Syzygium campanulatum korth methanolic extract inhibits angiogenesis and tumor growth in nude mice

Abstract

Background: Syzygium campanulatum Korth (Myrtaceae) is an evergreen shrub rich in phenolics, flavonoid antioxidants, and betulinic acid. This study sought to investigate antiangiogenic and anti-colon cancer effects of S.C. standardized methanolic extract.

Methods: Betulinic acid was isolated from methanolic extract by crystallization and chromatography techniques. S.C. methanolic extract was analyzed by UV-Vis spectrophotometry, FTIR, LC-MS, and HPLC. Antiangiogenic effect was studied on rat aortic rings, matrigel tube formation, cell proliferation and migration, and expression of vascular endothelial growth factor (VEGF). Antitumor effect was studied using a subcutaneous tumor model of HCT 116 colorectal carcinoma cells established in nude mice.

Results: Analysis by HPLC, LC-MS and FTIR confirm presence of betulinic acid in S.C. methanolic extract. Quantitative analysis by HPLC indicates presence of betulinic acid in S.C. extract at 5.42 ± 0.09% (w/w). Antiangiogenesis study showed potent inhibition of microvessels outgrowth in rat aortic rings, and studies on normal and cancer cells did not show any significant cytotoxic effect. Antiangiogenic effect was further confirmed by inhibition of tube formation on matrigel matrix that involves human endothelial cells (IC50 = 17.6 ± 2.9 μg/ml). S.C. extract also inhibited migration of endothelial cells and suppressed expression of VEGF. In vivo antiangiogenic study showed inhibition of new blood vessels in chicken embryo chorioallantoic membrane (CAM), and in vivo antitumor study showed significant inhibition of tumor growth due to reduction of intratumor blood vessels and induction of cell death.

Conclusion: Collectively, our results indicate S. campanulatum as antiangiogenic and antitumor candidate, and a new source of betulinic acid.

Figure 1

Different parts of Syzygium campanulatum Korth. Fruit buds (A), flowers (B), ripe fruits (C), and whole tree (D).

Figure 2

Chemical structure of betulinic acid.

Figure 3

HPLC analysis of S.C. methanolic extract. BA, UA and OA standard compounds (A), S. campanulatum methanolic extract (B), BA-rich fraction (C), and isolated BA (D).

Figure 4

In vitro inhibition of angiogenesis. Rat aortic rings (A): Untreated (1), suramin at 100 μg/ml (2), and S.C. methanolic extract at 100 μg/ml (3). Inhibition of HUVECs cell migration (B and C): zero time treatment (B1 and C1), methanolic extract at 100 μg/ml (B2) and at 50 μg/ml (C2), Untreated cells (B3 and C3). The lines are drawn for explanation.

Figure 5

Inhibition of tube formation on Matrigel matrix. Untreated cells (A), suramin (B), (C-G) S.C. methanolic extract at 100, 75, 50, 25 and 12.5 μg/ml, and analysis showing a dose dependent inhibition of tube formation (H).

Figure 6

In vivo inhibition of angiogenesis using Chicken embryo CAMs. Untreated CAM (A), treated with suramin at 25 μg/disc (B), treated with S.C. methanolic extract at 200 μg/disc (C) and 100 μg/disc (D).

Figure 7

Inhibition of the subcutaneous tumor growth in nude mice. A time course of tumor size measurement (A), cross sections of tumor tissues (B): untreated (1), and treated with S.C. methanolic extract at 0.25% w/w (2). Pictures were taken at 5× magnification. (*) refers to P < 0.05.