Study On Chemical Constituents And Quality Control Of Compound Kushen Injection

Compound Kushen Injection (CKI), namely Yanshu Injection, is a prescription prepa-ration recorded in the section14of the Drug Standards issued by Ministry of Health of the People’s Republic of China and prepared from both of Sophorae Flavescentis Radix and Heterosmilacis Japonica Rhizomae. CKI has the diverse activities of an-ti-tumor, analgesia and raising tumor patient’s immunity and was widely used for the treatment of the pain caused by the various cancer diseases. Sophorae Flavescentis Radix is the root of Sophora flavescens Ait.. Heterosmilacis Japonica Rhizomae is collected the dried tuber of H. japonica Kunth., H. yunnanensis Gapnep. or H. chinensis Wang., in the family of Liliaceae.1400g of Sophorae Flavescentis Radix and600g of Heterosmilacis Japonica Rhizomae were extracted by percolation with water containing1%acetic acid. The extract was subjected to alcohol precipitation repeatedly, filtered with activated carbon, adjusted pH with NaOH, and then diluted with injective water.In the present thesis, the isolation and structural elucidation of the chemical constitu-ents of CKI as well as HPLC and LC-MS analyses were carried out to find the effec-tive substances of CKI and establish the more feasible quality standards.2000mL of CKI sample was passed through HPD100macroporous resin column and the fractions were subjected repeatedly to silica gel column chromatography and puri-fication with Sephadex LH-20、ODS as well as semi-preparative liquid chromatog-raphy to afford11compounds. Their structures were identified on the basis of physo-chemical properties and spectroscopic analysis as piscidic acid, macrozamin, ox-ymatrine, oxysophocarpine, N-methylcytisine, matrine, sophocarpine, trifolirhizin, adenine, baptifoline and liriodendrin. Apart from the above eleven constituents, other twelve constituents with low anounts were identified by LC-MS, including5a,9a-hydroxymatrine, oxysophoranol, oxysophoridine,9a-hydroxysophocarpine, sophoridine, sophoranol, isosophocarpine,7,11-dehydromatrine,5,6-dehydrolupanine,9a-hydroxymatrine, lamprolobine and isomatrine. The HPLC fingerprint standard of CKI samples was preliminarily established in order to comprehensively reflect the type and quantity of the chemical composition in CKI sample and improve the product consistency from batch to batch. CKI samples were analyzed on a Phenomenex Luna C18column (5μm,4.6×250mm) using the mobile phase consisted of water containing0.01mol·L-1ammonium acetate (adjusted pH to8.0with NH3·H2O, solvent A) and acetonitrile-water containing0.01mol·L-1ammo-nium acetate (9:1, v/v, solvent B). A linear gradient program was used as follows:0min,100%A;60min,60%A. The flow rate was0.8mL·min-1and the column temperature was set at35℃. The HPLC chromatogram was monitored at225nm. Under the optimized HPLC conditions,27batches of CKI samples were analyzed and the data were evaluated by Similarity Evaluation System for Chromatographic Fin-gerprint of Traditional Chinese Medicine software. The similarity of each chromato-gram of24batches of CKI samples against the simulative median chromatogram is more than0.85. Eight main characteristic peaks, were found in each individual chro-matogram of CKI sample. The ratio of the area of each characteristic peak to total peak area was28.2%,3.3%,20.6%,7.9%,2.8%,10.3%,4.9%and1.9%, respec-tively and their sum accounted for80%of the total area. The proposed fingerprint standard for CKI sample was as followed:the sample chromatogram should be con-sistent with the standard one (mean chromatographic fingerprint) and the similarity, corrected by the characteristic peaks of oxymatrine and mean chromatographic fin-gerprint, should not be less than0.85.Currently, the total content of alkaloids determined by acid-base titration method is employed for the quality control of CKI, which is set to the least18mg alkaloids in1mL of injection. Apparently, this standard is not sufficient for the quality consisten-cy evaluation of CKI. In the present investigation, a HPLC method for the simultane-ous determination of six constituents in CKI samples was developed, including oxy-marine, oxysophocarpine, N-methylcytisine, matrine, sophocarpine and trifolirhizin as marker conpounds. The samples were analyzed on a Luna C18column (4.6mm×250mm,5um). The mobile phase consisted of water containing0.01mol/L ammonium acetate (adjust pH value to8with the ammonia water, A) and acetoni-trile-water containing0.01mol/L ammonium acetate (3:2, v/v, B). A linear gradient program was used as followed:0min,80%A;8min,50%A;10min,47%A;25min,40%A. The flow rate was at0.8mL·min-1. The detection wavelength was at225nm. This HPLC method was valided and could be used for the intermediate and finished product of CKI samples. According to the established method above,19batches of CKI samples were analyzed. The six marker compounds were found in the different batches of CKI samples, however, their contents were significantly different in the different batches of CKI samples, which could be related to the quality of the crude materials and the manufacturing process employed.