Studies On The Chemical Constituents And Bioactivities Of Panacis Majoris Rhizoma

Panacis majoris Rhizoma,which belongs to the Panax genus of the family Araliaceae,comes from the dried rhizomes of Panax japonicus C.A.Mey.var.major（Burk.）C.Y.Wu et K.M.Feng or Panax japonicus C.A.Mey.var.bipinnatifidus（Seem.）C.Y Wu et K.M.Feng.Pharmacological studies have shown that Panacis majoris Rhizoma has anti-tumor,anti-thrombotic,improving immunity and other effects.Previous phytochemical investigations on Panacis majoris Rhizoma had revealed that triterpenoid saponins are the main chemical components in this medicinal plant.To date,the domestic and overseas scholars have studied the chemical constituents and pharmacological activities of the other plants of Araliaceae in depth,such as Panax ginseng C.A.Meyer,Panax quinquefolium L.,and Panax notoginseng（Burk.）F.H.Chen.However,the development and utilization of Panacis majoris Rhizoma has been limited because of the lack of studies on it.In order to improve the quality standard,study the pharmacodynamic material basis,and develop the new medicinal use of Panacis majoris Rhizoma,we studied its chemical constituents and its bioactivities.Firstly,the ethanol extract of Panacis majoris Rhizoma was repeatedly subjected to column chromatography and preparative HPLC to yield 42 compounds and their chemical structures had been elucidated by means of MS,1D,2D NMR and other spectroscopic methods,including six new compounds and sixteen compounds obtained from this plant for the first time.The new compounds were identified as（20S,24 S,25R*）-6-O-[β-D-glucopyranosyl-（1 → 2）-β-D-glucopyranosyl]-dammar-20,24-epoxy-3β,6α,12β,25,26-pentaol（1）,（20S,24 R,25R）-6-O-[β-D-glucopyranosyl-（1 → 2）-β-D-glucopyranosyl]-dammar-20,24-epoxy-3β,6α,12β,25,26-pentaol（2）,（20S）-6-O-[β-D-glucopyranosyl-（1 → 2）-β-D-glucopyranosyl]-dammar-20,25-epoxy-3β,6α,12β,24α-tetraol（3）,6-O-[β-D-glucopyranosyl-（1 → 2）-β-D-glucopyr-anosyl]-dammar-3β,6α,12β,20 S,24R,25-hexaol（4）,6-O-[β-D-glucopyranosyl-（1 →2）-β-D-glucopyranosyl]-dammar-25（26）-ene-3β,6α,12β,20 S,24R-pentaol（5）,3-O-[β-D-glucopyranosyl-（1 → 2）-β-D-（6′-O-ethyl）-glucuronopyranosyl]-oleanolic acid-28-O-β-D-glucopyranoside（6）.The sixteen compounds obtained from Panacis majoris Rhizoma for the first time were identified as oleanolic acid-3-O-α-Larabinofuranosyl-（1 → 4）-β-D-glucuronopyranoside（12）,3-O-{β-D-glucopyranosyl-（1 → 2）-[α-L-arabinofuranosyl-（1 → 4）]-β-D-glucuronopyranosyl}-oleanolic acid-28-O-β-D-glucopyranoside（18）,6″-O-acetylginsenoside Rb1（20）,vinaginsenoside R4（21）,6″′-O-acetylginsenoside Re（27）,pseudoginsenoside RT2（32）,pseudoginsenoside F11（33）,vinaginsenoside R8（34）,notoginsenoside E（35）,vinaginsenoside R9（36）,vinaginsenoside R13（37）,ginsenoside I（38）,ginsenoside Re5（39）,ginsenjilinol（40）,quinquenoside L11（41）,yesanchinoside R2（42）.Other known compounds were identified as chikusetsusaponin V（7）,ginsenoside Ro methyl ester（8）,oleanolic acid-3-O-β-D-glucopyranosyl-（1 → 2）-β-D-glucuronopyranoside（9）,chikusetsusaponin IV（10）,araloside A methyl ester（11）,pseudoginsenoside RT1（13）,pseudoginsenoside RT1 methyl ester（14）,chikusetsusaponin IVa（15）,chikusetsusaponin IVa methyl ester（16）,oleanolic acid-28-O-β-D-glucopyranoside（17）,ginsenoside Rb1（19）,ginsenoside Rf（22）,20-glucoginsenoside Rf（23）,notoginsenoside R2（24）,notoginsenoside R1（25）,ginsenoside Re（26）,ginsenoside Rd（28）,majonoside R1（29）,24（R）-majonoside R1（30）,and majonoside R2（31）.The above-mentioned saponins are classified as dammarane-type,oleanane-type,and ocotillol-type glycosides based on the structural differences in their aglycones.In order to make clear the contents of three kinds of saponins,three HPLC detection methods combined with three new pretreatment methods were established respectively.（1）A method of ionic liquid aqueous two-phase extraction coupled with HPLC had been firstly developed for the analysis of five oleanane-type saponins in Panacis majoris Rhizoma.Through the optimization of experimental conditions,the pretreatment method established was as follows: when 0.5 m L [Hmim]Br,3.0 m L water and 2.5 g K2HPO4 were used as the extraction solvent,the analytes could be extracted from 0.2 g Panacis majoris Rhizoma.The LODs of five oleanane-typesaponins were in the range of 0.31⁰.88 μg·m L-1.The RSDs of stability experiments were in the range of 0.26%².69%.The recoveries of the method were in the range of90.00%¹06.00%.This method was proved to have high sensitivities,good stabilities and good recoveries which is suitable for extraction and determination of oleanane-type saponins in Panacis majoris Rhizoma.Under this experimental condition,the contents of five compounds were 47.10 mg·g^-1,24.00 mg·g^-1,0.35mg·g^-1,0.17 mg·g^-1 and 0.44 mg·g^-1,respectively;（2）A method of ionic liquid extraction and solid phase extraction coupled with HPLC had been firstly developed for the analysis of five dammarane-type saponins in Panacis majoris Rhizoma.Through the optimization of experimental conditions,the pretreatment method established was as follows: when 0.2 m L [Bmim]Br,0.8 m L water were used as the extraction solvent,and 3.0 m L 60% acetone-water as the eluent,the analytes could be extracted from 50.0 mg Panacis majoris Rhizoma.The LODs of five oleanane-type saponins were in the range of 0.43⁰.90 μg·m L-1.The RSDs of stability experiments were in the range of 0.63%¹.73%.The recoveries of the method were in the range of92.00%¹06.40%.This method was proved to have high sensitivities,good stabilities and good recoveries which is suitable for extraction and determination of dammarane-type saponins in Panacis majoris Rhizoma.Under this experimental condition,the contents of five compounds were 92.0 μg·g^-1,106.0 μg·g^-1,100.0 μg·g^-1,33.0 μg·g^-1 and 470.0 μg·g^-1,respectively;（3）A method of matrix solid-phase dispersion（MSPE）coupled with HPLC had been firstly developed for the analysis of five ocotillol-type saponins in Panacis majoris Rhizoma.Through the optimization of experimental conditions,the pretreatment method established was as follows:diatomaceous earth as the dispersant and methanol as the eluent.The RSDs of stability experiments were in the range of 0.57%¹.96%.The recoveries of the method were in the range of 85.71%¹03.00%.This method was proved to have high sensitivities,good stabilities and good recoveries which is suitable for extraction and determination of ocotillol-type saponins in Panacis majoris Rhizoma.Under this experimental condition,the contents of five compounds were 916.0 μg·g^-1,35.7 μg·g^-1,720.0 μg·g^-1,19.2 μg·g^-1 and 12.9 μg·g^-1,respectively.From the above determinationresults,we could know that the contents of the five oleanane-type saponins in Panacis majoris Rhizoma were higher than that of the other two types,and their total content was 7.02%.As compared to the traditional extraction method,the three pretreatment methods used in this paper were easier with less extraction time and lower organic solvent-consuming which were more suitable for the rapid extraction and analysis of effective ingredients in Panacis majoris Rhizoma.According to literature reports,the oleanane-type saponins containing C3-sugar chain and C28-COOH had better pharmacological effects.Based on this point,we assumed that C3,C28-bisdesmosides could be degraded into C3-monodesmosides,and the bioactivities of degradation products probably increased.So we studied the degradation of Panax Japonicus Total Saponins（PJTS）,and the chemical constituents of degradation products of Panax Japonicus Total Saponins（DPJTS）were isolated and identificated,and the comparative study on the bioactivities of PJTS and DPJTS were also investigated.PJTS were firstly preparated by macroporous resin,and the main constituents of PJTS were identified as chikusetsusaponin IVa（I）,chikusetsusaponin IV（II）,pseudoginsenoside RT1（III）,chikusetsusaponin V（IV）and 3-O-{β-D-glucopyranosyl-（1 → 2）-[α-L-arabinofuranosyl-（1 → 4）]-β-D-glucuronopyranosyl}-oleanolic acid-28-O-β-D-glucopyranoside（V）by HPLC analysis.All five compounds belonged to C3,C28-bidesmosidic oleanane-type saponins,and their contents in PJTS were 168.1mg·g^-1,115.7 mg·g^-1,65.4 mg·g^-1,270.9 mg·g^-1 and 79.3 mg·g^-1,respectively.The total content of compounds I^V in PJTS was 69.94%.In this paper,the alkali（sodium hydroxide）water solution was used to degrade the Panax Japonicus total saponins（PJTS）,and the degradation products of PJTS（DPJTS）were obtained.Moreover,the main constituents in DPJTS were isolated and identified.The results showed that the five C3,C28-bidesmosidic oleanane-type saponins（compounds I^V）in PJTS were completely turned into five C3-monodesmosidic oleanane-type saponins in DPJTS,and the main constituents of DPJTS were identified as oleanolic acid-3-O-β-D-glucuronopyranoside（P-1）,oleanolic acid-3-O-α-L-arabinofuranosyl-（1 → 4）-β-D-glucuronopyranoside（P-2）,oleanolic acid-3-O-β-D-xylopyranosyl-（1 → 2）-β-D-glucuronopyranoside（P-3）,oleanolic acid-3-O-β-D-glucopyranosyl-（1 → 2）-β-D-glucuronopyranoside（P-4）and oleanolic acid-3-O-β-D-glucopyranosyl-（1 → 2）-[α-L-arabinofuranosyl-（1 →4）]-β-D-glucuronopyranoside（P-5）,and their contents in DPJTS were 128.1 mg·g^-1,99.7 mg·g^-1,39.2 mg·g^-1,302.6 mg·g^-1 and 63.7 mg·g^-1,respectively.We investigated the protective effects of PJTS and DPJTS on myocardial ischemia reperfusion injury（MIRI）in rats comparatively,and the mechanism was also discussed.The results showed that both PJTS and DPJTS have protective effect on myocardial ischemia reperfusion injury in rats,and the protective effect may be related to the inhibition of platelet adhesion and aggregation,the inhibition of high blood viscosity and the enhancement of antioxidant capacity.The above effect of DPJTS was slightly better than that of PJTS,but there was no significant difference between DPJTS and PJTS.We did not find this phenomenon which the activities of monodesmosidic oleanane-type saponins were significantly stronger than that of bidesmosidic oleanane-type saponins,and a further study is needed to reveal the real reason.The literatures reported that PJTS have anti-thrombotic effects,and the results of our study also showed that PJTS have the activities of antiplatelet adhesion and aggregation.In order to investigate the relationship between PJTS and its antithrombotic effect,we studied the inhibitory effects of seventeen saponins in Panacis majoris Rhizoma on platelet aggregation induced by adenosine diphosphate（ADP）and arachidonic acid（AA）,and the structure-activity relationships were also discussed.The results showed that the saponins have different inhibitory activities,and the inhibitory activities were increased with the increase of the number of hydroxyl groups in the case of an equal number of sugars.In conclusion,we studied the chemical constituents and bioactivities of Panacis majoris Rhizoma in depth in this paper,and we have made a series of innovative research achievements in terms of the separation and identification of chemical constituents,the content determination,the preparation of DPJTS and the biological activities.This work provides a new scientific basis for the further development andutilization of Panacis majoris Rhizoma.