Revision For The Determination Method And Study On The Influencing Factors Of Transformation Of Saikosaponins In Chaigui Granules

Rationales:Chaigui granules is a new antidepressant Chinese medicine,which was obtained by to our research group after the prescription of Xiaoyao San was disassembled and reconstituted.It was consisted of 6 herbs,including Radix Bupleuri,Radix Angelicae Sinensis,Radix Paeoniae Alba,Rhizoma Atractylodis Macrocephalae,Herba Menthae and Radix Glycyrrhizae.In October 2018,the drug obtained clinical approval（approval No.2018l03149）.The quality control method and the internal control standard of chaigui granules was established in our previous study.However,by further tracking the content of SSa（saikosaponin a）in 14 batches of Radix Bupleuri and chaigui extract during storage,it was found that the content of SSa in some Radix Bupleuri could not meet the internal control standard,which indicated that the quality control method with SSa as index was limited,and that the content of SSa would change during the storage of the extract and transform to SSb₁（saikosaponin b₁）.Therefore,the previous study will revise the quality standards and draft instructions in the original application materials,establish HPLC and UPLC determination methods with four saikosaponins as quality control indicators,and complete the corresponding methodological investigation.In addition,further studies showed that SSa and SSd（saikosaponin d）in Chaigui granules were transformed to SSb₁and SSb₂（saikosaponin b₂）in different degrees in the production process.Therefore,this project will explore the influencing factors of its composition transformation and content change,the results will provide scientific basis for the improvement of quality control methods and quality standards of Chaigui granules.Objective:By establishing a new method for the determination of saikosaponins in chaigui granules,the quality standard and drafting instructions in the original application materials were revised and improved,and the factors influencing the transformation of saikosaponins in the preparation process were discussed.Methods:HPLC and UPLC were used to establish the content determination method of saikosaponin,furthermore,the linear range,specificity,precision,repeatability,reproducibility,durability and sample recovery were investigated.HPLC was used to detect the saikosaponin content of samples obtained in each step of large-scale production process,and the key steps of saikosaponin a and d transformation in the production process were explored.According to the extraction process of Chaigui granules,alcohol extracts and alcohol precipitates with different p H values were prepared.The content of saikosaponin in the extracts with different p H values was measured by HPLC,and the effect of p H value on the content and composition transformation of saikosaponin was studied.Result:1.HPLC experiment was performed on an Agela Venusil C18 Plus column（25cm×4.6mm,5μm）.The mobile phase consisted of acetonitrile（A）-water（B）in gradient mode.The detection wavelength was set at 210nm.The flow rate was 1 m L·min^-1.The column temperature was maintained at 30℃.The results showed that the separation of the four saikosaponins was good.The four saikosaponins showed good linear relationship in a certain range（Ra²=0.9999,Rb₁²=0.9999,Rb₂²=1,Rd²=1）.Good precision,stability and reproducibility was achieved with the RSDs below 3.0%.The average recoveries were among 99.7%-102.9%with RSDs below 3.0%.2.UPLC experiment was performed on an ACQUITY UPLC HSS T3 column（2.1×100mm,1.8μm）.The mobile phase consisted of acetonitrile（A）-water（B）in gradient mode.The detection wavelength was set at 210 nm.The flow rate was 0.2 m L·min^-1.The results showed that the separation of the four saikosaponins was good.The four saikosaponins showed good linear relationship in a certain range（Ra²=0.9999,Rb₁²=0.9999,Rb₂²=0.9999,Rd²=0.9999）.Good precision,stability and reproducibility was achieved with the RSDs below 3.0%.3.The results of the content determination of large-scale production samples showed that SSb₁was not detected in this batch of samples,indicating that SSa was not transformed.Part of SSd was transformed to SSb₂in ethanol extraction,and all of SSd was transformed to SSb₂in water extraction extract.It was found that the total amount of Saikosaponins in alcohol extract was about 4 times as much as that in water extract,and it was 5 times as much as that in alcohol precipitate,which indicated that most of Saikosaponins was extracted by ethanol.4.The results showed that the contents of SSa+SSb₁and SSd+SSb₂was decreased with the decrease of the p H value of ethanol extract and water extract,indicating that the acid condition would affect the dissolution of saikosaponin.The p H value of ethanol extract changed,SSa did not change to SSb₁.Howerer,under certain acid condition,the conversion rate of SSd to SSb₁decreased with the decrease of p H value.Conclusion:The established HPLC and UPLC methods were simple,rapid and reproducible,which can be used for the content determination of four saikosaponins in Chaigui granules.water extraction step may be the key step affecting the extraction of SSd.The p H value of the extract will not only affect the dissolution of saikosaponins,but also affect the conversion rate of saikosaponin d to saikosaponin b₁.