Studies On Chemistry And Pharmacokinetics Of Pungent Flavor Detached Constituent Of Radix Phytolacca

Radix Phytolaccae,as a traditional Chinese medicine,is the dried root of Phytolacca acinosa Roxb.and Phytolacca americana L.in Phytolaccaceae family.It was first carried out in "Shen Nong’s Herbal Classic" and concluded in Chinese Pharmacopoeia.According to my tutor’ hypothesis "one flavor one nature,one medicine has X flavor and Y nature（Y≤X）.The property of a medicine has nonlinear adduction.The material foundation of the nature and flavor of traditional Chinese medicine is resolvability and combinability." From the resolvability research on the nature and flavor material foundation of Phytolacca acinosa by our research group,five compositions obtained were fatty oils composition,polysaccharide composition,saponin composition,phenolic acid composition and tanin composition;the material foundation of "pungent" was saponin composition,it has obvious anti-inflammatory and immunomodulatory effects.In order to study the material foundation of the "pungent" detached composition of Phytolacca acinosa deeply,and toclarify the relationship between chemical characteristics and pharmacokinetics,the following study was carried out on the "pungent" detached composition（Triterpenoid saponin）of Phytolacca acinosa:1.In order to study the material foundation of the "pungent" detached composition of Phytolacca acinosa,11 compounds were isolated with hot water extraction,alcohol sedimentation,macroporous adsorptive resins,ODS and HPLC.The chemical structures were identified by ¹H-NMR and ¹³C-NMR data together with HR-ESI-MS mass spectrometry data.They were Esculentoside H（1）,Esculentoside L（2）,Esculentoside S（3）,Esculentoside F（4）,Esculentoside R（5）,Esculentoside A（6）,Esculentoside C（7）,Esculentoside B（8）,β-adenosine（9）,EriosematinF（10）,1,2,3,4-Tetrahydro-¹H-pyrido[3,4-b]indole-3-carboxylic acid（11）.compounds 9-11 were isolated from Radix Phytolaccae for the first time.2.In order to study the possibility of detached composition from Phytolacca acinosa,the chemical characteristics of the "pungent" detached composition of Phytolacca acinosa could determined by HPLC.An HPLC-ELSD method was first established for the simultaneous determination of Esculentoside A,Esculentoside B,Esculentoside C and Esculentoside H in saponins detached composition and Radix Phytolacca from different sourses.The Diamonsil C18（250 mm×4.6 mm,5μm）column was used.The mobile phase was methanol（A）and 0.1%acetic acid（B）with a gradient elution（0-20 min,50%A→90%A）,the flow rate was 1.0 mL·min^-1.The results indicate that the calibration curves of esculentoside A,esculentoside B,esculentoside C and esculentoside H showed good liner relationship between logarithm of the peak area and logarithm of the content of each compound over 0.719-8.628,0.251-4.518,0.258-3.102,0.503-6.036μg（r=0.9992-0.9996）,respectively.The contents in saponins detached composition were 39.36,10.98,7.62,41.56 mg·g^-1.The contents in 30 batches of samples of Phytolaccae Radix were 1.53-4.58 mg·g^-1 for esculentoside A,0.16-1.40 mg·g^-1 for esculentoside B,0.10-0.61 mg·g^-1 for esculentoside C,0.13-4.98 mg·g^-1 for esculentoside H.3.In order to demonstration the feasibility of developing "pungent" detached composition into new drugs,and to study the relationship between chemical characteristics and pharmacokinetics,the UPLC-MS-MS method for the determination of esculentoside A and esculentoside B in plasma samples was established,and the pharmacokinetic characteristics of esculentoside A and esculentoside B in rats were systematically studied.After liquid-liquid extraction with methanol,the analyte EsA,EsB and swertiamarin（IS）were separated on a ACTUITY UPLC? BEH（2.1 mm × 100 mm,1.7 μm）column with the mobile phase of acetonitrile（0.1%formic acid）（A）-water containing 0.1%formic acid（B）at a flow rate of 0.3 mL·min^-1.An ion trap mass spectrometer equipped with an ESI performed negative ion mode was used as the detector.The plasma concentration of EsA and EsB was determined,and its pharmacokinetic parameters were calculated.The results indicate that the calibration curves of esculentoside A and esculentoside B showed good liner over 1.26～100.80 ng·mL-1 and 1.20～96.38 ng·mL-1（r=0.9988,0.9986）,and the LLOQ was 1.26 and 1.20 ng mL-1,respectively.The extraction recovery of esculentoside A and esculentoside B was more than 65%and it was also stable.Pharmacokinetic parameters of the analytes after oral administration in rat plasma were as follows:AUC0-t were（273.33±71.98）ng·h·mL-1 and（92.48±21.75）ng·h·mL-1,AUC0-∞ were（301.93 ±78.84）and（105.63±28.79）ng·h·mL-1,Cmax were（101.46±3.65）and（20.44±0.78）ng·mL-1,Tmax were（0.75±0.20）and（0.56±0.13）h,T1/2 were（4.07±0.28）and（4.60±0.47）h.Under the selected conditions,the separation of esculentoside A,esculentoside B and the internal standard was good,and there is no obvious endogenous substance interference.This paper is a part of the 973 project "The theoretical research about the nature of TCM based on the efficacy of alleviating water retention-Study on the medicinal properties of traditional Chinese medicine with inducing diuresis to alleviate edema",It is a study on the development of new Chinese medicine under the guidance of the theoretical of Chinese medicine’s property,The modern chromatography method has been used to further confirm the material foundation of the "pungent" detached composition of Phytolacca acinosa,It provides an important reference for the deep development of the Phytolacca acinosa and the development of the theoretical on the nature and flavor of modern traditional Chinese medicine.