Evaluation of Anti-angiogenic Activity and Biological Safety of Limonoids from Selected Philippine Citrus Fruits

Abstract

Background and Objectives: Suha Citrus maxima (Burm.) Merr and kalamansi Citrofortunella microcarpa (Bunge) Wijnands are citrus fruits common to the Filipino diet and are found to contain bioactive phytochemicals, such as limonoids. Limonoids are triterpenoid bitter principles from Citrus fruits, predominantly found in the seeds of citrus fruits. Initially studied for their intrinsic bitterness, this group of phytochemicals was found to have a plethora of health-giving benefits including anti-microbial, anticarcinogenic, and hepatoprotective, among others. In this study, seed limonoids from suha and kalamansi, along with their major limonoids, limonin and nomilin, were evaluated for their angiogenic activities and biological safety.

Methodology: Limonoids were isolated and characterized from the seeds of suha and kalamansi along with their major limonoids, limonin, and nomilin. A modified duck egg chorioallontoic membrane (CAM) assay was aided by AngioQuant, a digital imaging software used to evaluate angiogenic activity. Inhibition of angiogenesis was evaluated by percent increase or decrease in mean length of blood vessels, mean size of blood vessels, and total number of blood vessel junctions. Zebrafish embryotoxicity assay was utilized to evaluate the toxicity of limonoids. Zebrafish embryos were exposed to the aforementioned limonoids at 100 ppm [maximum concentration for a Category 5 (practically non-toxic) substance] and were observed for 96 hours for the four apical signs of zebrafish lethality.

Results: Analysis with AngioQuant revealed that treatment of the duck egg CAM with limonin, nomilin, and seed limonoid mixtures of suha and kalamansi showed a decrease in the percent mean length and size of blood vessels, and the total number of blood vessel junctions comparable to that of quercetin, a known antiangiogenic compound (P<0.0001). Zebrafish exposed to the same phytochemicals at 100 ppm did not show any of the four apical signs of zebrafish lethality 96 hpf.

Conclusion: Limonin, nomilin, and the seed limonoid mixtures of suha and kalamansi inhibited angiogenesis in a dose-dependent manner, comparable to the anti-angiogenic effect of quercetin. These are bioactive, yet non-toxic phytochemicals.