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Anti-angiogenic effect of Nelumbo nucifera leaf extracts in human umbilical vein endothelial cells with antioxidant potential.

Lee JS, Shukla S, Kim JA, Kim M - PLoS ONE (2015)

Bottom Line: In this study, N. nucifera leaf extracts displayed potent antioxidant and inhibitory effects on VEGF-induced angiogenesis.N. nucifera exerted an inhibitory effect on VEGF-induced proliferation and tube formation, as well as CAM angiogenesis in vivo.Moreover, N. nucifera leaf extracts significantly blocked VEGF-induced ROS production in HUVECs, confirming their possible anti-angiogenic mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science and Technology, Yeungnam University, Gyeongsan, Gyeongbuk, 712-749, Republic of Korea.

ABSTRACT
Nelumbo nucifera Gaertn (Nymphaeaceae) has long been used as a traditional herb in Chinese, Japanese, Indian, and Korean medicinal practices since prehistoric times and flourishes today as the primary form of medicine. This study reports for the first time the potent ability of N. nucifera leaf extracts to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro and in vivo, as well as their antioxidant efficacy in various scavenging models and an analysis of their chemical composition. In vivo anti-angiogenic activity was evaluated in a chick chorioallantoic membrane (CAM) model using fertilized chicken eggs, in human umbilical vein endothelial cells (HUVECs) by using cell viability, cell proliferation and tube formation assays, and by determining intracellular reactive oxygen species (ROS) in vitro. The antioxidant efficacy of N. nucifera leaf extracts was determined in various scavenging models, including total phenolic and flavonoid content. The chemical composition of N. nucifera leaf extracts was determined by GC-MS analysis, which revealed the presence of different phytochemicals. The IC50 values for the DPPH radical scavenging activities of water and methanol extracts were found to be 1699.47 and 514.36 μg ml(-1), and their total phenolic and flavonoid contents were 85.01 ± 2.32 and 147.63 ± 2.23 mg GAE g dry mass(-1) and 35.38 ± 1.32 and 41.86 ± 1.07 mg QA g dry mass(-1), respectively. N. nucifera leaf extracts (10-100 μg ml(-1)) exhibited significant dose-dependent inhibition of VEGF-induced angiogenesis, as well as VEGF-induced proliferation and tube formation in HUVECs. In this study, N. nucifera leaf extracts displayed potent antioxidant and inhibitory effects on VEGF-induced angiogenesis. N. nucifera exerted an inhibitory effect on VEGF-induced proliferation and tube formation, as well as CAM angiogenesis in vivo. Moreover, N. nucifera leaf extracts significantly blocked VEGF-induced ROS production in HUVECs, confirming their possible anti-angiogenic mechanism.

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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.
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pone.0118552.g004: 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.

Mentions: Since ROS derived from growth factor-stimulated receptors are critically important in many aspects of vascular cell behavior including VEGF-induced angiogenesis [10], we examined whether N. nucifera leaf extracts inhibit VEGF-induced ROS production in HUVECs or not. Cells stimulated with VEGF showed intracellular ROS elevation as evidenced by cell labeling using the fluorescent probe DCF-DA (Fig. 4). During angiogenesis, a large increase in oxygen uptake resulted in the massive release of intracellular ROS in VEGF-treated cells (Fig. 4), compared with untreated control cells (Fig. 4). A significant increase in ROS production over basal levels was observed in VEGF-induced HUVECs (Fig. 4). However, pre-treatment with water and methanol leaf extracts significantly inhibited VEGF-stimulated intracellular ROS generation (Fig. 4).


Anti-angiogenic effect of Nelumbo nucifera leaf extracts in human umbilical vein endothelial cells with antioxidant potential.

Lee JS, Shukla S, Kim JA, Kim M - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4340789&req=5

pone.0118552.g004: 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.
Mentions: Since ROS derived from growth factor-stimulated receptors are critically important in many aspects of vascular cell behavior including VEGF-induced angiogenesis [10], we examined whether N. nucifera leaf extracts inhibit VEGF-induced ROS production in HUVECs or not. Cells stimulated with VEGF showed intracellular ROS elevation as evidenced by cell labeling using the fluorescent probe DCF-DA (Fig. 4). During angiogenesis, a large increase in oxygen uptake resulted in the massive release of intracellular ROS in VEGF-treated cells (Fig. 4), compared with untreated control cells (Fig. 4). A significant increase in ROS production over basal levels was observed in VEGF-induced HUVECs (Fig. 4). However, pre-treatment with water and methanol leaf extracts significantly inhibited VEGF-stimulated intracellular ROS generation (Fig. 4).

Bottom Line: In this study, N. nucifera leaf extracts displayed potent antioxidant and inhibitory effects on VEGF-induced angiogenesis.N. nucifera exerted an inhibitory effect on VEGF-induced proliferation and tube formation, as well as CAM angiogenesis in vivo.Moreover, N. nucifera leaf extracts significantly blocked VEGF-induced ROS production in HUVECs, confirming their possible anti-angiogenic mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science and Technology, Yeungnam University, Gyeongsan, Gyeongbuk, 712-749, Republic of Korea.

ABSTRACT
Nelumbo nucifera Gaertn (Nymphaeaceae) has long been used as a traditional herb in Chinese, Japanese, Indian, and Korean medicinal practices since prehistoric times and flourishes today as the primary form of medicine. This study reports for the first time the potent ability of N. nucifera leaf extracts to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro and in vivo, as well as their antioxidant efficacy in various scavenging models and an analysis of their chemical composition. In vivo anti-angiogenic activity was evaluated in a chick chorioallantoic membrane (CAM) model using fertilized chicken eggs, in human umbilical vein endothelial cells (HUVECs) by using cell viability, cell proliferation and tube formation assays, and by determining intracellular reactive oxygen species (ROS) in vitro. The antioxidant efficacy of N. nucifera leaf extracts was determined in various scavenging models, including total phenolic and flavonoid content. The chemical composition of N. nucifera leaf extracts was determined by GC-MS analysis, which revealed the presence of different phytochemicals. The IC50 values for the DPPH radical scavenging activities of water and methanol extracts were found to be 1699.47 and 514.36 μg ml(-1), and their total phenolic and flavonoid contents were 85.01 ± 2.32 and 147.63 ± 2.23 mg GAE g dry mass(-1) and 35.38 ± 1.32 and 41.86 ± 1.07 mg QA g dry mass(-1), respectively. N. nucifera leaf extracts (10-100 μg ml(-1)) exhibited significant dose-dependent inhibition of VEGF-induced angiogenesis, as well as VEGF-induced proliferation and tube formation in HUVECs. In this study, N. nucifera leaf extracts displayed potent antioxidant and inhibitory effects on VEGF-induced angiogenesis. N. nucifera exerted an inhibitory effect on VEGF-induced proliferation and tube formation, as well as CAM angiogenesis in vivo. Moreover, N. nucifera leaf extracts significantly blocked VEGF-induced ROS production in HUVECs, confirming their possible anti-angiogenic mechanism.

Show MeSH
Related in: MedlinePlus