Methanol extraction revealed anticancer compounds Quinic Acid, 2(5H)‑Furanone and Phytol in Andrographis paniculata

Abstract

Andrographis paniculata (Ap) has been a part of traditional medicine for its anti-inflammatory effects, treatment of snake bites and liver abnormalities. Several investigations have revealed its bioactive components to be andrographolides. The methanolic extracts of leaves from Ap were characterized, the non-andrographolides were identified and screened for anti-proliferative activity. The extracts showed significant toxicity against numerous cancer cells including HeLa, MCF7, BT549, 293 and A549 cells. Anti-proliferative activity and effect on cancer cell invasion (metastatic potential) and cell migration were examined. The extracts revealed significant inhibition of the ability of HeLa cells in repairing the gap created by a vertical wound made on a confluent cell monolayer. Similarly, a 40% reduction in cell migration was observed in presence of the extracts. Significant anti-angiogenic activity in terms of reduced blood capillary formation was observed on the Chorioallantoic membranes of embryonated hen eggs co-inoculated with HeLa cells and the extracts. Analysis of HeLa cells treated with the extracts using flow cytometry indicated the arrest of cell cycle progression at the G2/M phase. Variation in the Bax/Bcl-2 ratio and elevated caspase-3 levels by immunoblotting confirmed cell death induction via the apoptotic pathway. Investigation of the extracts by gas chromatography-mass spectrometry displayed the predominant components to be 2(5H)-Furanone (14.73%), Quinic acid (17.32%), and Phytol (11.43%). These components have been previously known to have anticancer activity, while being studied individually in other plants. This is the first study, to the best of our knowledge, on the anti-proliferative and anti-angiogenic activity of the non-andrographolide components from Ap.

Keywords: Andrographis paniculata; anti-angiogenic activity; anti-proliferative activity; apoptosis; cell cycle arrest.

Figure 1

A list of components identified by gas chromatography performed for crude methanolic extracts of Andrographis paniculata showing the major bioactive components.

Figure 2

Effect of Ap extracts on cell toxicity. Cytotoxicity of Ap extracts is expressed as percentage of viability of cells. Statistical analyses were performed by One-way ANOVA, followed by Tukey's post-hoc test. Data obtained from three individual experiments was compared between each of the treatments with their respective vehicle control (methanol) expressed as the mean ± SD. ^**P<0.01, ^***P<0.001 and ^****P<0.0001 vs. vehicle control. Ap, Andrographis paniculata; ns, non-significant.

Figure 3

Analysis of neo-vascularization of CAM layer after treatment with Ap extracts. CAM layer of embryonated chick egg was injected with (A) PBS (control) and (B) Hela cells; (C-E) 25, 50 and 100 µg of the Ap extract along with HeLa cells. The capillaries arising from the central blood vessel were enumerated and represented as % angiogenesis. (F) Graph showing percentage of angiogenesis. Statistical analyses were performed by One-way ANOVA followed by Tukey's post hoc test. Data from three individual experiments vs. the control group are expressed as the mean ± SD. ^***P<0.001 and ^****P<0.0001 vs. A (untreated). CAM, chorioallantoic membrane; Ap, Andrographis paniculata.

Figure 4

Effect of Ap extracts on HeLa cell migration and invasion in vitro. (A) Microscopic images at x40 magnification representing a single scratch made on the cell monolayer treated with Ap (25, 50, and 100 µg/ml) and untreated cells as control, images of the wound made by the scratch were captured at 0 and 24 h post-treatment using a phase-contrast microscope. The boundaries of the scratched wounds determined by dark lines. (B) Bar graph showing percentage of cell migration. Quantitative analysis of the rates of migration was performed using ImageJ software. Results represented in the graphs are the mean ± SD obtained from triplicate experiments. Data are expressed as the mean ± SD from three individual experiments vs. control group according to ordinary One-way ANOVA. (C) Transwell migration assay in Matrigel-coated Transwell chambers seeded with HeLa cells in the presence of different concentrations of Ap extracts. After 24-h treatment, images were captured at x400 magnification. (D) Percentage of invading cells in the presence of Ap extracts. Results shown in the graphs are presented as the mean ± SD obtained from triplicate experiments. Statistical analyses were performed by One-way ANOVA followed by Tukey's post hoc test. Data are expressed as the mean ± SD from three individual experiments against the control group. ^*P<0.05, ^**P<0.01 and ^****P<0.0001 vs. untreated cells. Ap, Andrographis paniculata.

Figure 5

Ap extracts arrest cell cycle progression in HeLa cells. Ap induced cell cycle arrest at G2/M phase in HeLa cells. Cells were stained with PI after treatment with Ap extracts for 24 h (A) Control, (B) 50 µg/ml (C) 100 µg/ml and (D) Paclitaxel. (E) Bar diagram showing groups B and C had significantly higher percentages of cells at G2/M phases than the other phases. Statistical analyses were performed by One-way ANOVA followed by Tukey's post hoc test. Data are expressed as the mean ± SD from three individual experiments against the control group. ^*P<0.05 vs. A (untreated cells). Ap, Andrographis paniculata.

Figure 6

Ap extracts induce cell death by apoptosis in HeLa cells. HeLa cells were treated with Ap extracts (25, 50 and 100 µg/ml) up to 24 h. (A) Whole-cell lysates were resolved on SDS-PAGE gel and probed with the indicated antibodies. β-actin was used as a loading control. (B-D) Protein quantification of the western blot results for caspase-3, Bax and Bcl-2 protein levels upon treatment with Ap. Typical results from three independent experiments (n=3). (B) Upregulation of Caspase-3 was detected by western blotting. (C) Downregulation of anti-apoptotic protein Bcl-2. (D) Upregulation of pro-apoptotic protein Bax. Data represent the mean ± SD from three individual experiments against the control group. Statistical analyses were performed by one-way ANOVA followed by Tukey's post hoc test. ^**P<0.01 and ^****P<0.0001 vs. untreated cells. Ap, Andrographis paniculata.