| Yangzheng Xiaoji(YZXJ)capsule is a traditional Chinese formulation,which is composed of 16 herbs.It is mainly applicable to the adjuvant treatment that can be used in primary hepatic cancer,which is caused by the asthenia of both the spleen and kidney,as well as internal obstruction of stasis and toxin.And it can improve the efficacy of TAE(transcatheter arterial embolization)and TACE(transcatheter arterial chemoembolization)as well.In addition,clinical studies have confirmed that YZXJ capsule can be used in the adjuvant treatment of several malignant solid tumors,such as lung cancer,gastric cancer,colorectal cancer and breast cancer,etc.Recent studies highlighting on pharmacological mechanisms of YZXJ indicated that this formulation can inhibit the adhesion,migration and invasion of tumor cells by acting on multiple signaling pathways around body,such as PI3K/Akt,FAK,HSP27,HGF and its receptor c MET;More than that,YZXJ works on anti-tumor by regulating related tumor factors and immune cells.Nonetheless,there is no field experiment of active ingredients or pharmacokinetic characteristic of YZXJ has been reported until now.This study is going to test chemical constituents of it,and analysis active ingredients of YZXJ by applying modern analytical methods.Besides that,the research provides experimental reference for quality control of YZXJ capsule by measuring contents of some active compositions.Furthermore,analysis of pharmacokinetics of different active compositions in mice would reveal the process of drug in the body metabolism,and make contribution to selecting suitable active components as PK-markers of YZXJ in the future.Objective: The methods were established for initial exploring the chemical compositions,contents of partial active ingredients as well as pharmacokinetics in mice,which provide the reference of quality control and PK-marker selection.Methods: 1.Identification of liposoluble constituents in YZXJ by GC-MS.The preparation process of YZXJ sample solution for liposoluble constituents’ analysis was as follows: YZXJ was dissolved by adding about 70oC petroleum ether,after the reflux extraction,evaporation and concentration,centrifugation,and then filtered by polypropylene membrane.Filtrate 1 μL was taken for analysis.In GC-MS condition,purity nitrogen was used as carrier gas with temperature-programmed.The date collection was in full scan mode with EI source and the scanning range of quality was m/z 40-650.2.Identification of water-soluble constituents in YZXJ by UPLC-Q-TOF-MS.The preparation process of YZXJ sample solution for water-soluble constituents’ analysis was as follows: YZXJ was added with 70% methyl alcohol,after reflux extraction,evaporation and concentration,centrifugation,and then filtered by polypropylene membrane.Filtrate10 μL was taken for analysis.In UPLC-Q-TOF-MS condition,15 mmol/L ammonium formate solution and acetonitrile were used as mobile phase with gradient elution.The data collection was in positive and negative mode with ESI source and the scanning range of quality was m/z 50-1500.3.Contents determination of the partial active constituents of YZXJ by HPLC.The contents of Scutellarin,Baicalein and AtractylenolideⅡwere determined by HPLC.At first,sample solution or a series of mixed standard solutions were prepared,and filtered by polypropylene membrane.Filtrate 20 μL was taken for analysis.In HPLC condition,acetonitrile and 1% acetic acid were used as mobile phase with gradient elution,and the test wavelength was settled at 275 nm.Then,the methodology should be validated by analysis the specificity,linearity,precision,repeatability,24 h stability and the average recovery under the same experimental condition.After all,the average contents of Scutellarin,Baicalein,AtractylenolideⅡin six batches of samples were determined.4.Exploring pharmacokinetic characteristics of some active constituents of YZXJ by UHPLC-MS.Pharmacokinetic characteristics of Scutellarin and Oleanolic acid of YZXJ in mice with UHPLC-MS was preliminary explored.To study it,the mice model was established,the preparation process of which was as follows: blood samples were collected by eyeball extirpating at different time points after oral administration of 3.90 g/kg YZXJ sample extract.Then,the plasma concentrations of two constituents were determined and the concentration-time curves were obtained.At last,pharmacokinetic parameters were analyzed by Win Nonlin6.4 software with non-compartmental model based on statistical moment theory.Results: 1.The result of liposoluble constituents’ analysis in YZXJ.A total of 42 liposoluble components were identified with GC-MS,including volatile oils,sesquiterpenoids,phenols and sterols,etc.Among this complex chemical compound system,only 17 of them could be defined,mainly were derived from Curcumae zedoary,Atractylodis macrocephalae rhizome,Poria,Cynanchum paniculatum.The percentage of the paeonol which was derived from Cynanchum paniculatum was the most,to the number of 11.49%;Lupeol,at the second place,was existed in much traditional Chinese medicine,such as Curcumae zedoary and Ligustri lucidi fructus,totally accounting for 3.71%;Neocurdione,which was from Curcumae zedoary,occupied 1.2% of all.2.The result of water-soluble constituents’ analysis in YZXJ.A total of 38 water-soluble constituents were identified with UPLC-Q-TOF-MS,mainly belonged to Astragali radix,Ligustri lucidi fructus,Ginseng radix et Rhizoma,Ganoderma,Atractylodis macrocephalae rhizoma,Scutellariae barbatae herba,Artemisiae scopariae herba.According to the genus of compositions,9 components of Ganoderma,6 components of Astragali radix,Ginseng radix et Rhizoma and Ligustri lucidi fructus,2 components of Scutellariae barbatae herba and Artemisiae scopariae herba,1 component of Atractylodis macrocephalae rhizome were identified.According to the classification of chemical structure,21 triterpenoids,11 flavonoids,each one of iridoid,lactone,phenylethanoid glycoside,phenolic acid,alkaloid and vitamin were identified.3.The result of content determination of the partial active ingredients in Scutellariae barbatae herba and Atractylodis macrocephalae rhizome.The contents of Scutellarin,Baicalein and AtractylenolideⅡ in YZXJ were determined by HPLC.The average contents of Scutellarin,Baicalein and AtractylenolideⅡ in six batches of samples were about 80.89,2.713,3.708 μg/g,respectively.4.The result of pharmacokinetic characteristics of Scutellarin and Oleanolic acid in mice.Pharmacokinetic characteristics of Scutellarin and Oleanolic acid were preliminary explored by UHPLC-MS.The results were as follow: both of Scutellarin and Oleanolic acid were absorbed rapidly after oral administration of YZXJ extract,the peak concentration of the two ingredients were both reached at around 10 min.About 8 hours after administration,there was secondary absorption of two ingredients,and the plasma concentration curves showed double-peak phenomenon.The half-life of Scutellarin and Oleanolic acid were at 5.69 h and 3.08 h respectively.The plasma concentration and content of Oleanolic acid in vivo were lower than those of Scutellarin,but Oleanolic acid’s clearance rate was faster and distribution in body tissues was wider.Conclusion: 1.The chemical constituents of YZXJ were analyzed by GC-MS and UPLC-Q-TOF-MS.There were 42 liposoluble constituents and 38 water-soluble constituents had been identified,some of these ingredients preformed anti-tumor activity in different degrees according to literature reports.2.The contents of partial active ingredients of Scutellariae barbatae herba and Atractylodis macrocephalae rhizome in YZXJ were determined by HPLC.The average contents of Scutellarin,Baicalein and AtractylenolideⅡ in six batches of YZXJ were about 80.89,2.713,3.708 μg/g respectively.3.The pharmacokinetic characteristics of Scutellarin and Oleanolic acid in mice had been initially explored by UHPLC-MS. |
