| Objective:HongHe capsule is a Chinese herbal preparation composed of multi-component.In order to better evaluate the quality of HongHe capsule preparation,this paper established the HPLC fingerprints of HongHe capsule preparation and studied the fingerprint correlation of the preparation,effective components,and raw herbal materials based on previous researches.Multiple components were simultaneously determinated to studied the external release rate of the HongHe capsules.And an analytical method to simultaneously determinate the multi-indicator of HongHe capsule in rat plasma was established to reflect the pharmacokinetic process of HongHe capsule in rat.All of the research could provide a basis for the quality assessment and dosage regimen of the HongHe capsule,and provide guidance for the rational use of drugs for clinical trials of HongHe capsules.Methods:1.The fingerprint correlation of the HongHe capsule preparation,effective components,and raw herbal materialsThe effective components were separated by HPLC with Inertsil ODS-3 column(5?m,4.6×250mm)for 125min;the mobile phases was acetonitrile and 0.01%H3P04 water in gradient elution;the flow rate was lmL/min;the column temperature was at 25?;the detection wavelength at 223nm.The fingerprint of the preparation,effective components,and raw herbal materials were established.The software "Similarity evaluation System for Chromatographic Fingerprint of TCMs(Version 2010)" was applied to analysis ten batches of HongHe capsules.In addition,by comparing the fingerprint correlation among HongHe capsules,the effective components from each raw materials,the raw materials and negative contrast preparations.2.Study on dissolution rates of multiple effective components in HongHe capsulesAccording to the 2010 edition of the "Chinese Pharmacopoeia",900mL 0.3%sodium dodecyl sulfate solution was used as dissolution media.The experiment used paddle method in condition of speed of 100r/min and temperature of 37±0.5?.With Chikusetsu saponin ?a,total saponins and the flavonoids in HongHe capsules as detection index,the HPLC was used to determined Chikusetsu saponin ? a,and total saponins and the flavonoids were determined by UV.3.Pharmacokinetic studies on multiple effective components of HongHe capsules in rats by UPLC-MS/MS An HPLC-MS/MS method for the determination of Chikusetsu-saponin ?a,hyperin,bergenin was used to determine the pharmacokinetics of HongHe capsule in rats following oral dose administration.The pharmacokinetics parameters were processed by DAS2.0 Pharmacokinetic software program,in dealing with the data of drug-time,to investigate the changes of the 3 components in rats.Results:1.The fingerprint correlation of the HongHe capsule preparation,effective components,and raw herbal materialsAll the compounds in HongHe capsule preparation were separated well by the HPLC fingerprint method.The methodological evaluation showed that the instrument precision was good and the method was reproducible and stable.The software"Similarity evaluation System for Chromatographic Fingerprint of TCMs(Version 2012)"was applied to analysis ten batches of HongHe Capsules.Twenty-eight mutual peaks were identified as the fingerprint peaks with Salidroside as the reference peak,and all of the detected peaks were separated effectively.Similarity analysis(SA)were done based on data of 10 samples.The results showed that the similarity of fingerprints of 10 batches of finished products was more than 0.96.In addition,by comparing the fingerprint correlation among HongHe Capsules,the effective components from each raw materials,the raw materials and negative contrast preparations,28 mutual peaks were classified.Each peak of the final product can correspond with that of the effective components from each raw materials and the raw materials.2.Study on dissolution rates of multiple effective components in HongHe capsulesThe contents of Chikusetsusaponin ?a were determined by HPLC,there was a good linearity over the range of 0.0163?0.3904mg/ml(r2=1),the average recovery was 97,58%,the RSD was 2.54%;Ginsenoside Re and rutin were used as the reference substances to establish ultraviolet spectrophotometry methods for content determination of the total saponins and the flavonoids.For Ginsenoside Re,there was a good linearity over the range of 0.04784?0.26312mg(r2=0.9993),the average recovery was 99.47%,the RSD was 2.93%;for rutin,there was a good linearity over the range of 0.03828?0.30624mg(r2=0.9992),the average recovery was 101.44%,the RSD was 1.43%.The methodological evaluation showed that the instrument precision was good and the method was reproducible and feasible,the tested samples in a certain time period have a good stability.At 45min,the cumulative dissolution rates of Chikusetsu saponin ?a were 91.29%?88.64%?91.72%;the cumulative dissolution rates of total saponins were 89.41%?87.68%?87.10%;the cumulative dissolution rates of flavonoids were 91.02%?90.77%?89.98%in three batches of HongHe Capsules.3.Pharmacokinetic studies on multiple effective components of HongHe capsules in rats by UPLC-MS/MSAn HPLC-MS/MS method was used to determine the content of Chikusetsu-saponin ?a,hyperin and bergenin in the HongHe capsule.There was good linearity in the range of 0.031?41.68ng/mL(r2=0.9921)for Chikusetsu-saponin ? a,in the range of 0.014?11.26ng/mL(r2=0.9981)for hyperin,in the range of 0.019?9.456ng/mL(r2=0.9992)for bergenin.For three ingredients,the RSD of middle and high concentration for the inter-and intra-day were less than n 13%,and the low concentration of RSD was less than 18%.The average recovery of all the ingredients were above 75%except that the middle concentration of Chikusetsu-saponin ?a was 58%.After oral administration,the plasma concentration curve of Chikusetsu-saponin ?a,hyperin,bergenin in rats were all multi-peaks.The Cmax of Chikusetsu-saponin ?a,hyperin and bergenin were 53.16?g/L,6.29?g/L,0.37?g/L respectively,the Tmax of Chikusetsu-saponin ?a,hyperin and bergenin were 49.17min,32.50min,54.00min respectively.And the t1/2 of Chikusetsu-saponin ?a,hyperin and bergenin were 437.99min,535.41min and 603.12min respectively.Conclusion:In the research,the HPLC fingerprint method was stable and reliable,and had good applicability.It could be used of the overall quality assessment and control of HongHe capsules.The results showed that the quality and preparation process of HongHe capsule preparations were stable.The dissolution method established by screening of dissolving condition was stable and reliable.The dissolution rate of all the major ingredients in HongHe capsule conformed to the requirement of pharmacopoeia.The UPLC-MS/MS method is rapid,high sensitivity and selectivity,and it could be successfully applied to the pharmacokinetic study of 3 analytes,namly Chikusetsu-saponin ?a,hyperin and bergenin,after the intragastric administration of HongHe capsules in rats.The results showed that all the ingredients can be quickly absorbed into the blood,and the blood drug concentration could maintain a period of time after the intragastric administration. |
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