Interaction Of Ilex Kudingcha C.J.Tseng And Bovine Serum Albumin And Construction And Characterization Of Nanoparticles

Ilex kudingcha C.J.Tseng is a tree plant in the genus Ilex holly.It is rich in pentacyclic triterpene saponins,flavonoids,steroids,polysaccharides and other chemical components.It has hypolipidemic,hypoglycemic,anti-tumor and antioxidant properties.And many other biological activities.Triterpene saponins are one of the main active components of Ilex Kudingcha,and they are also a hot spot in the research on the chemical constituents of Ilex plants in recent years.Serum albumin(SA)can carry many drug molecules through plasma storage and transportation to the receptor site to produce drug effects,which is an important step for drug molecules to exert drug effects.At present,there is no report on the research of the interaction mechanism between Ilex kudingcha C.J.Tseng saponin compound and bovine serum albumin(BSA).Therefore,it is of great significance to clarify the information on the binding status and type of action between Ilex kudingcha C.J.Tseng saponins and serum albumin,and its pharmacodynamics and pharmacokinetics.The poor water solubility and low bioavailability of kudingcha holly saponins directly affect its medicinal effects.Therefore,this paper selected 3monomer compounds from the 15 kinds of Ilex kudingcha C.J.Tseng saponins monomer compounds isolated and identified in the early stage of the laboratory,including a monomer compound with a closed ring structure in the aglycone and a pair of open ring structures in the aglycone.The interaction mechanism between these three monomer compounds and BSA was preliminarily discussed using various spectroscopy methods,and the characteristics of the transport of different structural types of compounds in the body were clarified.Nano formulations can improve the solubility and targeting of drugs,thereby improving the utilization rate of drugs.In this paper,two different carrier materials are used to prepare the nano formulations of Ilex kudingcha C.J.Tseng saponins.It explores to improve its bioavailability and provides an experimental basis for the development of new traditional Chinese medicine nano preparations.Using atmospheric column chromatography,semi-preparative high performance liquid chromatography and other means,the saponins were separated and purified from the n-butanol extraction part of the 70% ethanol extract of Ilex kudingcha C.J.Tseng dry leaves to obtain compound 2(446.3 mg),Compound 9(303.1 mg)and compound 10(276.9 mg)were identified as the structure of Kudinoside D,Kudinoside N and Kudinoside L,the aglycone of Kudinoside D has a closed ring structure,Kudinoside N and Kudinoside L are a pair of isomers,and the aglycone has an open ring structure.In order to study the mechanism of binding and transport between Kudinoside D,Kudinoside N,Kudinoside L and BSA at different temperatures(298K,310K)and the changes in the secondary structure of BSA,fluorescence spectroscopy was used to analyze the relationship between Kudinoside D,Kudinoside N,Kudinoside L and BSA;and circular dichroism was used to investigate the changes in the secondary structure of BSA.The results show that Kudinoside D,Kudinoside N,and Kudinoside L can effectively quench the endogenous fluorescence of BSA.Each of the three compounds forms a binding site with BSA,indicating that the open and closed ring structures of the Ilex kudingcha C.J.Tseng saponin compound aglycone do not affect its combination with BSA.In addition,after the combination of Kudinoside D,Kudinoside N,and Kudinoside L with BSA,the content of α-helix,β-sheet,β-turn and random coils in BSA all changed to different degrees,it shows that the combination of these three saponin compounds with BSA changes the internal environment of BSA.It is worth noting that the fluorescence quenching degree and binding constant of Kudinoside N to BSA are both smaller than Kudinoside L.This may be due to the fact that Kudinoside N has an extra rhamnose group on C-28,and the sugar chain is longer than Kudinoside L.It is related to the difficulty of combining with BSA.Using polylactic acid-glycolic acid copolymer(PLGA)and polyglutamic acid-L-phenylalanine ethyl ester copolymer(γ-PGA-PAE)as carriers,prepare nanoparticles loaded with Kudinoside D,Kudinoside N,and Kudinoside L,Screening the carrier based on drug loading and encapsulation efficiency;using dialysis method to optimize the nanoparticles prepared by γ-PGA-PAE;using transmission electron microscopy and dynamic nanoparticle size analyzer to analyze the γ-PGA-PAE nanoparticles Characterization of physical and chemical properties;use dialysis method to investigate the in vitro release characteristics of γ-PGA-PAE nanoparticles.The results show that the drug loading of nanoparticles prepared with γ-PGA-PAE as a carrier is 2 to 3 times higher than those prepared with PLGA as a carrier.The encapsulation efficiency of the former is as high as 7.82% higher than that of the latter.In addition,the results show that it is comparable to the precipitation Compared with the nanoparticles prepared by dialysis method,the encapsulation efficiency and drug loading capacity are 11.2%～14.26% and 1.38%～3.74% higher than those prepared by precipitation method,respectively.The in vitro characterization results of γ-PGA-PAE nanoparticles prepared by dialysis method showed that the average particle size ofγ-PGA-PAE-Kudinoside D nanoparticles was(75±25)nm,and the average particle size of γ-PGA-PAE-Kudinoside N nanoparticles.The average particle size ofγ-PGA-PAE-Kudinoside L nanoparticles is(90±25)nm.Studies have found that these drug-loaded nanoparticles have good nanometer size,are more concentrated,have a strong positive charge,and have good stability.In vitro drug release experiments show that γ-PGA-PAE-Kudinoside D nanoparticles,γ-PGA-PAE-Kudinoside N nanoparticles,and γ-PGA-PAE-Kudinoside L nanoparticles can significantly improve the water solubility of drugs and have a good sustained-release effect in vitro.