Study On Exploitation Of Pharmacy Of Aidi Lyophilizer

Aidi liquid injection was a clinical medicine commonly used in China for thetreatment of cancer, which was listed in ministerial standard. In this paperpreparation procedure, anti-tumor activity, quality control method andpharmacokinetics on a few active constitutes of Aidi lyophilizer were studied.The preparation procedure of Aidi lyophilizer was established using differentkinds of methods and techniques. The optimal extraction process of Aidilyophilizer was studied by orthogonal design and the contents of cantharidin,ginsenoside Rg₁, ginsenoside Re and isofraxidin as quality assessment indices.Three factors were studied in this experiment, including the solvent consumption,duration of extraction and times of extraction. D-101 macroporous resin and ultrafiltration were selected to optimize the refining process and lyophilization was alsostudied. The lyophilizer was yellowish freeze-dried powder and solved easily inwater.The anti-tumor activity of Aidi lyophilizer in vitro and vivo were studied. Theantiproliferative activities of Aidi lyophilizer against human hepatocarcinoma cellline BEL-7402, human cervix epithelial cell line HeLa, human malignantmelanoma cell line A375-S2, human gastric carcinoma cell line SGC-7901 andmurine fibrosarcoma cell line L929 were evaluated by MTT colorimetric assay invitro. The results showed Aidi lyophilizer possessed the antiproliferative activityagainst different tumor lines. IC₅₀ were 0.0162, 0.0796, 0.0982, 0.0119 and0.0144g·mL^-1, respectively. The inhibitory effects of Aidi lyophilizer on tumorgrowth were observed by the models of implanted sarcoma 180 (S180) andhepatoma 22 (H22) in mice and Lewis lung-cancer in C57BL/6 mice. The resultsshowed Aidi lyophilizer restrained the growth of tumor in mice in adose-dependent manner.TLC methods were developed for the identification of cantharidin in Mylabris, ginsenoside Rg₁ and ginsenoside Re in Radix Ginseng, astragaloside in RadixAstragali and isofraxidin in Radix Acanthopanacis. GC fingerprint was carried outon a DB-1 column with cantharidin as internal reference. Column temperaturemaintained 10min at 120℃, then raised to 250℃with rate of 3℃·min^-1, and thenraised to 280℃with rate of 2℃·min^-1, and maintained 30min at 280℃. HPLCfingerprint was separated on a Agilent Zorbax SB-C₁₈ column with syringin asinternal reference. GC and HPLC fingerprints were applied to analyze quality ofAidi lyophilizer and its intermediate.The determination of cantharidin was carried out by using GC-FID method,which showed a good linearity from 2.46 to 49.2μg·mL^-1 with a mean recovery of100.3%. The acetonitrile-water containing 0.05% phosphate acid (19:81) solutionwas adopted as the mobile phase for the determination of ginsenoside Rg₁ and Reon an Hypersil C₁₈ column. It showed good linearities from 20 to 200μg·mL^-1 forginsenoside Rg₁ with a mean recovery.of 98.2% and from 10 to 100μg·mL^-1 forginsenoside Re with a mean recovery of 97.2%. A ttPLC-DAD method wasdeveloped for simultaneous determination of .protocatechuic acid, syringin,chlorogenic acid, caffeic acid, liriodendrin and isofraxidin in RadixAcanthopanacis, collected from different batches. The determination wasaccomplished by a gradient elution system. The linear calibration curves wereplotted with satisfied relative coefficients. The average recoveries were in therange of 90.3～94.3 % with RSD less than 2.4 %. With the same gradient elutionsystem, calycosin-7-O-beta-D-glucoside, syringin, chlorogenic acid, caffeic acid,liriodendrin and isofraxidin were determined in Aidi lyophilizer. The linearcalibration curves were plotted with satisfied relative coefficients. The averagerecoveries were in the range of 96.6～98.2 % with RSD less than 2.3 %.A high performance liquid chromatography (HPLC) method was developed toquantify isofraxidin in rat plasma using liquid-liquid extraction and coumarin asinternal standard. The linear range was 0.05～8.0μg·mL^-1 and the low quantificationlimit was 0.05μg·mL^-1. The intra- and inter-day relative standard deviations (RSD)in the measurement of quality control (QC) samples 0.25, 2.0 and 6.0μg·mL^-1ranged from 5.7% to 6.4% and 6.3% to 7.9%, respectively. The accuracy was±5.1% in terms of relative error (RE). The mean extraction recovery was 84.9%. The corresponding pharmacokinetic parameters were obtained, t_1/2 was 4.26h, C_maxwas 5.47μg·mL^-1 and t_max was 0.24h.A simple and sensitive high-performance liquid chromatographic method wasdeveloped to simultaneously quantify syringin and chlorogenic acid in rat plasmausing wavelength-transfer technology. After deproteinized by methanol, the plasmasamples were analyzed with gardenoside as internal standards. The linear rangeswere 0.20～10μg·mL^-1 and 0.25～30μg·mL^-1, respectively. The lower limits ofquantification were 0.20μg·mL^-1 and 0.25μg·mL^-1, respectively. The method wasshown to be reproducible and reliable with intraday precision below 8.5% and6.1%, inter-day precision below 7.1% and 5.5%, accuracy within±7.1% and±8.6%,and mean extraction recovery excess of 92.1% and 80.9%, respectively, whichwere all calculated from the blank plasma sample spiked with syringin andchlorogenic acid at three concentrations of 0.20, 1.0 and 6.0μg·mL^-1 for syringinand 0.25, 2.0 and 20μg·mL^-1 for chlorogenic acid. Since the concentration ofsyringin in plasma samples was lower, it was determined only 1.5h afterintravenous administration of Aidi lyophilizer, t_1/2 of syringin and chlorogenic acidwere 0.49h and 0.82h, respectively.A specificity and accuracy high performance liquid chromatographic methodwas developed to quantify calycosin-7-O-beta-D-glucoside in rat plasma usingliquid-liquid extraction and vanillin as internal standard. The linear range was0.11～17.6μg·mL^-1 and the low quantification limit was 0.11μg·mL^-1. The intra-andinter-day relative standard deviations (RSD)in the measurement of quality control(QC) samples 0.11, 0.22, 1.32 and 8.80μg·mL^-1 ranged from 4.1% to 6.3% and4.3% to 6.2%, respectively. The accuracy was from -6.7% to 4.3% in terms ofrelative error (RE). The validated method was used to take a limited view of thepharmacokinetic profile of calycosin-7-O-beta-D-glucoside in rat plasma aftertaken Aidi lyophilizer. The main pharmacokinetic parameters were:AUC_0-t=1.38μg·h·mL^-1, AUC_0-∞=1.64μg·h·mL^-1 and t_1/2=0.70h, respectively.