Exploration The Application Of New Oral Drug Formulation Of Nanocrystals Self-stabilized Pickering Emulsions In Component-based Chinese Medicine Of Tongmai Recipe

Nanocrystals self-stabilized Pickering emulsion（NSSPE）is a new oral drug delivery system designed by our research group,which uses nanocrystals of insoluble ingredients as solid particle stabilizers.It has been successfully applied to monomers of Traditional Chinese medicine,such as silybin and puerarin.As far as I am concerned,whether NSSPE could be applied to complicated traditional Chinese medicine with multi components is not yet known.In this study,the Tongmai Recipe which is composed of Puerariae Radix,Salvia miltiorrhiza Bge,and Chuanxiong Rhizoma at a mass ratio of 1:1:1 was used as a model recipe,and its main effective ingredients were combined to form a component-based Chinese medicine.The aim of the study was to investigate the feasibility of applying NSSPE to component-based Chinese medicine,and to explore the effect of NSSPE on the absorption of different effective ingredients of Chinese medicines.The research methods and results were as follows:（1）Construction and in vitro evaluation of NSSPE of component-based Chinese medicine of Tongmai RecipeA HPLC method was established for simultaneous determination of puerarin,ferulic acid,salvianolic acid B,ligustilide,and tanshinoneⅡA in Tongmai Recipe.The ratio of puerarin,ferulic acid,salvianolic acid B and tanshinoneⅡA in the components of traditional Chinese medicine was determined to be 1:0.035:0.7:0.03 according to their content ratio in the Tongmai preparations on market and Salvia Miltiorrhizae Radix et Rhizoma.Influences of pH and concentration of nanocrystals suspension and the ratio of Ligusticum chuanxiong oil and Fabrafil M 1944 CS in the mixed oil phase on the construction of NSSPE of component-based Chinese medicine of Tongmai Recipe were investigated.The drug content in the emulsion and the separation of the oil phase after standing and centrifugation were used as evaluation indexes.The results showed that a relatively good NSSPE of component-based Chinese medicine of Tongmai Recipe could be prepared at the following condition:pH of the nanocrystals suspension was 6.5,the concentration of the total ingredients was 13.24 mg·m L^-1,and the oil phase was a mixed oil of Ligusticum chuanxiong oil:Fabrafil M 1944 C=9:1（v/v）.For NSSPE of component-based Chinese medicine of Tongmai Recipe,the emulsion droplet size was1.43±0.09μm,which was larger than 1.26±0.03μm of the blank emulsion,the zeta potential was-54.17±0.85 m V,which was similar to 59.70±0.50 m V of the nanocrystals suspension,and both were lower than-31.10±0.36 m V of blank emulsion.The stability of NSSPE was significantly better than nanocrystals suspension and blank emulsion.Fluorescence inverted microscope（FIM）observed a circle of bright green fluorescence of puerarin,ferulic acid,or tanshinoneⅡA on the surface of the droplets of NSSPE.Scanning electron microscope（SEM）observed that the surface of droplets of NSSPE had obvious adsorbent,which was different from the smooth surface of blank emulsion.These results confirmed that the constructed NSSPE of component-based Chinese medicine of Tongmai Recipe had a microstructure of NSSPE,i.e.the nanocrystals of insoluble ingredients adsorbed on the interface of oil droplets.It could be seen that it was feasible to apply NSSPE to component-based Chinese medicine with many active ingredients.（2）The effect of freeze drying on solidification of NSSPE of component-based Chinese medicine of Tongmai RecipeIn view of the fact that solid preparations have better stability and more dosage forms than liquid preparations,NSSPE of component-based Chinese medicine of Tongmai Recipe was solidified by freeze-drying method.With the appearance and redispersibility of freeze-dried emulsion as evaluation indexes,the kinds and amount of freeze-drying protectant were selected.Finally,the freeze-dried emulsion with good appearance and redispersibility could be obtained when freeze-drying protectant was PVP K30-maltose（1:9,w/w）and the amount was 8%.The appearance,particle size,zeta potential and contents of puerarin and salvianolic acid B in the reconstituted emulsion were not significantly different from the original emulsion,but contents of ferulic acid,ligustilide and tanshinoneⅡA in the reconstituted emulsion decreased by16.72%,9.53%and 19.32%,respectively.Similar morphology and structures of reconstituted emulsion droplets to original emulsion droplet were observed by FIM and SEM,which indicated that freeze drying did not destroy the structure of the emulsion droplets of NSSPE.The in vitro dissolution experiment was carried out by the paddle method with simulated artificial gastric fluid（0～2 h）and artificial intestinal fluid（2-8 h）as releasing media,.The results showed that the cumulative release rates of puerarin and tanshinoneⅡA of original NSSPE increased from 72.57%and 18.18%of the crude materials to96.80%and 24.01%,but there was no significant difference for ferulic acid,salvianolic acid B and ligustilide.Freeze drying did not influence the release of puerarin,ferulic acid and tanshinoneⅡA in NSSPE greatly,but significantly reduced the release of salvianolic acid B,especially in simulated gastric fluid NSSPE of component-based Chinese medicine of Tongmai Recipe.The release NSSPE of component-based Chinese medicine of Tongmai Recipe of ligustilide from freeze dried NSSPE in simulated intestinal fluid was 80%higher than from the original NSSPE.The stability of freeze-dried NSSPE was investigated at 40,4 and-20℃,respectively.The appearance,redispersibility,particle size,Zeta potential and drug content were measured at 0,1,2,3 and 6 m.The results showed that the freeze-dried NSSPE could be stable for 6 m at-20℃.The higher the temperature was,the more components lost.（3）Caco-2 cell absorption of NSSPE of component-based Chinese medicine of Tongmai RecipeThe Caco-2 cell model was used to study the oral absorption of NSSPE of component-based Chinese medicine of Tongmai Recipe.The MTT experiment,cell uptake experiment and bidirection transport experiment.On the basis of screening the pH and concentration of NSSPE,the uptake and transports of each component in the crude materials,nanocrystals,original NSSPE and reconstituted NSSPE were compared,and endocytosis pathways of NSSPE in Caco-2 cell was investigated.The MTT experiment showed that when the concentration reached 200μg·m L^-1which was calculated as Pu concentration,NSSPE was toxic to Caco-2 cells.After being prepared into reconstituted emulsion,the toxicity was further enhanced.In the investigated pH range of 5.0 to 7.4 and the concentration range of 25 to 100μg·m L^-1,the changing trends of the uptake and transport of each component in NSSPE are not completely consistent.On the whole,Caco-2 cells with pH 5.0 and concentration of 100μg·m L^-1 had higher uptake rate,higher apparent permeability coefficient（Papp）and lower efflux rate（ER）in AP→BL direction.The results of uptake and transport experiments of different preparations showed that:（1）Compared with the crude materials+oil group,the uptake rates of puerarin,ferulic acid,salvianolic acid B,ligustilide,and tanshinoneⅡA in the original emulsion of NSSPE increased by 78.72%,269.18%,161.50%,90.22%and 131.06%,respectively.The Papp of puerarin,salvianolic acid B and tanshinoneⅡA increased significantly,but the ER decreased;the Papp of ligustilide respectively increased from 0（difficult to transport across the membrane）to 0.79（AP→BL）and 0.74（BL→AP）;the bidirection transport of ferulic acid has no obvious change.（2）Compared with the nanocrystals+oil group,the uptake rates of puerarin,ferulic acid,ligustilide,and tanshinoneⅡA in NSSPE increased by 31.73%,198.52%,114.10%,58.22%,respectively,and salvianolic acid B did not change significantly.There was no significant change in the Papp of puerarin,ferulic acid,and salvianolic acid B in bidirection transport;the Papp of bidirection transport of tanshinoneⅡA increased by about 3.3 times,so there was no significant change in ER;ligustilide was also difficult to transport across the membrane become a certain transmembrane transport rate.（3）Compared with the no oil group,only the uptake rate of ferulic acid（this is due to the ferulic acid contained in the oil phase）and the Papp of TanshinoneⅡA AP→BL increased significantly when the oil phase was added to the crude materials or nanocrystals.It is suggested that the effect of NSSPE in promoting the absorption of each component is not simply due to the addition of the oil phase,but is also related to its special emulsion droplet structure.（4）After NSSPE freeze-dried emulsion was redissolved,the uptake rates of ferulic acid,ligustilide and tanshinoneⅡA were significantly lower than those of the original emulsion,while puerarin had no significant difference,while salvianolic acid B increased slightly.The Papp for the bidirection transport of the five components was significantly reduced,especially puerarin and salvianolic acid B decreased by about 50%;but the ER of each ingredient did not change significantly.It is suggested that the absorption of each ingredient of NSSPE becomes worse after freeze-drying,which may be related to the loss of Ligusticum chuanxiong oil during freeze-drying.The results of NSSPE entry pathway showed that genistein（caveolin mediated endocytosis inhibitor）inhibited the uptake of five components in NSSPE,but only affected the transport of ferulic acid and tanshinoneⅡA in AP→BL,the Papp decreased by 15.88%and 24.35%,respectively.However,chlorpromazine（an inhibitor of clathrin mediated endocytosis）and amiloride（an inhibitor of clathrin mediated macropinocytosis）had no effect on the uptake and transport of the ingredients.It is suggested that the endocytosis of NSSPE by Caco-2 cells is related to the caveolin-mediated pathway.In this study,nanocrystals of NSSPE of component-based Chinese medicine of Tongmai Recipe which were composed of puerarin,ferulic acid,salvianolic acid B,and tanshinoneⅡA,the main effective ingredients of Tongmai Recipe,were used as aqueous phase and stabilizers,and Ligusticum Chuanxiong oil,the main effective component of Chuanxiong Rhizoma,was used as the main oil phase to prepare NSSPE with many active ingredients.Compared with the crude materials,the oral absorption of the five effective ingredients had been improved,especially for salvianolic acid B,ligustilide and tanshinoneⅡA.The reasons were related to the existing form of nanocrystals,as well as the dissolution in the oil phase in the form of molecules.The oral absorption mechanism was mainly passive diffusion,and caveolin-mediated endocytosis also played a certain role.