via a mitochondrion-independent pathway.

Compound 5 (C 35 H 56 O 9 ) was obtained as an amorphous powder. The 1 H-NMR spectrum of 5 displayed signals arising from two tertiary methyl groups at δ H 1.41 and 1.00 (each s, Me-19, Me-18); a secondary methyl group at δ H 1.26 (d, J = 6.8 Hz, Me-21); two methyl groups on a double bond at δ H 1.78 (Me-27) and 1.73 (Me-26); two olefinic protons at δ H 5.82 (br t, J = 7.2 Hz, H-24) and 5.65 (br d,J = 4.7 Hz,; an anomeric proton at δ H 5.03 (br s, H-1 of Rha); and the methyl group of an acetyl moiety at δ H 2.03 (s). These spectral features of 5 showed similarity to those of 18, and alkaline degradation of 5 with 3% NaOMe in MeOH yielded 18. A long-range correlation was observed between the H-2 of Rha at δ H 5.65 (br d, J = 2.2 Hz) and the carbonyl carbon of the acetyl group at δ C 170.6 in the HMBC spectrum of 5, providing evidence that the acetyl group was located at C-2 of the α-L-rhamnopyranosyl moiety. The structure of 5 was defined as (22S)-3β,11α,22-trihydroxycholesta-5,24-dien-16β-yl 2-O-acetyl-α-L-rhamnopyranoside.
Compound 6 (C 43 H 68 O 15 ), for which 1 H-NMR analysis showed two anomeric proton signals at δ H 5.07 (d, J = 7.7 Hz, H-1 of Glc) and 5.03 (br s, H-1 of Rha), was assumed to be a bisdesmosidic cholestane glycoside closely related to 2. The two compounds differed only in the signals of the methyl groups assignable to Me-26 and Me-27. The two three-proton doublets for Me-26 and Me-27 observed in the 1 H-NMR spectrum of 2 were displaced by two three-proton singlets at δ H 1.77 and 1.73 in that of 6, suggesting that 6 was the C-24/C-25 dehydro derivative of 2. This was confirmed by alkaline methanolysis of 6, followed by enzymatic hydrolysis using β-D-glucosidase, to yield 18 and D-glucose. The linkage position of the glucosyl moiety of 6 was ascertained by an HMBC correlation between H-1 of Glc and C-3 of the aglycone at δ C 78.1. The structure of 6 was elucidated as (22S)-3β-[(β-D-glucopyranosyl)oxy]-11α,22-dihydroxycholesta-5,24-dien-16β-yl 2,3-di-O-acetyl-α-L-rhamnopyranoside.
Compound 11 (C 33 H 54 O 8 ) was a cholestane rhamnoside with similar spectral properties to those of 18. However, the molecular formula of 11 lacked one oxygen atom in comparison to 18, and the 13 C signals assignable to C-11 differed between the two compounds. The hydroxymethine carbone signal observed at δ C 68.1 (C-11) in the 13 C-NMR spectrum of 18 was displaced by a methylene carbon signal at δ C 21.1 in that of 11, allowing 11's structure to be determined as (22S)-3β,22-dihydroxycholesta-5,24-dien-16β-yl α-L-rhamnopyranoside.    Compound 12 had the molecular formula C 43 H 68 O 14 , possessing two fewer hydrogen atoms than 10.

Apoptosis-Inducing Properties of 11 in HL-60 Cells
As described above, the new cholestane rhamnoside (11) showed potent cytotoxicity against HL-60 cells. Therefore, the apoptosis-inducing properties of 11 in HL-60 cells were evaluated. After HL-60 cells were exposed to 11 at a concentration of 10 µM for 24 h, the cells were stained with 4 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed via fluorescence microscopy. The cells exhibited nuclear chromatin condensation and nuclear disassembly as shown in Figure 2, which are the representative morphological features of apoptotic cells. To obtain further evidence for the apoptosis-inducing activity of 11, cell cycle distribution, DNA fragmentation, and caspase-3 activity were evaluated in 11-treated HL-60 cells. When HL-60 cells were cultured with 11 at 10 µM for 20 h, the sub-G1 and G2/M phase populations, of which the vehicle control were 3.3 ± 0.20% and 22.4 ± 0.10%, increased to 12.3 ± 0.70% and 32.2 ± 2.44%, respectively ( Figure 3). This showed that 11 arrested HL-60 cell proliferation in the G2/M phase and induced apoptotic cell death. Agarose gel electrophoresis of the DNA fraction of HL-60 cells treated with 11 at 10 µM for 20 h displayed an apoptotic DNA ladder pattern, as shown in Figure 4. Furthermore, caspase-3, a key enzyme in the execution phase of the apoptotic pathway, was markedly activated in HL-60 cells treated with 11 at 10 µM for 20 h ( Figure 5). These results indicated that HL-60 cell death was partially mediated by 11 via the induction of apoptosis. As described above, the new cholestane rhamnoside (11) showed potent cytotoxicity against HL-60 cells. Therefore, the apoptosis-inducing properties of 11 in HL-60 cells were evaluated. After HL-60 cells were exposed to 11 at a concentration of 10 µM for 24 h, the cells were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed via fluorescence microscopy. The cells exhibited nuclear chromatin condensation and nuclear disassembly as shown in Figure 2, which are the representative morphological features of apoptotic cells. To obtain further evidence for the apoptosis-inducing activity of 11, cell cycle distribution, DNA fragmentation, and caspase-3 activity were evaluated in 11-treated HL-60 cells. When HL-60 cells were cultured with 11 at 10 µM for 20 h, the sub-G1 and G2/M phase populations, of which the vehicle control were 3.3 ± 0.20% and 22.4 ± 0.10%, increased to 12.3 ± 0.70% and 32.2 ± 2.44%, respectively ( Figure 3). This showed that 11 arrested HL-60 cell proliferation in the G2/M phase and induced apoptotic cell death. Agarose gel electrophoresis of the DNA fraction of HL-60 cells treated with 11 at 10 µM for 20 h displayed an apoptotic DNA ladder pattern, as shown in Figure 4. Furthermore, caspase-3, a key enzyme in the execution phase of the apoptotic pathway, was markedly activated in HL-60 cells treated with 11 at 10 µM for 20 h ( Figure 5). These results indicated that HL-60 cell death was partially mediated by 11 via the induction of apoptosis.

Apoptosis-Inducing Properties of 11 in HL-60 Cells
As described above, the new cholestane rhamnoside (11) showed potent cytotoxicity against HL-60 cells. Therefore, the apoptosis-inducing properties of 11 in HL-60 cells were evaluated. After HL-60 cells were exposed to 11 at a concentration of 10 µM for 24 h, the cells were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed via fluorescence microscopy. The cells exhibited nuclear chromatin condensation and nuclear disassembly as shown in Figure 2, which are the representative morphological features of apoptotic cells. To obtain further evidence for the apoptosis-inducing activity of 11, cell cycle distribution, DNA fragmentation, and caspase-3 activity were evaluated in 11-treated HL-60 cells. When HL-60 cells were cultured with 11 at 10 µM for 20 h, the sub-G1 and G2/M phase populations, of which the vehicle control were 3.3 ± 0.20% and 22.4 ± 0.10%, increased to 12.3 ± 0.70% and 32.2 ± 2.44%, respectively ( Figure 3). This showed that 11 arrested HL-60 cell proliferation in the G2/M phase and induced apoptotic cell death. Agarose gel electrophoresis of the DNA fraction of HL-60 cells treated with 11 at 10 µM for 20 h displayed an apoptotic DNA ladder pattern, as shown in Figure 4. Furthermore, caspase-3, a key enzyme in the execution phase of the apoptotic pathway, was markedly activated in HL-60 cells treated with 11 at 10 µM for 20 h ( Figure 5). These results indicated that HL-60 cell death was partially mediated by 11 via the induction of apoptosis.

Apoptosis-Inducing Properties of 11 in HL-60 Cells
As described above, the new cholestane rhamnoside (11) showed potent cytotoxicity against HL-60 cells. Therefore, the apoptosis-inducing properties of 11 in HL-60 cells were evaluated. After HL-60 cells were exposed to 11 at a concentration of 10 µM for 24 h, the cells were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed via fluorescence microscopy. The cells exhibited nuclear chromatin condensation and nuclear disassembly as shown in Figure 2, which are the representative morphological features of apoptotic cells. To obtain further evidence for the apoptosis-inducing activity of 11, cell cycle distribution, DNA fragmentation, and caspase-3 activity were evaluated in 11-treated HL-60 cells. When HL-60 cells were cultured with 11 at 10 µM for 20 h, the sub-G1 and G2/M phase populations, of which the vehicle control were 3.3 ± 0.20% and 22.4 ± 0.10%, increased to 12.3 ± 0.70% and 32.2 ± 2.44%, respectively (Figure 3). This showed that 11 arrested HL-60 cell proliferation in the G2/M phase and induced apoptotic cell death. Agarose gel electrophoresis of the DNA fraction of HL-60 cells treated with 11 at 10 µM for 20 h displayed an apoptotic DNA ladder pattern, as shown in Figure 4. Furthermore, caspase-3, a key enzyme in the execution phase of the apoptotic pathway, was markedly activated in HL-60 cells treated with 11 at 10 µM for 20 h ( Figure 5). These results indicated that HL-60 cell death was partially mediated by 11 via the induction of apoptosis.

The Pathway of Apoptosis Induced by 11
Apoptotic HL-60 cell death, induced by 11, was examined to ascertain whether this occurred via a mitochondria-dependent or -independent pathway. When stained with MitoCapture TM dye, apoptotic cells emit green fluorescence, whereas non-apoptotic cells with healthy mitochondria emit red fluorescence. As shown in Figure 6, HL-60 cells treated with cisplatin at 33 µM for 6 h showed a decrease in red fluorescence intensity compared to that of control cells. However, treatment of HL-60 cells with 11 at 10 µM for 6 h did not decrease the intensity of red fluorescence, indicating that 11 caused no disruption of the mitochondrial membrane potential in HL-60 cells ( Figure 6). An increase in mitochondrial membrane permeability and a decrease in mitochondrial membrane potential resulted in the rapid release of caspase activators, such as cytochrome c, into the cytoplasm. Treating HL-60 cells with 11 at 10 µM for 6 h had no significant effect on cytosolic cytochrome c levels ( Figure 7). Taken together, these results suggested that 11 exerts apoptotic effects in HL-60 cells through a mitochondria-independent pathway.

The Pathway of Apoptosis Induced by 11
Apoptotic HL-60 cell death, induced by 11, was examined to ascertain whether this occurred via a mitochondria-dependent or -independent pathway. When stained with MitoCapture TM dye, apoptotic cells emit green fluorescence, whereas non-apoptotic cells with healthy mitochondria emit red fluorescence. As shown in Figure 6, HL-60 cells treated with cisplatin at 33 µM for 6 h showed a decrease in red fluorescence intensity compared to that of control cells. However, treatment of HL-60 cells with 11 at 10 µM for 6 h did not decrease the intensity of red fluorescence, indicating that 11 caused no disruption of the mitochondrial membrane potential in HL-60 cells ( Figure 6). An increase in mitochondrial membrane permeability and a decrease in mitochondrial membrane potential resulted in the rapid release of caspase activators, such as cytochrome c, into the cytoplasm. Treating HL-60 cells with 11 at 10 µM for 6 h had no significant effect on cytosolic cytochrome c levels ( Figure 7). Taken together, these results suggested that 11 exerts apoptotic effects in HL-60 cells through a mitochondria-independent pathway.

The Pathway of Apoptosis Induced by 11
Apoptotic HL-60 cell death, induced by 11, was examined to ascertain whether this occurred via a mitochondria-dependent or -independent pathway. When stained with MitoCapture TM dye, apoptotic cells emit green fluorescence, whereas non-apoptotic cells with healthy mitochondria emit red fluorescence. As shown in Figure 6, HL-60 cells treated with cisplatin at 33 µM for 6 h showed a decrease in red fluorescence intensity compared to that of control cells. However, treatment of HL-60 cells with 11 at 10 µM for 6 h did not decrease the intensity of red fluorescence, indicating that 11 caused no disruption of the mitochondrial membrane potential in HL-60 cells ( Figure 6). An increase in mitochondrial membrane permeability and a decrease in mitochondrial membrane potential resulted in the rapid release of caspase activators, such as cytochrome c, into the cytoplasm. Treating HL-60 cells with 11 at 10 µM for 6 h had no significant effect on cytosolic cytochrome c levels ( Figure 7). Taken together, these results suggested that 11 exerts apoptotic effects in HL-60 cells through a mitochondria-independent pathway.

The Pathway of Apoptosis Induced by 11
Apoptotic HL-60 cell death, induced by 11, was examined to ascertain whether this occurred via a mitochondria-dependent or -independent pathway. When stained with MitoCapture TM dye, apoptotic cells emit green fluorescence, whereas non-apoptotic cells with healthy mitochondria emit red fluorescence. As shown in Figure 6, HL-60 cells treated with cisplatin at 33 µM for 6 h showed a decrease in red fluorescence intensity compared to that of control cells. However, treatment of HL-60 cells with 11 at 10 µM for 6 h did not decrease the intensity of red fluorescence, indicating that 11 caused no disruption of the mitochondrial membrane potential in HL-60 cells ( Figure 6). An increase in mitochondrial membrane permeability and a decrease in mitochondrial membrane potential resulted in the rapid release of caspase activators, such as cytochrome c, into the cytoplasm. Treating HL-60 cells with 11 at 10 µM for 6 h had no significant effect on cytosolic cytochrome c levels ( Figure 7). Taken together, these results suggested that 11 exerts apoptotic effects in HL-60 cells through a mitochondria-independent pathway.

Plant Material
The fresh bulbs of O. saundersiae were obtained from Sakata Seed Corporation (Kanagawa, Japan) in 2013. A voucher specimen has been deposited at the herbarium of this university (KS-2013-006).

Structural Characterization
Supplementary Materials: The following are available online. 1 H-and 13 C-NMR of compounds 1-12.