| Gentiopicroside is one of the active gentianaceous ingredients.Researches have shown that gentiopicroside has a good hepatoprotective,choleretic,anti-inflammatory,analgesic and stomachic effect.In the current Chinese Pharmacopoeia(Edition 2010),only Jiaolong Capsule has been recorded,which is made of single herb Gentiana macrophy lla and is mainly used for the treatment of gastric diseases.And other gentianaceous herbs are formulated into compound preparations with other herbs in the clinical practice.The gentiopicroside monomer has a good protective and choleretic effect alone as preparation,but there is still no marketed product.The only the freeze-dried gentiopicroside powder for injection has been developed,but is now still in the clinical research stage.Due to the special structure having a split ring alkenyl ether terpene lactone structure,leading to its instability,Gentiopicrin is prone to degradation in the body of a strong acid and weak base.Meanwhile,due to the Gentiopicroside molecules linking part of glucose,Gentiopicroside is one of the active gentianaceous ingredients.Researches have shown that gentiopicroside has a strong polarity and good water solubility,so the absorption of the drug needs to penetrate the biofilm.It requires a certain amount of lipophilic,which means that oil-water partition coefficient is between-1 and 2.Gentiopicrin is of short oral half-life,low bioavailability,fast clearing,and short mean residence time.Therefore,aiming at the above insufficiency of gentiopicroside,this study made structural modification of gentiopicroside,attempting to make acetylation on the hydroxyl groups on the sugar molecule gentiopicroside and add gentiopicroside fat-soluble,in order to improve Gentiopicroside glycoside oral absorption and bioavailability.The study was conducted mainly on the following aspects:1 Gentiopicroside is a natural active ingredient widely distributed in gentians.The Gentiana scabra Bunge and Gentiana macrophylla with relatively high content of gentiopicroside were chosen in this study as the sources of herbs.It was detected by determining the content of different batches that Liaoning Gentiana scabra Bunge had the highest content of 30.41mg/g,the average content of the three batches of Gansu Gentiana macrophylla was approximately 25mg/g and the average content of the two batches of Guizhou Gentiana scabra Bunge was the lowest,12.37mg/g.2 The study selected the Gentiana macrophylla to extract Gentiopicroside;reflux extracted three times with 70%ethanol.The extraction rate was 87.36%.Referring to the experimental pre-the“gentian glycosides refined purification technology”,Gentiopicroside crude extract concentrate was refined by D101 macroporous resin and Gentiopicroside crude content of 51.03%;Gentiopicroside crude was purified by silica gel column chromatography and ethanol recrystallization method and Gentiopicrin purity of 91,74%.3 This study selected acetylation routes to modify Gentiopicroside structure.Taking reaction time,reaction temperature,the ratio of ingredients as factors to desigh the L9(34)orthogonal experiment and inspecting the significant affect factor,the best Gentiopicroside acetylation reaction process was as follows:pyridine as catalyst,acetic anhydride as acetylating agent,acetic anhydride and Gentiopicroside to the feed ratio of 10:1,the reaction at room temperature for 12 hours.The reaction crude product was purified by silica gel column chromatography,first eluting with 3BV chloroform,and then the 5BV chloroform/methanol 70:1 eluting.The target eluting was dryed and obtained a white solid.Purified product was identified by UV,mass spectrometry,el.The modification was identified as tetra acetylated Gentiopicroside.4 Through tetra acetylated Gentiopicrin physicochemical properties study,it was found that its melting point was 137.9?.Apparent solubility in water was 0.1lmg/mL,which increased with increasing pH value.Tetra acetylated Gentiopicrin was insoluble in water,slightly soluble in ethanol and methanol,soluble in acetonitrile and chloroform.The apparent oil/water partition coefficient 1gP was 1.40,which reduced with increasing pH value.In stability test,tetra acetyl Gentiopicroside were high temperature,high light and acid-sensitive and insensitive to humidity.Micromeritic basic properties measurement results showed that tetra acetyl Gentiopicroside had poor liquidity.5 The absorption characteristics of Gentiopicroside and tetra acetyl Gentipicroside were investigated by in vivo and in vitro intestinal of SD rats.Gentiopicroside blood samples using SPE solid phase extraction pretreatment,the extraction recoveries of high,medium and low concentration were 76.94%,74.63%and 71.49%respectively.Tetra acetyl Gentiopicroside blood samples using chloroform extraction pretreatment,the extraction recoveries of high,medium and low concentration were 76.81%,74.13%and 71.62%respectively.The blood samples were detected by HPLC,but did not detect Gentiopicroside or tetra acetyl Gentiopicroside.Taking the valgus intestinal sac for vitro intestinal absorption,the study results shew that the absorption percentage of GTD and tetra acetyl Gentiopicroside were low in each intestine part.However,compared with GTD,the absorption percentage of tetra acetyl Gentiopicroside increased by 28.22%,42.78%and 29.91%respectively in three bowel.After statistical analysis,compared with GTD,the apparent permeability coefficient of tetra acetyl Gentiopicroside significantly increased in jejunum and ileum,added 43.04%and 28.83%,respectively.but the apparent permeability coefficient in duodenum did not significantly increase,an increase of only 10.34%.The introduction acetyl into Gentiopicroside,its absorption was raised.6 The paper made a preliminary pharmacodynamics study on the tetra-acetylated gentiopicroside.High,medium and low doses of tetra-acetylated gentiopicroside were given to hepatitis model made by carbon tetrachloride.The contents of serum alanine aminotransferase(ALT)and aspartic acid aminotransferase(AST)were measured after blood sampling.The result revealed that tetra-acetylated gentiopicroside can significantly relieve the increasing of carbon tetrachloride-induced hepatitis aminotransferase and a dose-effect relationship was showed.As the dose of tetra-acetylated gentiopicroside increased,ALT and AST declined more obviously.Comparing with the gentiopicroside group,its hepatoprotective effect was equal or even better.In short,although the synthesized tetra-acetylated gentiopicroside is a bit different from gentiopicroside in structure,its original nucleus has not changed and it still has hepatoprotective effect.7 By modifying the structure of gentiopicroside and after the acetylation of sugar hydroxyl groups,the fat-solubility and oil-water partition coefficient of gentiopicroside have been changed to some extent.After intestinal absorption experiment it was found that the absorption of gentiopicroside was increased and its bioavailability improved.However,its efficacy did not change significantly still with hepatoprotective effect,which laid a certain material base for the follow-up research of tetra-acetylated gentiopicroside.8 From the absorption of tetra-acetylated gentiopicroside in the intestinal tract,it was indicated that its absorption was poor under acidic conditions,but it was better absorbed in the jejunum and ileum comparing with the absorption in other intestinal segments.From this point it can regarded that as to the future development of tetra-acetylated gentiopicroside enteric coating may be considered for oral agent,which is convenient for administration,to avoid degradation under acidic conditions in the stomach and thereby to improve the bioavailability during oral administration. |
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