Cytotoxic and Angiosuppressive Potentials of Zehneria japonica (Thunb. ex Murray) S.K. Chen (Cucurbitaceae) Crude Leaf Extracts

Abstract

Background and Objectives: Zehneria japonica belongs to the Cucurbitaceae family which is one of the most important plant families. It is commonly known as "Pipinong-gubat," widely distributed in Central Luzon regions and in areas along streams and clearings at low and medium altitudes in the Philippines. This study aimed to evaluate the potential cytotoxic and angiosuppressive properties of Zehneria japonica (Thunb. ex Murray) S.K. Chen (Cucurbitaceae) leaf extracts.

Methodology: The Z. japonica semi-crude extracts were obtained by sequential extraction using hexane, ethyl acetate, and n-butanol. A modified duck egg chorioallantoic 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. Moreover, the cytotoxic effects of
the extracts were determined through MTT Assay. Osteosarcoma (U2Os) and hepatocellular carcinoma (HepG2) cells were used as cancer representatives while human umbilical vein endothelial cells (HUVEC) were used as the normal cell control.

Results: Analysis with AngioQuant revealed that treatment of the duck egg CAM with Z. japonica semi-crude extracts suppressed angiogenesis with IC₅₀ values of 1,810.00 μg/mL, 192.50 μg/mL, and 147.70 μg/mL for hexane, ethyl acetate, and n-butanol, respectively, with Celecoxib (20 μg/mL) as the positive control. For MTT assay, Z. japonica extracts exhibited strong cytotoxic effect against U2Os with an IC₅₀ values of 19.65 μg/mL, 9.89 μg/mL, and 31.04 μg/mL for the hexane, ethyl acetate, and n-butanol extracts, and no cytotoxic effects against HepG2 with IC₅₀ values of 770.90 μg/mL, 130.10
μg/mL and 231.60 μg/mL for the hexane, ethyl acetate, and n-butanol extracts. Doxorubicin (0.544 μg/mL) was used as the positive control. The extracts also inhibited the growth of the normal cells, with IC₅₀ values of 69.46 μg/mL, 42.23 μg/mL and 63.44 μg/mL for the hexane, ethyl acetate, and n-butanol extracts. There were no mortality and toxic symptoms observed for 14 days after the administration of the crude butanolic extract of Z. japonica in six female Sprague- Dawley rats.

Conclusion: Z.japonica crude leaf extracts exhibited angio-suppressive activity through CAM assay. In MTT assay, the extracts exhibited strong cytotoxicity in U2Os (IC₅₀ ≤20 μg/mL), no cytotoxic effect in HepG2 (IC₅₀ >100 μg/mL) cells, and mild cytotoxic effect in HUVEC (IC₅₀ 40-60 μg/mL). Phytochemical screening through TLC revealed that the extracts contain alkaloids, anthrones, flavonoids, and sterols.