| The use of Chinese medicine often take the compound form,herb couple is the smallest unit of compound form of TCM?Traditional Chinese Medicine?,is summed up by a common form of traditional Chinese medicine clinical use and proved to be effective,although the composition is simple,but with the characteristics of compatibility,can produce synergistic effect or reduce the toxicity effect.Ligusticum chuanxiong and Angelicae Dahuricae are commonly used for the treatment of migraine.This study uses drug serum chemical analysis and pharmacokinetics research methods,study on the absorption and metabolism of Ligusticum chuanxiong and Angelicae Dahuricae,to explore and clarify intestinal absorption characteristics and pharmacokinetics of the main active ingredients of Ligusticum chuanxiong and Angelicae Dahuricae,in order to reveal the pharmacodynamic material basis and lay the foundation for the development of drug preparation.1.Objective1.1 To establish High Performance Liquid Chromatography?HPLC?method for the determination of the content of the main active ingredients of Ligusticum chuanxiong and Angelicae Dahuricae.1.2 Intestinal absorption of the main active ingredients of Ligusticum chuanxiong and Angelicae Dahuricae by in situ single pass intestinal perfusion model in rat1.3 To establish a GC-MS quantitative method for analysis of rat plasma to investigate the pharmacokinetic parameters of medicinal herbs used insingle or compound.2 Methods2.1 The preparation of extracts of Ligusticum chuanxiong and Angelicae Dahuricae,using quantitative HPLC established in Chuanxiong ferulic acid and ligustilide and imperatorin,isoimperatorin,xanthotoxol analysis method,determination of content of different batches of medicinal materials.2.2 Using in situ single pass intestinal perfusion model in rat,The HPLC method established in the first part was used to determine the changes in the content of active ingredients in the irrigation fluid and calculate the Ka and Peff.To analyze the absorption changes of the ingredients of Ligusticum chuanxiong and Angelicae Dahuricae in various intestinal segments,different mass concentrations and protein inhibitors.2.3 GC-MS detection methods be used,the qualitative and quantitative analysis of two kinds of volatile components in the extract,and then establish a quantitative analysis method in rat plasma.Blood samples were collected from the posterior orbital venous plexus of 10 min,30 min,1 h,1.5 h,2 h,3 h,4 h,6 h,8 h,12 h,24 h after intragastric administration in SD rats.The plasma concentration was measured after the sample was processed,and the pharmacokinetic characteristics of single herb and compound conditions were investigated.3.Results3.1 Established with methanol-water as the mobile phase quantitative gradient elution HPLC method for analysis of ferulic acid and ligustilide in Ligusticum chuanxiong.Using acetonitrile-water as mobile phase quantitative gradient elution HPLC method for analysis of xanthotoxol,imperatorin and isoimperatorin in Angelicae Dahuricae.The method validation of the above two methods,the specificity,good linearity,precision,repeatability and recovery rate to meet the requirements.Using the method of two kinds of medicinal materials of each five batches were determined,the results showed that significant differences in ligustilide in different batches of Ligusticum chuanxiong,content of the three substances in Angelicae Dahuricae between different batches were significant difference.3.2 This part show ligustilide and ferulic acid in Ligusticum chuanxiong were all absorbed in the different parts of intestine,the absorption of ligustilide is better than ferulic acid.The Ka of ligustilide in duodenal was 3.58×10-3 s-1,the different parts of intestinal absorption order was ileum>duodenum?jejunum>colon,increase its absorption parameters with concentration decreased.The absorption parameters increased significantly with probenecid and Vera Pammy?P<0.05?,and the absorption of probenecid group was significantly higher than Vera Pammy group.The Ka of ferulic acid in duodenum was 1.23×10-3 s-1,and the absorption of jejunum and ileum was not significantly different from duodenum,but the Ka in colon was significantly decreased to0.78×10-3 s-1.There was no significant difference in the absorption of the three groups at high,medium and low concentrations,but decreased significantly at high doses.The absorption of ferulic acid in extract group was significantly higher than the monomer group?P<0.05?,containing probenecid group significantly increased.The Ka of imperatorin in Angelicae Dahuricae in duodenal was 1.34×10-3 s-1,the absorption was significantly better than isoimperatorin and xanthotoxol?P<0.05?,the three substances in the intestinal absorption has no specific location.With the increase of the quality,the absorption of three components in Angelicae Dahuricae has significant difference?P<0.05?.There is no significant difference in the absorption of the three components in monomer group and extract group.With verapamil added to liquid flow in medicine,imperatorin and isoimperatorin absorption has no significant change,but the Ka of xanthotoxol were significantly increase to 0.75×10-3 S-1?P<0.05?.With probenecid was added in solution,the absorption of three components also has no significant difference.3.3 The qualitative and quantitative analysis of Ligusticum chuanxiong and Angelicae Dahuricae by GC-MS,45 compounds were identified in the extract of Ligusticum chuanxiong,multi esters,phthalide compounds,including Z ligustilide the highest accounted for 30.34%and 3-n-Butylphthalide relative content is 10.16%.34 substances were identified in the extract of Angelicae Dahuricae,most of which were lipids and alcohols.Established an internal standard GC-MS quantitative method for analysis of ligustilide and 3-n-Butylphthalide in Ligusticum chuanxiong,imperatorin and byakangelicin in Angelicae Dahuricae,in rat plasma samples,the method has a good linear relationship,and the precision and recovery also meet the requirements,and has no biological substances would interfere with the accurate determination.Pharmacokinetic study results show that the single use of ligustilide in Ligusticum chuanxiong Cmax=13903.0 ngˇmL-11 compared with combined use has no significant difference,the single use of 3-n-Butylphthalide Cmax=1928.8 ngˇmL-1 compared with combined use has no significant difference.The imperatorin in Angelicae Dahuricae Angelicae Dahuricae with combined use,AUC,Tmax and Cmax were significantly increased compared with the single use.With combined use,the MRT of byakangelicin increased to 5.7 h,and the Tmax increased to 3.0 h.The plasma concentration time curve of ligustilide and byakangelicin rose a second time,but it was disappeared in combined use. |
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