| Glycyrrhiza Radix et Rhizoma,known as licorice,is one of the most popular Chinese herbal medicines.It can replenish the spleen and qi,clear away heat and detoxification,eliminate phlegm,relieve cough,emergency,pain,and reconcile various drugs as well.Glycyrrhiza uralensis Fisch.,as the main base plant of licorice in the pharmacopoeia,is widely used in the clinic with various biological activities,and of great medicinal and edible value.To date,various chemical components have been isolated and identified from Glycyrrhiza Radix et Rhizoma and many biological activities have been discovered.But few researches have been focused on its alkaloid components.Besides,aerial parts of G.uralensis are currently used as fuel,feed,or treated as waste,which have not been fully developed and utilized.Therefore,in-depth study of the chemical components and biological activities of G.uralensis is extremely important for the overall optimization of licorice resources.In recent few years,the neuroprotective effects of the active ingredients in G.uralensis have attracted widespread attention.However,the researches on this subject mostly focused on the flavonoids from roots and rhizomes.There are few reports on neuroprotective activity of aerial parts.This experiment is to isolate and identify the chemical constituents of the n-butanol portion of G.uralensis so as to further study the chemical components and neuroprotective activities of licorice and its aerial parts.Then the anti-neuroinflammatory and antioxidant activities of the monomer compounds isolated from roots,rhizomes and aerial parts have been studied.The chemical constituents were investigated by many chromatographic methods.Finally 23 compounds were isolated from the n-butanol soluble part.Then the structures were determined via spectral data and physic-chemical evidence,such as HR-ESI-MS and NMR data analysis.The 23 glycoside compounds are as follows:3,4-dihydroxyquinoline 4-O-β-D-glucopyranoside（1）,liquiritin（2）,isoliquiritin（3）,neoisoliquiritin（4）,liquiritin apioside（5）,neoliquiritin（6）,isoliquiritin apioside（7）,ononin（8）,licuraside（9）,（2S）-liquiritigenin-7-O-β-D-glucopyranosyl-（1→2）-β-D-apiofuranoside（10）,（2R）-liquiritigenin-7-O-β-D-glucopyranosyl-（1→2）-β-D-apiofuranoside（11）,liquiritigenin 7,4’-di-O-glucopyranoside（12）,isoliquiritigenin4,4’-di-O-glucopyranoside（13）,3-O-β-D-glucuronopyranosyl-glycyrrhetinic acid（14）,6,7-dihydroxyliquiritigenin-4’-O-glucopyranoside（15）,glycyrrhizic acid（16）,7-hydroxy-4’-O-[β-D-apiofuranosyl-（1→2）-O-β-D-glucopyranosyl]flavones（17）,2’,4’-dihydroxy-4-O-[β-D-glucopyranosyl-（1→6）-O-β-D-glucopyranosyl]chalcone（18）,licorice saponin E2（19）,22β-acetoxy-glycyrrhizin（20）,licorice saponin H2（21）,glycyrrhizic acid 6"-methyl ester（22）,and glycyrrhizic acid 6’-methyl ester（23）.Compounds 1-23 are all glycoside compounds.In addition,the alkaloid components contained in licorice and the aerial parts were compared and analyzed by UPLC,with the isolated alkaloid compound 1 as reference.It was found that the alkaloid components contained in the aerial parts were more plentiful than those in the licorice.Then the anti-neuroinflammatory and antioxidant activities of the compounds,the extracts R（herbal extract）and L（aerial parts extract）were conducted.The compounds included isolated compounds 1-23 and those isolated from licorice in the early stage of our laboratory.They are licostilbene A（1’）,licostilbene B（2’）,licofuranol A（3’）,licofuranol B（4’）,ɑ,ɑ’-dihydro-3,5,4’-trihydroxy-4,5’-diisopentenylstilbene（5’）,glycypytilbene B（6’）,luteolin（7’）,diosmetin（8’）,echinatin（9’）,3’,4’-dimethylquercetin（10’）,3,3’-dimethylquercetin（11’）,chrysoeriol（12’）,glycypytilbene A（13’）,β-sitosterol（14’）,liquiritigenin（15’）,isoliquiritigenin（16’）,and glycycoumarin（17’）.Among them compounds 1’-14’are from the aerial parts.For anti-neuroinflammatory activity studies,it showed that the chemical components of licorice and its aerial parts both exerted anti-neuroinflammatory activities by inhibiting nitric oxide（NO）release in lipopolysaccharide（LPS）-induced murine microglial BV-2 cells.Compounds 1’-17’had a significant inhibitory effect,with the inhibition rate reaching more than 70%at a concentration of 10μg/m L.The anti-inflammatory activity of the flavonoid glycosides compound was lower than that of triterpene saponins overall among compounds 1-23.The anti-inflammatory activity was obvious in only 4 of the 15 compounds（2-4,7）at the highest concentration of 50μg/m L（inhibition rate>50%）.The mean NO inhibition rate of triterpene saponins（16,19-23）was higher than those 4 flavonoid glycosides（2-4,7）at the equal concentrations.For antioxidant activity researches,it showed that the new compound 1 had a significant effect on the H2O2-and 6-OHDA-induced cell damage（P<0.05）,displaying good neuroprotective effect.Meanwhile,the number and the efficiency of compounds among 1-23 against H2O2-induced cell damage were both more and better than 6-OHDA-induced.Compounds 1’-17’and extracts R and L had good neuroprotective activities except for coylidin（12’）.The cell viability was restored to over 50%.According to the activity comparison of compounds with the same basic parent structure（2,5,12,15’;3,4,16’）,the preliminary structure and relationships（SAR）analysis showed that there was a negative correlation between the number of sugar moieties and the neuroprotective activities of compounds.And the categories of sugar moieties might have impact on their antioxidant activities.In summary,in this study the chemical constituents from G.uralensis were isolated and identified.The alkaloid components of both licorice and its aerial parts were compared and analyzed.As for compounds and extracts derived from licorice and aerial parts,the research on anti-neuroinflammatory and antioxidant activities in vitro was carried out.The preliminary SAR of some monomer compounds was discussed.The findings would provide a scientific basis for the development of leading compounds for neurodegenerative diseases,and be of significance for promoting the optimal utilization of licorice resources. |
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