Study On Synthesis And Nano Drug Delivery System Of Rhein Amide Derivatives

As the main effective component of Chinese herbal rhubarb and aloe,the source of rhein is very wide.In recent years,more and more studies have shown that rhein has a lot of activities,such as antitumor,antibacterial,antiviral,hepatoprotective,antifibrosis,glucose-lowering and lipid-modifying effect,especially in the treatment of osteoarthritis and diabetic nephropathy,the effect of rhein is outstanding.At the same time,the toxicity of rhein is also very low.However,its low water solubility and poor bioavailability greatly limit the development and clinical application of rhein.This researth firstly studied the structure transformation of rhein.Six kinds of amino acid were chosen to generate a series of rhein amide derivatives by the acylation reaction with rhein.The nuclear magnetic resonance hydrogen spectrum,mass spectrum and fourier transform infrared spectrum analysis had been studied to confirm the structure of each compound.The differential scanning calorimetry was used to determine the melting point of each compound.In addition,the methods for in vitro analysis of six rhein amide derivatives were established,and the purity,solubility and apparent oil/water partition coefficients of each compound were determined by HPLC method.The HepG2 cells were chosen to study the preliminary pharmacological of six rhein amide derivatives,and the positive control drug was 5-fluorouracil.IC50 value of rhein,5-fluorouracil and six rhein amide derivatives had been determined at 12 h,24 h,48 h,72 h,respectively.The results showed that within 48 hours,the activities of four kinds of rhein amide derivatives were better than rhein.Of those,rhein-phenylalanine had the best activity,which followed by rhein-isoleucine,rhein-leucine and rhein-valine.When the culture time was extended to 72 h,the activity of rhein-leucine was the strongest,which followed by rhein-isoleucine and rhein-phenylalanine.After culture of 72 h,the effect of rhein-glutamic acid on the proliferation was promoted.SD rats were chosen as the animal model to analyze the pharmacokinetics of six rhein amide derivatives.The results showed that plasma concentrations of rhein-leucine,rhein-isoleucine and rhein-valine were significantly higher than that of rhein.The plasma concentration of rhein-phenylalanine was slightly higher than rhein.The plasma concentrations of rhein-glycine and rhein-glutamic acid were significantly lower than that of rhein.Through the comprehensive comparison of solubility,oil/water partition coefficient and pharmacokinetics of each compound,the rhein-isoleucine and rhein-phenylalanine were chosen finally to be prepared into nanosuspensions,which would further improve the efficacy of compounds.In the study of preparation,the methods of preparation were compared and selected,respectively.Some important factors which influence the preparation process had been studied,such as the content of stabilizer,homogen number,homogen pressure,and so on.The particle size,zeta potential and polydispersity index(PDI)were used as the index of investigation.The optimal prescription were as follows:Taking rhein-isoleucine(rhein-phenylalanine)50 mg,dissolved in 4 ml of methanol as oil phase.Taking poloxamer 188 25 mg and 25 mg of lecithin in 100 ml beaker,dissolved in 40 ml of pure water by ultrasonic as water phase.The water phase was placed in an ice water bath,and the oil phase was added dropwise into water phase under stirring.After dispersing completely,making the solution continuously stirred for 30 min,and then removing the methanol by a rotary evaporator.Under 20000 rpm condition,the solution was sheared for 3 min by high speed shearing machine to obtain a coarse nanosuspension.After placing for 10 min to remove the bubble,the coarse nanosuspension was transferred into a high pressure micro jet.Under 4660 psi condition,the coarse nanosuspension was processed for 3 times,followed by 20 times under the pressure of 11650 psi to obtain the final nanosuspension.In order to accomplish long-term storage and facilitate the administration of animals,the rhein-isoleucine nanosuspension and rhein-phenylalanine nanosuspension were transformed into dry powders by freeze-drying method.The proportion of cryoprotectant was studied.The results showed that when adding 5%mannitol as cryoprotectant,the effect of freeze-drying product was the best.In order to compare the pharmacokinetics and bioavailability of raw compounds and its nanosuspensions,the in vivo experiments of rhein-isoleucine nanosuspension and rhein-phenylalanine nanosuspension were studied.The analysis method of compounds in vivo was established,and the concentration-time curve in plasma was studied by high performance liquid chromatography.The results showed that after prepared into nanosuspensions,the bioavailability of rhein-isoleucine was improved by about 2 times.The AUC0-∞ of rhein-isoleucine suspension group and rhein-isoleucine nanosuspension group were 250.01 mg/L.h and 545.82 mg/L·h,respectively.The half-life was increased from 4.73 h of rhein-isoleucine suspension group to 8.18 h of rhein-isoleucine nanosuspension group.However,the bioavailability of rhein-phenylalanine was improved by about 3.5 times.The AUC0-∞of rhein-phenylalanine suspension group and rhein-phenylalanine nanosuspension group were 19.97 mg/L·h and 70.28 mg/L·h,respectively.The half-life was increased from 3.53 h of rhein-phenylalanine suspension group to 9.90 h of rhein-phenylalanine nanosuspension group.In conclusion,the rhein-isoleucine and rhein-phenylalanine could improve the antitumor activity and solubility of rhein.After making into nanosuspensions,the pharmacokinetics of rhein-isoleucine and rhein-phenylalanine were improved.The results showed that the nanosuspensions could improve the bioavailability and extend the release of rhein-isoleucine and rhein-phenylalanine.The reaserch provides a way for the structural modification of natural products and study of insoluble drug development,which has important significance for the clinical application of rhein.