Anticancer Effects of Five Biflavonoids from Ginkgo Biloba L. Male Flowers In Vitro

Abstract

Ginkgo biloba L., an ancient dioecious gymnosperm, is now cultivated worldwide for landscaping and medical purposes. A novel biflavonoid-amentoflavone 7''-O-β-D-glucopyranoside (1)-and four known biflavonoids were isolated and identified from the male flowers of Ginkgo. The anti-proliferative activities of five biflavonoids were evaluated on different cancer lines. Bilobetin (3) and isoginkgetin (4) exhibited better anti-proliferative activities on different cancer lines. Their effects were found to be cell-specific and in a dose and time dependent manner for the most sensitive HeLa cells. The significant morphological changes validated their anticancer effects in a dose-dependent manner. They were capable of arresting the G2/M phase of the cell cycle, inducing the apoptosis of HeLa cells dose-dependently and activating the proapoptotic protein Bax and the executor caspase-3. Bilobetin (3) could also inhibit the antiapoptotic protein Bcl-2. These might be the mechanism underlying their anti-proliferation. In short, bilobetin (3) and isoginkgetin (4) might be the early lead compounds for new anticancer agents.

Keywords: Ginkgo biloba flowers; anticancer; biflavonoids; bilobetin; isoginkgetin.

Figure 1

Structures of compounds 1–5 (A). Key heteronuclear multiple bond correlation (HMBC) correlations () and nuclear overhauser enhancement spectroscopy (NOESY)correlations () of compound 1 (B).

Figure 2

Effects of compounds 3 (A) and 4 (B) on viability of HeLa cell at different concentrations for different incubation time. HeLa cells were incubated with compound 3 or 4 at the concentration of 3.1 μM to 100 μM, and then the cytotoxic activity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 24 h, 48 h, and 72 h, respectively. The values are expressed as mean ± SD of triplicate experiments.

Figure 3

Morphological structure of HeLa cancer cells through Giemsa staining after 48 h of treatment with different concentration of compounds 3 or 4. (A) control (untreated), (B) compound 3 (15 μM), (C) compound 3 (20 μM), (D) compound 3 (25 μM), (E) compound 4 (10 μM), (F) compound 4 (15 μM), and (G) compound 4 (20 μM). The arrows show the obviously changed cells.

Figure 4

Cell cycle analysis of HeLa cells treated with compounds 3 and 4 (5 μM, 10 μM, and 20 μM) for 24 h by flow cytometry.

Figure 5

Histograms (A,B) showed the percentage of compounds 3 and 4 treated cells in different phases of the cell cycle, respectively. The values are expressed as mean ± SD of triplicate experiments. * p < 0.05, ** p < 0.01 compared with the control group.

Figure 6

Apoptosis was assessed using Annexin V- fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining after HeLa cells were treated with compounds 3 and 4 (5 μM, 10 μM, and 20 μM) for 24 h, measured by flow cytometry.

Figure 7

Histograms showed the apoptosis rate (early and late apoptosis) of HeLa cells treated with compounds 3 and 4. The values are expressed as mean ± SD of triplicate experiments. ** p < 0.01 compared with the control group.

Figure 8

Western blots were performed to analyze relative protein expression levels involved in the cell apoptosis including Bax, Bcl-2, pro-caspase-3, and cleaved caspase-3(A,B). β -actin was used as a control. HeLa cells were treated with different concentrations (5, 10, 20 μM) of compound 3 or 4 for 24 h. The obvious change could be seen in the histogram in a concentration-dependent manner (C,D), * p < 0.05, ** p < 0.01, compared with the control group.