METHODS: C. nutans leaves was extracted with 50-100% ethanol or deionised water at 1% (w/v). Human umbilical veins endothelial cell (HUVEC) proliferation was examined using MTT assay. The in vitro anti-angiogenic effects of C. nutans were assessed using wound scratch, tube formation and transwell migration assays. The VEGF levels secreted by human oral squamous cell carcinoma (HSC-4) cell and HUVEC permeability were also measured. Besides, the rat aortic ring and chick embryo chorioallantoic membrane (CAM) assays, representing ex vivo and in vivo models, respectively, were performed.
RESULTS: The MTT assay revealed that water extract of C. nutans leaves exhibited the highest activity, compared to the ethanol extracts. Therefore, the water extract was chosen for subsequent experiments. C. nutans leaf extract significantly suppressed endothelial cell proliferation and migration in both absence and presence of VEGF. However, the water extract failed to suppress HUVEC transmigration, differentiation and permeability. C. nutans water extract also did not suppress HSC-4 cell-induced VEGF production. Importantly, C. nutans water extract significantly abolished the sprouting of vessels in aortic rings as well as in chick embryo CAM.
CONCLUSION: In conclusion, these findings reveal potential anti-angiogenic effects of C. nutans, providing new evidence for its potential application as an anti-angiogenic agent.
METHODS: The aqueous ethanolic leaf extracts of C. caudatus were characterized by NMR and LC-MS/MS. The total phenolic content and α-glucosidase inhibitory activity were evaluated by the Folin-Ciocalteu method and α-glucosidase inhibitory assay, respectively. The statistical significance of the results was evaluated using one-way ANOVA with Duncan's post hoc test, and correlation among the different activities was performed by Pearson's correlation test. NMR spectroscopy along with multivariate data analysis was used to identify the metabolites correlated with total phenolic content and α-glucosidase inhibitory activity of the C. caudatus leaf extracts.
RESULTS: It was found that the α-glucosidase inhibitory activity and total phenolic content of the optimized ethanol:water (80:20) leaf extract of the plant increased significantly as the plant matured, reaching a maximum at the 10th week. The IC50 value for α-glucosidase inhibitory activity (39.18 μg mL- 1) at the 10th week showed greater potency than the positive standard, quercetin (110.50 μg mL- 1). Through an 1H NMR-based metabolomics approach, the 10-week-old samples were shown to be correlated with a high total phenolic content and α-glucosidase inhibitory activity. From the partial least squares biplot, rutin and flavonoid glycosides, consisting of quercetin 3-O-arabinofuranoside, quercetin 3-O-rhamnoside, quercetin 3-O-glucoside, and quercetin 3-O-xyloside, were identified as the major bioactive metabolites. The metabolites were identified by NMR spectroscopy (J-resolve, HSQC and HMBC experiments) and further supported by dereplication via LC-MS/MS.
CONCLUSION: For high phytomedicinal quality, the 10th week is recommended as the best time to harvest C. caudatus leaves with respect to its glucose lowering potential.