Preparation And Characterization Of Myricetin Supramolecular Aggregates And Its Antialcoholic Effect

Research background and purpose:Excessive alcohol consumption often induces lesions in several organs,such as gastric ulcer,liver cirrhosis,nerve damage,cardiovascular disease,and even cancer,etc.There are no specific therapeutic drugs for acute or chronic alcoholism in clinical practice.It has been reported that myricetin（MYR）has a wide range of pharmacological effects,such as anti-platelet activating factor,antioxidant,anti-tumor,anti-photoaging,anti-inflammatory,hepatoprotective,etc.However,few people has been known about its antialcoholic effect.Due to its structural similarity with dihydromyricetin,our group found that MYR has great potential to combat alcoholism and protect against alcoholic tissue damage based on pre-experiments.However,due to its poor solubility,stability,intestinal permeability and oral bioavailability,the effectiveness of MYR in the treatment of various diseases and alcoholism is greatly limited.The purpose of this study was to prepare a novel supramolecular aggregate（MYR-SA）using Kolliphor?HS15（HS15）as a surfactant to significantly increase the oral bioavailability of MYR by improving its solubility and stability,thereby improving its effectiveness in the treatment of alcoholism.This formulation also provides an experimental basis for the future development of oral MYR new formulations and offers new ideas and options for the treatment of alcoholism.Materials and Methods:1.Preparation and characterization of MYR-SAA method was established for the determination of MYR in vitro and in vivo samples by high performance liquid chromatography（HPLC）.Kolliphor?HS15 supramolecular aggregates containing MYR were prepared by thin film hydration,and characterized by particle size distribution analyzer,transmission electron microscopy and differential scanning calorimetry.It is verified that MYR-SA improves the solubility,stability and release rate of MYR by measuring the solubility,encapsulation rate,release rate in vitro,and investigating the stability of different p H buffers,simulated gastrointestinal fluid,and liver homogenate.2.Examination of intestinal permeability of MYR-SAA HPLC method for the determination of MYR in intestinal perfusion was established.A rat unidirectional in vivo intestinal perfusion model was used to investigate the absorption of MYR,MYR-SA,MYR+HS15 and MYR+VER in rat jejunal segments.This will verify whether MYR-SA has the ability to increase the intestinal permeability of MYR.3.Pharmacokinetic study of MYR-SA in ratsA method for the determination of MYR concentrations in rat plasma by Liquid chromatograph-mass spectrometer was established.The drug concentrations in rat plasma at different time points were determined after oral administration of MYR suspension and MYR-SA.The pharmacokinetic curves were plotted,pharmacokinetic parameters were calculated by non-atrial model of DAS software,and statistical analysis was performed to evaluate the pharmacokinetic characteristics of MYR-SA and to verify its ability to improve oral bioavailability.4.Pharmacodynamic study of MYR-SA in the treatment of alcoholism（1）The alcohol concentration in blood between different administration groups were examined after gavage of 50%ethanol solution（0.14 m L/10 g）in SD rats to evaluate the ability of MYR-SA in decreasing blood alcohol concentration.A mouse alcohol intoxication model was established by gavage of 62%ethanol solution（0.16 m L/10 g）in Kunming mice.Drunken mouse status,the onset and duration of intoxication were observed and recorded for different administration groups.The enzyme activities of Alcohol dehydrogenase（ADH）and Aldehyde Dehydrogenase（ALDH）in the liver of mice between different groups were also measured to assess the effectiveness of MYR-SA in promoting ADH and ALDH activity to alleviate intoxication.（2）To evaluate the protective effect of MYR-SA on acute alcoholic gastric mucosal injury in mice,a model of acute alcoholic gastric mucosal injury was established by gavage of anhydrous ethanol（0.10 m L/10 g）in Kunming mice.The morphology,degree of injury,and pathological sections of the gastric mucosa of mice in different administration groups were observed.The levels of superoxide dismutase（SOD）and malonaldehyde（MDA）in the gastric tissues of mice were measured to determine whether MYR-SA could protect the gastric mucosa from alcohol damage by enhancing antioxidant capacity.Result:1.The MYR-SA produced in our laboratory was sphere-like with an average particle size of 11.75±0.18 nm and a PDI of 0.059±0.020.The differential scanning calorimetry results showed that the two heat absorption peak of MYR was masked.MYR-SA could significantly improve the solubility,dissolution rate,and stability of MYR in simulated gastrointestinal fluid and liver homogenate.2.In the one-way intestinal perfusion experiment,the intestinal absorption rate constant absorption rate constant and effective permeability coefficient of MYR-SA were10.05 and 11.05 times higher than those of MYR suspension,respectively.It indicates that MYR-SA can significantly enhance the intestinal permeability of MYR.3.The results of pharmacokinetic experiments showed that the C_max of MYR-SA in blood（195.07 ng/m L）was 11.35 times higher than that of MYR suspension（17.19 ng/m L）,and the AUC_{（0²4 h）}value（333.56 ng/m L*h）was 2.17 times higher than that of MYR suspension（154.05 ng/m L*h）.It indicates that MYR-SA has higher bioavailability.4.In the experiment of MYR against alcohol intoxication,MYR-SA could significantly prolong the intoxication time and shorten the duration of intoxication state in drinking mice,significantly reduce the blood alcohol level and significantly increase the hepatic ADH and ALDH enzyme activity in drinking rats.Thus,MYR-SA exhibited better anti-alcoholic effect.5.In the protective experiment of MYR against acute alcoholic gastric injury,compared with the model group and MYR suspension group,MYR-SA significantly reduced the pathological damage of gastric mucosa in drinking mice by significantly increasing the SOD level and decreasing the MDA level（P<0.01）.Conclusion:The MYR-SA prepared in our laboratory greatly improves the solubility,release rate of MYR and its stability in simulated gastrointestinal fluid and liver homogenate.Meanwhile,MYR-SA can promote the absorption of MYR in the intestine due to its extremely small particle size and effective inhibition of intestinal P-gp.As a result,MYR-SA increased the oral bioavailability of MYR by 2.17-fold,which significantly improved the efficacy of MYR in counteracting alcoholism and protection of gastric mucosal tissues from alcohol damage.Therefore,MYR-SA will provide an experimental basis for the development of oral MYR formulations and provide new ideas and options for clinical applications such as alcoholism treatment.