Study On The Extraction,Separation And Analysis Of Hydrophilic Ginsenosides

Panax ginseng C.A.Mey.belongs to genus Panax of family Araliaceae and is commonly known as the"King of Herbs",which has the effects of calming the nerves,relieving palpitation,improving eyesight and intel igence.Ginsenosides are the main active ingredients of Panax ginseng C.A.Mey.,which have anti-inflammatory,anti-oxidation,anti-tumor,and protective pharmacological effects such as nerve cel s and cardiovascular system.So far,chemists have discovered more than 100 ginsenosides from the plants of the genus Panax.Most of them are hydrophobic compounds,and only a small part are hydrophilic compounds.Hydrophilic ginsenosides are difficult to be separated.Although the contents of hydrophilic ginsenosidesare high in the plants of the genus Panax,the numbers of the related research reports are far less than those of hydrophobic ginsenosides.In this paper,the extraction,separation,structure identification and analytical methods of hydrophilic ginsenosides were studied.The purpose was to clarify the distribution of hydrophilic ginsenosides in the plants of the genus Panax and explore theircorrelationship with the pharmacological effects of Panax ginseng C.A.Mey..In this paper,the hydrophilic ginsenosides in the roots of Panax ginseng C.A.Mey.were extracted and separated.Eight monomers were obtained from 70%methanol extract,and the chemical structures of seven monomers were identified by NMR,MS,IR and other spectroscopy methods.Two of them were new compounds,and one compound was isolated from Panax ginseng C.A.Mey.for the first time,and four were known compounds.The new compounds were identified as 3-O-[β-D-glucopyranosyl-（1→2）-β-D-glucopyranosyl]-20-O-[β-D-xylopyranosyl-（1→4）-α-L-arabinopyranosyl-（1→6）-β-D-glucopyranosyl]-dammar-23-ene-3β,12β,20S,25-tetraol and 3-O-{[β-D-gluco-pyranosyl-（1→2）-[β-D-xylopyranosyl-（1→6）]-β-D-glucopyranosyl]}-20-O-[β-D-xylopyranosyl-（1→2）-α-L-arabinofuranosyl-（1→6）-β-D-glucopyranosyl]-dammar-24-ene-3β,12β,20S-triol.The compound isolated from ginseng for the first time is Chikusetsusaponin VI.,and the known compounds are ginsenoside Ro,Ra₁,Ra₂ and Ra₃,respectively.In this paper,HPLC method for determination of ginsenoside Ra₁,Ra₂,Ra₃,Rb₁and Rc was established and methodological investigation was finished.First,with reference to the high performance liquid chromatography conditions given byginseng content determination item inthe 2015 Chinese Pharmacopoeia,the separation effect of different types of C-18 columns on the above ginsenosides was investigated.It was found that no matter how the proportion of the mobile phase was adjusted,the C-18 column could not separate the ginsenoside Ra₁ and Rc,nor could separate the ginsenoside Ra₃ and Rb₁,but the amide column could completely separate the four components.Therefore,a new HPLC method for the simultaneous determination of ginsenoside Ra₁,Ra₂,Ra₃,Rb₁ and Rc in ginseng by amide column was established.The linear response ranged from 0.4¹9.7μg for ginsenoside Ra₁,0.2⁹.9μg for ginsenoside Ra₂,0.4¹9.6μg for ginsenoside Ra₃,0.4⁶9.4μg for ginsenoside Rb₁ and 0.4⁶9.6μg for ginsenoside Rc.The RSDs value of precision,repeatability,stability and sample recovery rate are all less than 2%,indicating that the method has good precision and high accuracy.The contents of ginsenoside Ra1,Ra2,Ra3,Rb1 and Rc in three kinds of genus Panax（Panax ginseng C.A.Mey.,Panaxnotoginseng（Burk.）F.H.Chen and Panaxquinquefolium L.）were determined by this method.The results showed that the contents of ginsenoside Ra₁,Ra₂,Ra₃,Rb₁ and Rc in Panax ginseng C.A.Mey.were0.107%,0.068%,0.048%,0.428%and 0.422%,respectively;the contents of ginsenoside Ra₁,Ra₂,Ra₃,Rb₁ and Rc in Panaxnotoginseng（Burk.）F.H.Chen were0.008%,0.004%,0.027%,0.875%and 0.010%,respectively;the contents of Rb₁ and Rc were 0.630%and 0.066%in Panaxquinquefolium L.,respectively;ginsenoside Ra₁,Ra₂,Ra₃ were not detected in Panaxquinquefolium L..The above results indicate that the content of ginsenoside Rb₁ in Panax ginseng C.A.Mey.determined by pharmacopoeia method may be higher.Therefore,the contents of ginsenoside Rb₁ and Rc in ginseng were determined by two different columns（amide column and C-18 column）,and the results were compared and analyzed.The results showed that the contents of ginsenoside Rb₁ and Rc in Panax ginseng C.A.Mey.measured by C-18 column were higher than that of ginsenosideRb₁ and Rc measured by amide column.When the contents of ginsenoside Rb₁ and Rc in ginseng were determined by the pharmacopoeia method,the peaks of ginsenoside Rb₁ and Ra₃ were overlap,and the peaks of Rc and Ra₁ were overlap too,resulting in a higher measurement result.In addition,the above results also show that the amide column is more suitable for the separation and determination of hydrophilic ginsenosides than the C-18 column.In summary,seven hydrophilic ginsenoside monomers were identified from Panax ginseng C.A.Mey.,including four known compounds,two new compounds and one compound isolated from ginseng for the first time,which enriched the variety and quantity of ginsenosides.A new HPLC method for simultaneous determination of ginsenoside Ra₁,Ra₂,Ra₃,Rb₁ and Rc in ginseng by amide column was established.The defects of the method for determination of ginsenoside specified in the Chinese Pharmacopoeia were found,which proved that the method would result in higher determination of ginsenoside Rb1 and Rc.The results of this study provide a new scientific basis for improving the determination method of ginsenoside Rb₁ in Panax species and its preparations in the Chinese Pharmacopoeia.At the same time,a new HPLC content determination method for simultaneous determination of ginsenoside Ra₁,Ra₂,Ra₃,Rb₁ and Rc in Panax species is provided.