Study On Chemical Constituents From EtOAc Fraction Of Panax Japonicus And Their Anti-inflammatory Activity

Objective In order to clarify the material basis of the anti-inflammatory activity,to enrich the chemical diversity,provide theoretical basis and data support for establishing quality evaluation system of Panax japonicus.The phytochemical technology were applied to separate the chemical constituents of the ethyl acetate portion of the rhizome of Panax japonicas.The anti-inflammatory activity of these isolated compounds were evaluated.Meanwhile,the qualitive and quantitive analysis for chemical constituents of Panax japonicus were investedated using HPLC method.Methods（1）Normal-and reverse phase-silica gel column chromatography,combined with macroporous resin chromatography,were used to isolate and purify chemical constituents from the ethyl acetate portion of the rhizome of Panax japonicus.The spectroscopic methods(¹H-NMR,¹³C-NMR,2D-NMR,etc.)were used to identify the structures of the obtained compounds.（2）Lipopolysaccharide-stimulated murine macrophage RAW264.7 model was used for evaluating the anti-inflammatory effect of these isolated compounds from Panax japonicus.（3）The similarity of chemical composition of Panax japonicas from different habitats was compared and calculated using an application program named the similarity evaluation system of chromatographic fingerprints of traditional Chinese medicine.Additionally,the retention time of characteristic peaks in fingerprint chromatogram of Panax japonicas were determined by HPLC.（4）Chikusetsusaponin IVa was selected as the internal reference,and the relative correlation factors（RCFs）of other seven components were calculated to achieve quantitative analysis of multi-components by singlemarker（QAMS）.The ruggedness of RCFs was tested on different instruments and columns.Moreover,results of the QAMS were compared with the external standard method.Results（1）In this study,A total of 13 compounds were isolated and identified from the ethyl acetate portion of the rhizome of Panax japonicus.They were chikusetsu saponin-1（compound 1）,20（R）-ginsenoside-Rg₂（compound 2）,20（R）-ginsenoside-Rh₁（compound 3）,（3R,9R,10R）-panaxytriol（compound 4）,20（S）-notoginsenoside-R₂（compound 5）,20（S）-ginsenoside-Rh₁（compound 6）,20（S）-ginsenoside-Rg₂（compound 7）,ginsenoside-Rk₃（compound 8）,ginsenoside-Rg₆（compound 9）,2α,3β-dihydroxy-olean-12-en-28-O-β-D-glucopyrauoside（compound 10）,ginsenoside-Rg₁（compound 11）,oleanic acid-α-L-arabinofuranosyl-（1→4）β-glucopyranoside（compound 12）,and ginsenoside-R_f（compound 13）.（2）The anti-inflammatory activity of the isolated compounds were evaluated.As a result,compound 12 had obviously inhibitory effects on NO and IL-6production in LPS-stimulated RAW264.7 macrophages,and the IC₅₀ was 36.58μM and41.22μM,respectively.（3）In their respective linear ranges,the RCFs of chikusetsusaponin IVa and ginsenoside Re,notoginsenoside R₂,chikusetsusaponin V,chikusetsusaponin IV,ginsenoside Rg₆,ginsenoside Rk₃,and ginsenoside 3 were 0.84,0.59,0.74,0.33,0.63,0.535,and 0.28,respectively.Meanwhile,it had a good reproducibility under different chromatographs and different columns;and there was no significant difference between the calculated value of the different columns and the measured value of the external standard using QAMS.（4）The content range of each target constituents in 6batches of Panax japonicas was 6.81～27.92 mg/g,1.63～13.80 mg/g,1.24～8.49 mg/g,20.51～115.55 mg/g,61.15～119.71 mg/g,0.20～1.55 mg/g,0.13～2.15 mg/g,4.24～14.28mg/g.Conclusion Based on the isolated compounds,we evaluated their anti-inflammatory activities,compared the similarities and analyzed the quality differences among 6 batches Panax japonicus form different habitats.At the same time,the QAMS method was applied to the determination of the medicinal ingredients of Panax japonicas.Finally,the above methods can more accurately describe the quality of Panax japonicus.