| 10% |
With polyphenol oxidase; In aq. phosphate buffer; for 2.0h;pH 6.0;Enzymatic reaction; |
Ina typical experiment, EC (290 mg) and EGC (306 mg) were added to a mixture of acetone and phosphate buffer (pH 6.0) (1:10 v/v,100 ml), C. sinensis cell culture (50 ml including 10.2 g cells), and 3% H2O2 (0.8 ml). The mixture was stirred for 4 min and thenextracted using CH3COOEt. Further, the organic layer was driedover anhydrous MgSO4 and concentrated in vacuo to provide TF(395 mg) with 70% yield and 100% conversion. In the HPLC analysis,12 peaks for EC and ECG were virtually absent, but a peak for TF was observed. |
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With dihydrogen peroxide;herseradish peroxidase; In acetone; for 0.75h;pH 5.0;Phosphate citrate buffer; Enzymatic reaction; |
ECG (1 g) and EGCG (1 g) were dissolved in a mixture of acetone-pH 5.0 phosphate citrate buffer (1:10, v/v, 50 mL), which contained 4 mg horseradish peroxidase. While being stirred, 2.0 ml of 3.13% H2O2 was added four times during 45 minutes. The reaction mixture was extracted by ethyl acetate (50 ml×3). After concentration, the residue was subjected to Sephadex LH 20 column eluted with acetone-water solvent system (45%). 100 mg theaflavin 3,3'-digallate was obtained. [0088] 1H NMR (CD3OD, 600 MHz): deltaH 7.79 1H s, 7.76 1H s, 7.47 1H s, 6.88 2H s, 6.80 2H s, 6.07 1H d, J=2.4 Hz, 6.03 2H d, J=2.4 Hz, 6.00 1H d, J=2.4 Hz, 5.86 1H brs, 5.76 1H m, 5.67 1H m, 5.21 1H s, 3.17 1H dd, J=4.8, 16.8 Hz, 3.09 1H dd, J=4.8, 17.4, 2.91 2H m. |
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polyphenol oxidase; at 20℃; for 6.0h;pH 5.0;Phosphate-citrate buffer; Enzymatic reaction; |
EC (1 g, 3.5 mmol) and EGC (1 g, 3.3 mmol) were dissolved into the 200 mL of phosphate-citrate buffer (50 mM, pH 5.0) along with 2 g of crude PPO enzyme. The enzymatic oxidation was carried out at room temperature for 6 hour with stirring. The reaction solution was then subjected to fractionation with the same volume of ethyl acetate with three times. Then, the organic layer was concentrated under reduced pressure. The resulting residues were subjected to Sephadex LH-20 column chromatography eluting with gradient of ethanol to 20% of acetone in ethanol. Among the collected 14 fractions (each c.a. 90 mL), 810 fractions were combined, and concentrated under reduced pressure. The resulting residue was subjected to further purification on a RP-18 silica gel column eluting with gradient of 40%50% of aqueous methanol. During elution, 38 fractions (each c.a. 13 mL) were received. Among them, 1017 fractions were combined, and concentrated under reduced pressure, and were subjected to freeze-drying. It yielded deep-reddish color of compound 1 (280 mg). Along with the same enzyme reaction and isolation procedure, compound 2 was obtained from EC and EGCG reaction. The enzymatic oxidation of EGC and ECG, ECG and EGCG reaction yielded compound 3 and compound 4, respectively. |
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