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Abstract
Natural products in Chonnam, Korea were screened via anti-angiogenesis experiments, and 1 candidate product was identified, Corni fructus, which exerted dose-dependent inhibitory effects against angiogenesis, adipogenesis, and cell adhesion. C. fructus extract (CFE) exhibits an angiogenesis inhibitory effect superior to that of the EGCG from green tea leaves. The expression level of angiogenesis and adipogenesis-related signal molecules in the western blotting was reduced by increasing the amount of added CFE. Moreover, a diet supplemented with CFE was deemed more effective in inducing weight loss in LB mice than a representative synthetic diet drug, orlistat, which incidently caused the side effect of denuding the mice of their hair. These results indicate that C. fructus may prove to be a useful anti-adipogenic compound, and these in vitro results may be reflected later under in vivo conditions.
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Fig. 1.
Anti-angiogenesis experiment of 18 natural products with the positive control, EGCG. HUVEC was cultured on Matrigel-coated wells, and the tube formation of HUVEC cells was randomly photographed using a digital camera, and analyzed by the Scion Image software.
Fig. 2.
Cytotoxicity in HUVEC cells with different concentrations of C. fructus extract (CFE). HUVEC was seeded onto 96-well plates and treated with various doses (0.1, 0.5, 1, 5, 10, 25, 50, and 100 mg/l) of CFE for 48 hrs. MTT solution was added to the wells, and the 96-well plates were incubated. The absorbances of the 96-well plates were measured with a microplate reader at 540 nm. The value was converted to cell viability based on the control.
Fig. 3.
CFE was subjected to preparative size exclusion column of Asahipak GS-310. CFE was chromatographed on an Asahipak GS-310 column eluted with methanol at a flow rate of 5 ml/min, and monitored at 307 nm. CFE was separated into six fractions. Each fraction was evaluated via anti-angiogenesis experiments. HUVEC was cultured on Matrigel-coated wells, and the tube formation of HUVEC cells was randomly photographed using a digital camera, and analyzed by the Scion Image software.
Fig. 4.
Anti-angiogenesis effect of SHE fractions 1, 5, and 6. Anti-angiogenesis experiments were conducted with different concentrations (1, 5, 10, and 25 ppm) of fractions 1, 5, and 6. The pictures show the cellular morphology of HUVEC cells treated with different concentrations of fractions 1, 5, and 6.
Fig. 5.
Various doses (1, 5, 10, and 20 mg/l) of CFE addition on U937 cell adhesion. (A) Cell adhesion of U937 on IL-1β and CFE stimulated HUVEC, (B) Cell adhesion of PMA- and CFE-stimulated U937 on HUVEC, (C) Cell adhesion of CFE-stimulated U937 on IL-1β- and CFE-stimulated HUVEC cells. Each treatment in the cell adhesion experiments was conducted as described in the METHODS section.
Fig. 6.
CFE inhibits the interaction of PI3-kinase with VE-cadherin and Akt upon cell activation with VEGF. Proteins from HUVEC cells were immunoprecipitated with PI3-kinase antibody and immunoblotted with antibodies to VE-cadherin and Akt. β-actin was employed as a positive control.
Fig. 7.
The effect of CFE addition in the course of 3T3-L1 adipocyte differentiation. 3T3-L1 was treated with CFE at various concentrations (1, 5, 10, and 25 mg/l). After differentiation, lipid accumulation was stained with Oil red O solution and the morphological changes were observed via microscopy. The Oil red O stained lipid was quantified with a microplate reader at 520 nm.
Fig. 8.
(A) CFE inhibits the interaction of PI3-kinase with VE-cadherin and Akt upon cell activation with VEGF. Proteins from HUVEC cells were immunoprecipitated with PI3-kinase antibody and immunoblotted with antibodies to VE-cadherin and Akt. β-actin was employed as a positive control. Expression of PPARγ on 3T3-L1 preadipocyte with CFE (A) and expression of SREBP-1 and PPARγ on 3T3-L1 adipocytes with CFE (B). Protein from 3T3-L1 was treated with different concentrations (1, 5, and 10 mg/l) of CFE, and extracted with RIPA buffer for western blotting.
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