| The natural product（-）-epigallocatechin-3-gallate（EGCG） is a polyphenolic compound found in green tea（Camellia sinensis）. Many studies have reported that EGCG possesses many pharmacological properties like antioxidant, anti-inflammatory, antibacterial activity, UV-protective effect, cancer chemoprevention, anti-HIV, alleviation of metabolic syndrome（overweight, obesity and type-2 diabetes）, cardiovascular disease prevention, neuroprotective effect. However, the application of EGCG is restricted by its hydrophilicity and instability leading to low bioavailability. EGCG can be structurally modified to improve its stability and lipophilicity, and enhance its bioavailability. Ester-based pro-drugs are classical means to improve the lipophilicity and oral bioavailability as well as additional benefits.In this work, a series of O-acyl derivatives of EGCG were synthesized. The stability of EGCG, EGCG-octaacetate（AcEGCG）, EGCG-octapropionate（PrEGCG） and EGCG-octabutyrate（BuEGCG） was estimated in artificial simulation gastric and intestinal fluids. The hydrolysis properties of the three derivatives were examined in mouse liver microsomes. And the effect on cyclophosphamide-induced hepatotoxicity of EGCG and the three derivatives were examined in mice. More details are as follows:（1） Synthesis of EGCG derivatives: Anhydrides or acylchloride as the acylating agent react with EGCG in the presence of N, N-dimethylaminopyridine（DMAP） or pyridine. The reaction solution was stirred at designed temperature until thin-layer chromatography（TLC） showed the reaction had been completed. The solution was washed with distilled water, saturated aqueous NaHCO3 and distilled water. The ethyl acetate layer was dried by MgSO4 and filtered and crude EGCG derivatives were obtained by evaporation the solvent under vacuum distillation. The crude EGCG derivatives were purified by silica gel column chromatography. The chemical structures of the EGCG derivatives were confirmed by MS and 1H and 13 C NMR.（2） Stability of EGCG, AcEGCG, PrEGCG and BuEGCG was estimated in artificial simulation gastric and intestinal fluids by HPLC: The hydrolysis properties of the three derivatives were examined in mouse liver microsomes, and substrate depletion approach was used to calculate the enzyme kinetic parameters Km and Vmax.The results showed that EGCG and its derivatives were stable in artificial simulation gastric fluids. EGCG was unstable in artificial simulation intestinal fluids and it was not detectable. In summary, the stability of EGCG and its derivatives in artificial simulation gastric and intestinal fluids was in the order of BuEGCG > PrEGCG > AcEGCG > EGCG. In the metabolic system, AcEGCG was hydrolyzed by liver microsomes with high affinity(Km = 0.2098 mmol·L-1). The Vmax value of AcEGCG was 1.41-fold of PrEGCG and 9.38-fold of BuEGCG. And AcEGCG showed highest Km/Vmax values among all the substrates tested, and BuEGCG showed lowest Km/Vmax values. These data indicated that BuEGCG is relatively slowly hydrolyzed in liver microsomes.（3） The protective role of EGCG, AcEGCG, PrEGCG and BuEGCG against cyclophosphamide（CP）-induced hepatotoxicity in mice: The animals were divided into ten groups（n=10, normal control group, cyclophosphamide group, EGCG and its derivatives at low-dose groups, EGCG and its derivatives high-dose groups, respectively）. Mice were pretreated with EGCG and its derivatives for 9 days, 1h after the last dose, mice were injected with CP（300 mg/kg）. The blood was collected 24 h after the last treatment. And the mice were sacrificed and their livers were excised and processed immediately for biochemical analysis. The levels of AST, ALT, LDH in serum and MDA, GSH, SOD, CAT, GSH-PX in hepatic homogenate were evaluated and histopathology studies were carried out according to the standard procedures. The results showed that CP caused a significant increase in serum AST, ALT, LDH and MDA in hepatic momogenate, and remarkable reduction in GSH, SOD, CAT, GSH-PX levels in hepatic homogenate when compared with the control group. Pretreatment with EGCG and acyl-EGCGs significantly restored the serum AST, ALT, LDH in serum, and MDA GSH, SOD, CAT, GSH-PX in hepatic homogenate almost to the normal. It was also found that the liver was seriously injured by CP injection as the degeneration, necrosis and inflammatory cell infilatraion were obviously seen. These morphorlogy changes of liver tissue, however, were mitigated at various degrees by the administration of EGCG and acyl-EGCGs.In conclussion, acyl-EGCGs are more stable than EGCG in artificial simulation gastric and intestinal fluids. And they can be hydrolyzed by liver microsomes. Acylation modification of EGCG may be a useful method of decreasing its presystemic metabolism and improving its bioavailability. Acyl-EGCGs are more effective than EGCG in protecting against cyclophosphamide（CP）-induced hepatotoxicity in mice. EGCG and its derivatives may be promising modulators in ameliorating CP- induced hepatotoxicity. |
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