Study On The Processing-induced Transformation Dynamics Of Ginsenosides And Quality Criteria Of Red Ginseng

Panax ginseng（PG）,the roots and rhizomes of Panax ginseng Meyer belonging to Araliaceae family,red ginseng（RG）are prepared by steaming and drying by fresh ginseng.PG and RG as the dietary supplements and traditional Chinese medicine have been recorded Chinese Pharmacopoeia（Ch P）of edition in 2020.After preparation,PG and RG have differential characteristics in pharmacological effects,such as antioxidative,anticancer,antiasthmatic,and immunomodulation activities.Phytochemical researches indicate that dammarane saponins as the main bioactive compounds of ginseng include protopanaxadiols,protopanaxatriols,oleanolic acids,C-17 side chain.Recently,the following questions need to be investigated.（1）The few studies focus on the reason for the dynamic content of main bioactive ginsenosides during processing,especially for oleanane type.（2）In ChP of edition in 2020,it was unreasonable that white and red ginseng had same biomarkers for quality evaluation.（3）The extraction condition of white and red ginseng in Ch P were heavy workflow and consumption solvent.And the bioactive ginsenosides had long analytical time.The aim of this study is to improve quality criteria of red ginseng.It is to develop the analytical method of marker of PG and RG and to study the dynamic ginsenosides during steaming ginseng using quality-by-design（Qb D）,two-dimension liquid chromatography,UPLC-QTOF-MS^E,LC-MS/MS and multivariate statistical analysis.Ginsenoside Rb₁ as an internal standard,quantitative analysis of multi-components by single marker（QAMS）and the value of ginsenoside Ro/Re were used to the quality evaluations of PG and RG.UPLC-QTOF-MS^E and UNIFI were used to identify ginsenosides of different processing ginseng.The results indicated that 60 ginsenosides including 24protopanaxatriol,14 protopanaxadiol,18 ginsenosides with C-17 modified side chain and 4 oleanolic acid type were identified,and 12 pair ginsenosides isomer.It is found that ginsenosides with tetrasccharide（ginsenoside Rb₁,Rb₂,Rc）,trisaccharide（ginsenoside Rd,Re）and disaccharide（ginsenoside Rg₁,Rg₂,Rf）of fresh ginseng changed into monosaccharide（ginsenoside Rh₁,Rk₃,Rh₄,Rh₂）,disaccharide（ginsenoside Rg₆,F₄,Rg₅,Rk₁,Rg₃,Rs₃,Rs₄,Rs₅）.A total of 12 pair ginsenoside isomers(20（R/S）-Rh₁,20（R/S）-Rh₂,20（R/S）-Rg₃,20（R/S）-Rs₃,Rk₁ and Rg₅、R_S5 and R_S4、Rs₅ and Rs₄ isomer、Rs₆ and Rs₇、F₄ and Rg₆、Rk₃ and Rh₄、Rk₂ and Rh₃、Rg₉and Rg₈ after processed ginseng.Chemical markers of PG and RG were screened by UPLC-QTOF-MS^E and multivariate statistical analysis.A total of eight markers were validated by UPLC-MS.Multiple chromatographic technologies,thin layer chromatography,high performance liquid chromatography and liquid chromatography-mass spectroscopy were used to separate and collect malonyl ginsenoside Rb₁.The analytical methods（mobile phase,column temperature,flow rates,gradient programs）of ginsenosides Rg₁、Re、Rb₁、Ro、Rc、Rb₂ was developed by Qb D and two-dimension liquid chromatography.The results showed that a convenient chromatography method regarding selective column（Acclaim RSLC Polar Advantage II 2.2μm,100×2.1 mm）,temperature（25℃）,mobile phase（0.1%H₃PO₄）,flow rate（0.35 m L/min）,final（45%）and initial（19.5%）organic solvent,and gradient program were optimized by quality-by-design Fusion AE.The column of Accucore C30（2.2μm,2.1×100 mm）with different chemical phase from 1D column in this study,which enhanced the orthogonality of 2DLC systems.The analytical method was developed for ginsenosides of PG and RG based on optimized method.The results indicated that ginsenosides Rg₁,Re,Rb₁,m-Rb₁,Ro,Rc and Rb₂ of PG were separated during 85 min,and ginsenosides Rg₁,Re,Rb₁ and Ro were separated within 50 min.In addition,the optimized extraction method was 1 g ginseng dissolved by 25 m L 70%methanol with ultrasonicate for 50min.Multivariate statistical analysis and LC-MS/MS were used to evaluate ginsenosides of different processing.Multivariate statistical analysis including t-SNE,PLS-DA and RF were used to evaluate processing method.T-SNE analysis indicated that the chemical components of ginseng changed with temperature.PLS-DA and RF analysis indicated temperature is important factor for the processed ginseng.Besides,it suggested that process method of P.ginseng in this program was stable and reliable.Totally,17 ginsenosides of different processed ginseng were determined simultaneously by LC-MS/MS.The results suggested that it be found that the amount of ginsenosides Re,Rg₁,Rf,notoginsenoside R₁ decreased with ginsenoside Rg₂ and Rh₁ increasingly after processing 1 h.For PPT type,ginsenoside Rb₁,Rb₂,Rc,Rd,Rh₂increased at first,and the maximum content were at 110℃.Then,they decrease to temperature increasingly,and which were not detected at 140℃.A method was developed by precursor ion scan to study the content of neutral ginsenosides increasingly.At the beginning,the malonyl ginsenoside converted into neutral ginsenoside during processing.QAMS and the value of ginsenosides Ro/Re were used to evaluate PG and RG,respectively.An optimized extraction method was developed to determine ginsenosides Rg₁,Re,Rb₁ and Ro content by HPLC in 43 batches of red ginseng from different origins,growing years and manufacturers.The results indicated the content of ginsenoside Ro was in the range of 0.11%to 0.43%,and the average content was 0.26%,which was higher than that of ginsenoside Rg₁ and Re.The ratio of ginsenoside Ro and Re as a threshold could be used to discriminate red ginseng from different growing years.In addition,100%,94.4%and 46.6%of red ginseng from six,five and four years exceeded the threshold of 1.3.It is recommended that ginsenosides Ro/Re was over 1.3to limit red ginseng less than four years.HPLC-CAD and QAMS were used to determine ginsenosides Rg₁,Re,Rb₁,m-Rb₁,Ro,Rc,and Rb₂.