A new cytotoxic diterpenoid glycoside from the leaves of Blumea lacera and its effects on apoptosis and cell cycle

Abstract A new diterpenoid glycoside, 6E,10E,14Z-(3S)-17-hydroxygeranyllinalool-17-O-β-d-glucopyranosyl-(1 → 2)-[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside (1) together with the known diterpenoid glycoside (2) and two known flavonoid glycosides (3, 4) were isolated from the methanol extract of Blumea lacera leaves. The structures were determined by the interpretation of their spectroscopic data and comparison with the literature. All compounds were isolated for the first time from B. lacera and evaluated for their cytotoxic activity. Only the new compound (1) showed strong cytotoxic activity with the lowest IC50 value (8.3 μM) being displayed against MCF-7 breast cancer cells. In apoptosis and cell cycle analysis, 1 revealed strong apoptotic activity against MCF-7 cells (45.5% AV+/PI−) after 24 h, but showed no arresting of any of the cell cycle phases in MCF-7.

an anthelmintic, astringent, diuretic, anti-scorbutic, anti-dysentric, anti-microbial, anti-inflammatory and carminative agent, as well as for the treatment of cholera and catarrhal infections (Ghani 2003;Singh & Parthasarathy 2012). Recently, we have reported on the cytotoxic activity of methanolic and water extracts of B. lacera against a number of human cancer cell lines (Uddin et al. 2011).
Previous studies have reported the isolation of a total of 21 compounds from different parts of B. lacera, belonging to natural product classes such as flavonoids, terpene glycosides, phenol glycosides, sterols, essential oils, coniferyl alcohol derivatives, terpenoid ketones and steroidal glycoalkaloids (Bohlmann & Zdero 1969;Pal et al. 1972;Rao et al. 1977;Agarwal et al. 1995;le & Muoi 2003;Ragasa et al. 2007;Akter et al. 2015). Monoterpene glycosides and flavonoids isolated from B. lacera leaves have anti-bacterial and anti-fungal activities (Ragasa et al. 2007). In addition, anti-fungal activity of metabolites from Blumea balsamifera and anti-microbial activity of B. balsamifera extract and essential oil have been reported (Ragasa et al. 2005;Sakee et al. 2011). however, the specific compounds responsible for the cytotoxic activity associated with B. lacera extracts are yet to be determined.
herein, we report on the isolation of two diterpenoid glycosides and two flavonoid glycosides, including the new diterpenoid glycoside 6E,10E,14Z-(3S)-17-hydroxygeranyllinalool- (1) from the methanolic extract of B. lacera leaves for the first time. All four isolated compounds were evaluated for cytotoxicity against two healthy (NIh3T3 and VERO) and four human cancer (AGS, hT-29, MCF-7 and MDA-MB-231) cell lines. Compound 1, the new diterpenoid glycoside, was further evaluated for its apoptosis and cell cycle arresting potential against MCF-7 breast cancer cells.
In our study, only compound 1 showed strong cytotoxicity especially against the cancer cell lines with IC 50 values of 8.3, 17.4 and 24.9 μM against MCF-7, MDA-MB-231 and AGS cells, respectively. Activity against healthy NIh3T3 and VERO cells was also detected (IC 50 values: 41.4 and 31.9 μM). Of the other isolated compounds, only 2 exhibited any cytotoxic activity, but the activity was marginal and only against colon cancer cells (hT-29, IC 50 102.0 μM). In comparison to the cytotoxic compound 1, the slightly toxic compound 2 has an extra β-d-glucopyranose sugar attached at the C-3 position of the aglycone, whereas compound 1 has a hydroxyl group (Figure 1 and 2). Evidently, the absence of the β-d-glucopyranose on the C-3 oxygen accounts for the significantly increased cytotoxic activity detected for compound 1.
No reports exist on the cytotoxic activity of acyclic diterpene glycoside analogues of 1 such as lyciumosides (I-III) and capsianosides (I, III and V), isolated from Lycium chinense and Capsicum annuum (Terauchi et al. 1998;De Marino et al. 2006), respectively. however, for the pentacyclic triterpenoid betulinic acid, it has been reported that structural modifications at C-3 lead to alterations of its cytotoxic potency and selectivity (Kuo et al. 2009).
Similarly, to our knowledge no reports exist about investigations into apoptosis induction and cell cycle arresting potential of specific diterpenoid glycosides analogous to 1. however, other terpenoids have been reported to inhibit cancer cell proliferation through apoptosis induction and cell cycle arrest (Kuttan et al. 2011;huang et al. 2012).
To further evaluate the mechanism of action for compound 1, we assessed its apoptosis-inducing potential and found a significant (p < 0.05) increase in the number of cells (by 45.5% at 8.3 μM) that underwent apoptosis (AV + /PI) compared to paclitaxel (39.9% at 0.023 μM), following 24 h treatment (Supplementary material, Figure S4). When prolonging the treatment of MCF-7 cells with 1 to 48 h, no increase in apoptosis (% AV + /PI − ) was observed, whereas the positive control, paclitaxel, showed a slight increase in apoptosis (AV + /PI) from 39.9 to 50.3% from 24 to 48 h.
Compound 1 was also studied for its cell cycle arresting potential (Supplementary material, Figure S5) but showed only a slight arrest of the cell cycle at G1 (G0/G1) phase, with no strong effect on other phases of MCF-7 cell cycle (Supplementary material, Figure S5).

Conclusion
Our study revealed that the cytotoxic activity associated with compound 1 was most likely attributed to its apoptosis-inducing capacity, as only a slight G1 (G0/G1) phase arrest in cell cycle analysis was observed. however, further investigations such as caspase activity, expression of Bcl-2 and Bcl-xl and regulation of hER2/neu are required to identify the exact molecular mechanism of the strong cytotoxic activity observed for compound 1.

Supplementary material
Experimental and supplementary data associated with this article can be found in the online version along with the original spectra of compound 1.