Alcoholic Extract of Eclipta alba Shows In Vitro Antioxidant and Anticancer Activity without Exhibiting Toxicological Effects

Abstract

As per WHO estimates, 80% of people around the world use medicinal plants for the cure and prevention of various diseases including cancer owing to their easy availability and cost effectiveness. Eclipta alba has long been used in Ayurveda to treat liver diseases, eye ailments, and hair related disorders. The promising medicinal value of E. alba prompted us to study the antioxidant, nontoxic, and anticancer potential of its alcoholic extract. In the current study, we evaluated the in vitro cytotoxic and antioxidant effect of the alcoholic extract of Eclipta alba (AEEA) in multiple cancer cell lines along with control. We have also evaluated its effect on different in vivo toxicity parameters. Here, we found that AEEA was found to be most active in most of the cancer cell lines but it significantly induced apoptosis in human breast cancer cell lines by disrupting mitochondrial membrane potential and DNA damage. Moreover, AEEA treatment inhibited migration in both MCF 7 and MDA-MB-231 cells in a dose dependent manner. Further, AEEA possesses robust in vitro antioxidant activity along with high total phenolic and flavonoid contents. In summary, our results indicate that Eclipta alba has enormous potential in complementary and alternative medicine for the treatment of cancer.

Figure 1

Calibration curve of standards: gallic acid (a), quercetin (b), and ascorbic acid (c) (each point (R² values) represents the mean data set of n = 3).

Figure 2

Intracellular ROS level of HEK 293 cells. Cells are treated with different concentration of AEEA for 24 h. Results are represented as mean ± SD. Statistical significance determined as compared to control (0.05 ≥ p). ∗ represents p ≤ 0.05; ∗∗∗ represents p ≤ 0.001.

Figure 3

AEEA induced growth inhibition in different cancer cell lines. MDA-MB-231 (breast), HeLa (cervical), SK-OV-3 (ovary), SW620 (colon), DU145 (prostate), A549 (lung), and PANC-1 (pancreatic) were treated with different concentrations of AEEA for 48 h and cytotoxicity was measured as described in Materials and Methods. Percentage cell viability was plotted in graphs (a). Brightfield microscopy. Effects of AEEA on the morphological changes in different cancer cells after 24 h treatment (400 μg/mL) were monitored by phase contrast microscopy (b).

Figure 4

Cytotoxic effects of AEEA on human breast cancer cell line MCF 7, mouse breast cancer cell line 4T1, and normal epithelial cell line of African green monkey Vero. MCF 7, 4T1, and Vero cells were treated with different concentrations of AEEA for 48 h and cytotoxicity was measured as described in Materials and Methods.

Figure 5

Detection of apoptosis in AEEA treated MDA-MB-231 using the Annexin V FITC. MCF 7 and MDA-MB-231 cells were treated with different concentrations of AEEA for 24 h and stained with Annexin V FITC following standard protocol and observed under fluorescence microscope.

Figure 6

Detection of DNA damage by AEEA induced apoptosis in tumor cells by Hoechst 33342 staining. MCF 7 cells were seeded in 24-well culture plate and allowed to grow for 24 h. After treatment of 24 h with different concentrations of Eclipta alba extract cells were stained with Hoechst 33342 following standard protocol and observed under fluorescence microscopy.

Figure 7

Determination of mitochondrial membrane potential (ΔΨm). Rhodamine 123 (RH-123), a mitochondria specific fluorescent dye which stains live cells, is used to determine the mitochondrial membrane potential by measuring fluorescence intensity under microscope. MCF 7 cells were treated with different concentrations of AEEA for 24 h and stained with Rhodamine 123 following standard protocol and observed under fluorescence microscopy. (a) Control (without treatment); (b–d) treatment with various concentrations of Eclipta alba extract. ∗ represents p ≤ 0.05.

Figure 8

Cell migration inhibition by Eclipta alba extract. A scratch wound healing assay was performed on MCF 7 and MDA-MB-231 cells treated with different concentration of AEEA to determine the cell migration ability. (a) Showing scratch wounds in MDA-MD-231 cells at time 0. (b–e) Representing the status of wound at 24 h after the initiation of the scratch when the cells were treated with the vehicle control, DMSO (b), or different concentrations of AEEA (c–e). Wounds were created and AEEA was added immediately. Wounds were evaluated at 24 h after AEEA administration. ^∗Indicating values that were significantly different (p < 0.05) from the DMSO control.

Figure 9

Mass fingerprinting chromatogram of SIE in positive ion (M + 1) mode.