FIGURE 4.

Normal phase silica gel chromatogram of FE ethyl acetate partition fraction. 24 g of FE-E-EA was subjected to normal phase silica gel chromatography and eluted stepwise with solvent of increasing polarity; 20% ethyl acetate in n-hexane (20% EA/Hx), 40% ethyl acetate in n-hexane (40% EA/Hx), 60% ethyl acetate in n-hexane (60% EA/Hx), 80% ethyl acetate in n-hexane (80% EA/Hx), 100% ethyl acetate (100% EA), and 50% methanol in ethyl acetate (50% EA/Me) as described under “Experimental Procedures.” A, activity was assayed for each fraction (0.01 mg/ml each fraction was tested for its ability to potentiate receptor endocytosis elicited by 1 nm GLP-1) and was expressed as % of the response by 0.75 μm of GLP-1(7–36)-amide. B, increased cAMP production in RINm5F cells stably expresses RG-cAMP sensor. Activity was expressed as increased cAMP production (% of the maximal response) and was calculated as 100×(A − B)/(C − M), where A = BRET ratio by 1 nm GLP-1(7–36)-amide; B = BRET ratio by 1 nm GLP-1(7–36)-amide in the presence of 0.01 mg/ml indicated fraction; C = BRET ratio by 10 pm GLP-1(7–36)-amide, and M = BRET ratio by 0.25 μm GLP-1(7–36)-amide. Black triangles and red diamonds represent activity and weight of each fraction, respectively.