Table 1.
DPPH and nitrite radical scavenging activities and reducing power of water and methanol extracts from N. nucifera leaves.
Fig 1.
Effects of water and methanol extracts of N. nucifera leaves on cell viability and VEGF-induced proliferation.
(a) Cell viability: HUVECs were treated with various concentrations of extracts. After 48 h, cell viability was measured using the MTT assay. *: p<0.05 compared with untreated control (0 μg ml−1). (b) VEGF-induced proliferation: HUVECs were co-treated with various concentrations of extracts and VEGF for 48 h. The number of viable cells was measured by using the MTT assay. *: p<0.05 compared with untreated control; #: p<0.05 compared the VEGF-treated group. All tested concentrations (10, 50, and 100 μg ml−1) of the water as well as the methanol extracts displayed statistically significant differences with respect to each other.
Fig 2.
Inhibitory effects of water and methanol extracts of N. nucifera leaves on tube formation in HUVECs.
(a) HUVECs (5 x 104 cells) were plated on wells that had been previously coated with 40 μL of growth factor-reduced Matrigel basement membrane matrix. Cells were then treated with extracts in the presence of VEGF (20 ng ml−1). After 14 h, cells were photographed with a digital camera under a phase contrast microscope at 40x magnification. (b)The bar graph represents the relative area covered by the tube network, and data are shown as the mean ± SD. *: p<0.05 compared with untreated control; #: p<0.05 compared with VEGF-treated group.
Fig 3.
Inhibitory effects of water and methanol extracts of N. nucifera leaves on VEGF-induced angiogenesis.
(a) The CAM of a 10-d-old chick embryo was separately exposed to PBS (control) and VEGF (20 ng ml−1) by means of filter disks. After 30 min, extracts were introduced on top of the CAMs. After 72 h of incubation, the CAM tissue directly beneath each filter disk was resected, and digital images of the CAM sections were captured. (b) The bar graph represents the number of new branches formed from existing blood vessels. Photographs were imported into an image software program to visualize the new vessel branch points. Data are shown as the mean ± SD. *: p<0.05 compared with untreated control; #: p<0.05 compared with VEGF-treated CAM samples. None of the tested concentrations (10, 50, and 100 μg ml−1) of the water as well as the methanol extract display statistically significant differences with respect to each other.
Fig 4.
Inhibitory effects of water and methanol extracts of N. nucifera leaves on VEGF-induced ROS generation in HUVECs.
(a) Serum-starved cells were pretreated with extracts for 30 min and then treated with VEGF (20 ng ml−1) for 15 min. ROS generation was determined by measuring DCF-DA fluorescence. The fluorescence intensity was determined by analyzing the captured images with the Image Inside program. (b) Data are mean ± SD values from four independent experiments. *: p<0.05 compared with untreated control; #: p<0.05 compared with VEGF-treated CAM samples. All tested concentrations (10, 50, and 100 μg ml−1) of the water as well as the methanol extract display statistically significant difference with respect to each other.
Table 2.
GC-MS chemical composition analysis of the water and methanol extracts of N. nucifera leaves.