Hepatoprotective Mechinism Of Phillygenin From The Flowers Of Osmanthus Fragrans

Liver diseases are a major problem of worldwide proportions. However, the number of drugs actually used successfully in humans is limited. Therefore, people need to find an effective, low side effects natural hepatoprotective drug. Osmanthus concrete is a food additive, and preliminary study from our group member has found that Osmanthus concrete showed a great hepatoprotective effect on CCl4-induced hepatic injury in mice. Beside the unclear hepatoprotective components and mechanism and the limited pharmaceutical study, it’s hard to develop Osmanthus concrete to a clear effective component and mechanism hepatoprotective drug or functional food. Current study will focus on clarifying the hepatoprotective component from Osmanthus concrete, investigating pharmaceutical study of the hepatoprotective component, including pharmacokinetics, drug metabolism, drug-plasma protein binding and the effect of drug on CYP450enzymes, to evaluate whether this component can be a good pharmacokinetic properties new candidate compound, which will provide a theoretica for drug design and optimization, and is extremely important for the development a safer and more effective drug. The results of the research are as follows:(1) Investigate the hepatoprotective effective of the main component of Osmanthus concrete. A main component has been purified from Osmanthus concrete which is identified as PHI. Set up CCI4liver injury mice model to evaluate the hepatoprotective effect of phillygein. The results show that low, medium, and high dosage of PHI can significantly prevent the increasing of serum enzymatic activities of ALT and AST, the formation of hepatic MDA and the depression of SOD in the CCl4-intoxicated mice liver. And the results shows the same benificial with positive control drug-biphenylbiester. Histological examination demonstrated that PHI could attenuate the area and extent of necrosis, reduce the immigration of inflammatory cells;(2) Study the possible hepatoprotective mechanism of PHI. PHI can inhibit the activity of CYP2E1with Kivalue of0.44μmol/L in vitro. PHI can bleached the DPPH and ABTS radical immediately in vitro, which suggests that PHI could be classified as dynamic antioxidants. These results indicate that PHI may protect CCl4-induced hepatotoxicity in mice by inhibit the activity of CYP2E1and its antioxidant activity; (3) Investigate the pharmacokinetics of PHI in mice. Results showed that after oral administration (po,24mg/kg), PHI was quickly absorbed, reaching maximum level at30min with Cmax value of0.7μg/mL and absorbtion half-life value of54.8min, slowly distributed to tissues with distribution half-life value of150min, and the clenrance and elimilation half-life are345mL/(min·kg) and240min respectively. After intravenous administration (iv,12mg/kg), PHI can be quickly distributed to tissue with distribution half-life of3.78min. Oral bioavailability of PHI was56.4%, which means PHI has good oral bioavailability.(4) Investigate the metabolites of PHI both in vivo and in vitro, and study the stability of PHI in different species (mouse, rat, dog, monkey and huamn) liver microsomes. Two metabolites, hydroxylated and demethylated PHI were identified in rat urine and rat microsome. PHI shows good stability in rat, mice, monkey and human liver microsome with the t1/2>1h. The half life of PHI after incubated with dog microsomes is54min. These results indicate that PHI has two metabolites but the parent drug may still the main compound to show the heportecttive effect, and there is little species differences;(5) Investigate the human plasma protein binding of PHI and its interaction with human serum albumin. Protein binding results show that the binding rate of PHI with human plasma is concentration-dependent and the binding rate is above90%when the concentration of PHI is between the ranges of10-20μmol/L. The interaction with human serum albumin study shows that the fluorescence quenching of HSA by PHI resulted mainly from static mechanism. Van der Waals forces and hydrogen bonds play a role in stabilizing the HSA-PHI complex. Metal ions and ethanol may decrease the interaction of PHI on HSA and increase the concentration of free PHI.(6) Investigate the effect of PHI on CYP450enzymes. The results show that PHI can inhibit the activity of CYP2E1and CYP3A4in vitro with the Ki values of0.44and6.5μmol/L, respectively, both are competitive inhibition. The induction of PHI on primary human hepatocytes results shows that PHI do not have obviously induced activity on liver CYP450enzymes.