Antiangiogenic Activity of n-hexane Insoluble Fraction and Its Tylophorine Component from Ficus septica Leaves in Chicken Chorioallantoic Membrane Induced by bFGF

Abstract

Objective: Ficus septica is an Indonesian medicinal plant traditionally used to treat various illness, including cancer. The n-hexane insoluble fraction of the ethanolic extract of F. septica leaves (HIFFS) shows a potential anticancer activity against breast cancer cell line T47D. Considering that angiogenesis is a pivotal factor in malignant cancer growth, progression, and invasion, we aimed to investigate the antiangiogenic effect of HIFFS on chicken chorioallantoic membrane (CAM) induced by bFGF. We also evaluated tylophorine, the cytotoxic alkaloid of F. septica.

Methods: Chicken CAM was used to assess the antiangiogenic effect. Fertilized chicken eggs were induced with basic fibroblast growth factor (bFGF) ex ovo. Prior to bFGF induction, HIFFS (2.33, 4.65, 6.98, and 9.30 μg/mL) or tylophorine (9.20 µM) was added (10 µL) to a paper disk and implanted to the CAM. After 48 h of incubation, each treatment group was photographed, and the number of new blood vessel was calculated and compared with that in the solvent-treated group to determine the antiangiogenic activity. Histology of the CAM was evaluated after hematoxylin-eosin and Mallory acid fuchsin staining.

Results: We found that HIFFS at low concentrations (2.33, 4.65, 6.98, and 9.30 μg/mL) inhibited angiogenesis activity (31.87, 41.99, 53.65, and 70.08, respectively) in chicken CAM induced by bFGF. Tylophorine (9.20 µM) also showed similar antiangiogenesis activity in the same model. Histopathology analysis revealed that HIFFS and tylophorine reduced the number of new blood vessels in CAM induced by bFGF.

Conclusion: HIFFS and tylophorine showed antiangiogenic effect on chicken CAM induced by bFGF. This finding emphasized the potential of F. septica as a candidate anticancer agent.

Keywords: Angiogenesis; Anticancer; CAM; alkaloid; histopathology.

Figure 1

HPLC Profile of HIFFS and Tylophorine. A, HIFFS; B, Tylophorine. Stationary phase, Symmetry Shield RP-18 (reverse phase); mobile phase, methanol; flowrate, 1 mL/min; detector, UV 275 nm; run time, 15 min

Figure 2

Representative Photographs Showing CAMs with the Old and New Blood Vessels. The images were taken from the ventral side on day 8 of incubation. A, solvent; B, Tylophorine 9.2 µM; C, D, E, and F were HIFFS at concentrations of 9.30, 6.98, 4.65, and 2.33 µg/mL, respectively. i, implanted paper disc; blue light arrow, main blood vessel; black arrows, new blood vessel

Figure 3

Antiangiogenic Activity of HIFFS and Tylophorine on Chicken CAM Induced by bFGF. The antiangiogenic activity was evaluated according to the ability to inhibit the formation of new blood vessels. The solvent was PBS containing PEG 400; tylophorine was tested at 9.2 µM; bFGF (30 ng/µL) was used to induce angiogenesis in the CAM. The data are means ± SD from three independent experiment (n = 3). *significant (p > 0.05) using one-way ANOVA, followed by LSD post hoc

Figure 4

Representative Histological Cross-section Images of the CAM Containing Blood Vessels. The photographs were taken under a microscope on day 8 of incubation, and the CAMs were stained with hematoxylin–eosin (left panel), and Mallory acid fuchsin (right panel). A and B, solvent; C and D, Tylophorine (9.2 µM); E and F, HIFFS 9.30 µg/mL. Blue light arrow, main blood vessel; black arrow, new blood vessel; magnification, 40 × 10