The Active Constituents Of Radix Isatidis On The Basis Of Liposome Ultrafiltration

Objective: The methods to optimize of liposomes and using sedimentation centrifugal ultrafiltration,combining with HPLC and LC-ESI-MS technique, screening the composition of Radix isatidis which can be absorbed by liposome.In vitro of gastrointestinal microenvironment, It infered that which can be affected by the environment of gastrointestinal tract, the main catalysis of enzyme,the processes of metabolism and transport.Finally, it determined the active ingredient of Radix isatidis. Methods:(1) To use the method of reverse-phase evaporation, Using cholesterol, soybean lecithin as the carrier, orthogonal design was adopted to optimize soybean lecithin / cholesterol ratio, hydration time, hydration medium volume and hydration medium PH in preparation process. EE used as evaluation criteria, two representative compounds—cytidine and adenosine selected as testing indexes. An HPLC method was developed to determine the best preparation.(2) The best preparation of Radix isatidis liposomes, using HPLC-MS technique, furtherly researching on the composition of Radix isatidis which can be wrapped by liposomes.(3) In the Radix liposomes for the study, arginine, cytidine, uridine, epigoitrin and adenosine to have been detected. The technology of centrifugal sedimentation – ultrafiltration, analysising with HPLC combined LC-ESI-MS, in the the simulation environment of gastrointestinal tract(p H1.0~8.0), examining the affecting enzymes, and whether there were metabolites. Final it determinated of reasonablely Radix isatidis active ingredient.(4) Establish model for absorption and transport of Caco-2 cells. Adapt the method of MTT to observe the safety of the tested compound for Caco-2 cells. Inspect the established model with the indicators of trans-epithelial electrical resistance, alkaline phosphatase and permeability of sodium fluorescein. Respectively use HPLC method to inspect the influence of concentration time, temperature, p H value and inhibitor to the absorption of the tested compound. Measure the apparent permeability coefficient and efflux rate of the tested compound on different sides. Establish model for absorption and transport of Caco-2 cells. Measure the apparent permeability coefficient and efflux rate of the tested compound on different sides. Established the model of Caco-2 cells, study the rotor volume of compound from the AP side�' BL side and BL side �' AP side. Calculated apparent permeability coefficients of compound(Papp(AP-BL)、Papp(BL-AP))and ER(ER=Papp(BL-AP)/Papp(AP-BL)).Results:(1) Determined to use reverse-phase evaporation to prepare Radix isatidis liposomes: the ratio of soy lecithin to cholesterol is 6:1, the hydration time is 25 minutes, the amount of PBS phosphate buffer solution is 10 m L, the value of p H hydration medium is 6.86.The linear ranges were 10.27~500.3 μg·m L-1(r=0.9998,n=6) for cytidine and 10.36~493.6 μg·m L-1(r=0.9997, n=6) for adenosine. Their recoveries 、 precision and atability of cytidine and adenosine were good.(2) Furtherly analysis, the final test in Radix isatidis liposomes have five ingredients: arginine, cytidine, uridine, epigoitrin,adenosine.(3) By the LC-MS data analysis it found that arginine, cytidine and adenosine have been impacted the the environmental the gastrointestinal tract except epigoitrin.The new substances have been produced: arginine producing new substances as ornithine, cytidine producing new substances as cytosine, adenosine producing new substances as adenine. Then through the analysis of HPLC, arginine and cytidine are mainly affected by trypsin under gastrointestinal tract, adenosine is mainly affected by pepsin under gastrointestinal tract; Finally it determined by LC-MS technique, arginine was affected by trypsin, the metabolism was ornithine; adenosine was affected by pepsin, the metabolism is adenine.(4) After 21-day culture and differentiate of the Caco-2 cells used for the transport experiment, the cells are similar to intestinal epithelial cells in shape; the transcellular transportation of fluorescein was less than 0.5 %/h, transepithelial electrical resistance were more than 200 ?·cm2, the cells meet polarity differentiation and form even single cell layer, and its compactness, integrity and functions were all excellent. The absorption of Caco-2 cells to tested compound presents linear relation in 60 min, the absorption time of medicines was 60 min; as the temperature increases, the absorption amounts of arginine and adenosine in isatidis radix both decrease, while the absorption amount of adenosine in isatidis radix increases as p H value increases. P-gp inhibitor verapamil can significantly increase the absorption of the Caco-2 cells to arginine and adenosine in isatidis radix(P<0.05). The absorbent containing of arginine and adenosine in Radix isatidis was good(Papp(AP-BL)>10-6 cm/s、Papp(BL-AP)>10-6 cm/s), the ER value of adenosine was greater than 1.5, Apparent permeability of the bidirection transport was excellent, and direction differences existing. Conclusion: In this study, by the means of ultrafiltration precipitation techniques, combined with the analysis means of HPLC and LC-ESI-MS, preliminarily screening five ingredients in Radix isatidis liposomes: arginine, cytidine, uridine, epigoitrin,adenosine. Through the metabolism in vitro gastrointestinal microenvironment and transporting model of Caco-2 cells, finally it screened identification of reasonable and effective active ingredients in Radix isatidis: arginine, adenosine. Compared with conventional screening methods in medicinal ingredient, afiltration mass spectrometry as the method of reactive component screening had the technical advantages of high throughput, sensitive, and rapid. It could be suited for early screening of the active ingredient. Combined with gastrointestinal micro-environment in different p H and the model of cell, finally confirmating of the active ingredient. The method was novel, scientific and reasonable. This article also provided a new screening ideas of active ingredient,such as Radix isatidis,the complex herbal ingredients.Also provided a reference for clinical application.