Studies On The Chemical Constituents From The JieGengGanCao Decoction With Inhibiting HNE Activities

The imbalance between HNE and its endogenous inhibitors is resulted by excessive releasing of human neutrophil elastase（HNE）induced by bacterial or viral infections.Excessive HNE enhances cell membrane permeability,increases tissue fluid penetration,and then triggers inflammation.Which is one of the important mechanisms leading to pulmonary inflammation.In our preliminary work,experimental model had being established for screening HNE activity in vitro and in vivo.It was found that the pentacyclic triterpenoids from Traditional Chinese Medicine had different degrees of HNE inhibitory activity,and alleviated the permeability of pulmonary capillaries and lung injury.We found that the pentacyclic triterpenoids and HNE were reversible competitive binding modes through enzyme kinetics experiments and nuclear magnetic resonance studies,which could overcome the side effects of the chemical drug such as sivelestat sodium.Jie Geng Gan Cao decoction is consisted of Platycodonis Radix and Glycyrrhizae Radix et Rhizoma.It was used for treating lung diseases in<Shang Han Lun>,an ancient classics on Traditional Chinese Medicine.This decoction had the functions of clearing the lung,relieving sore-throat,clearing heat and detoxicating,and it was used for the treatment of pulmonary diseases such as sore throat,cough,pulmonary sputum in the modern clinical usage.It was reported that pentacyclic triterpenes were the main active constituents of Platycodonis Radix and Glycyrrhizae Radix et Rhizoma,with good inhibitory effect on lung inflammation according to many literatures.Based on these,Jie Geng Gan Cao decoction was chosed as the research objective of this subject.It was found that the ethanol extracts of Platycodonis Radix and Glycyrrhizae Radix et Rhizoma was superior to the water extracts according to the HNE inhibitory activities in vitro.Among them,the petroleum ether fraction,dichloromethane fraction,and ethyl acetate fraction all significantly inhibited HNE activities.The HNE inhibition had no remarkable difference between the single and mixed extracts.Meanwhile,there was no significant difference among the bioactive fractions between single and mixed extracts through the thin layer chromatography analysis.So we seperatedly studied the Platycodonis Radix and Glycyrrhizae Radix et Rhizoma next.Basing on bioassay-guided isolation method,18 compounds were obtained from the ethyl acetate fraction of 95%ethanol extract of Glycyrrhizae Radix et Rhizoma.It included 2 fatty acids,2 pentacyclic triterpenoids,11 flavonoids,etc.The total saponin from Platycodonis Radix was acid-hydrolyzed to obtain the total sapogenin,then the total sapogenin was separated and purified to obtain 5 pentacyclic triterpenoids.The compounds were linoleic acid（1）,n-hexadecane acid（2）,medicarpin（3）,7-hydroxy–2′,4′-dimethoxy isoflavan（4）,betulinic acid（5）,licoisoflavavone C（6）,licoisoflavonone（7）,1-methoxyphaseollin（8）,licoisoflavone B（9）,isoderrone（10）,gancaonin X（11）,glyasperin K（12）,semilicoisoflavone（13）,glycyrol（14）,glyasperin C（15）,3-oxo-18β-glycyrrhetinic acid（16）,2′,4",4-trihydroxy-3-methoxychalcone（17）,ulexone A（18）,polygalic acid（19）,platycogenic acid A lactone（20）,platycodin（21）,3-oxo-18β-D-glucopyranosyl platicogenic acid alactone（22）,polygalic acid-3-O-glucoside（23）.In this paper,it was found that the bioactive fractions all originated from the small polar extracts.Basing on bioassay-guided isolation method,18 compounds were obtained from the ethyl acetate fraction of 95%ethanol extract of Glycyrrhizae Radix et Rhizoma.According to the HNE inhibitory activities experiment,three compounds were found with effective HNE inhibition including linoleic acid（1）and3-oxo-18β-glycyrrhetinic acid（16）and ulexone A（18）.Linoleic acid（1）had the highest HNE inhibitory activity,the HNE inhibition rate was 93.74%in the final concentration of 100μM,and the IC₅₀ was 12.33μM.The compound 16 and 18 were84.45%、81.86%respectively.The content of fatty acids was higher than 0.15%in glycyrrhiza.The cell membrane was a lipid bilayer composed of hydrophilic exterior heads and a hydrophobic interior,hydrophobic group ended with two fatty acid chain.Polyunsaturated fatty acids played an important role in maintaining the structure and function of cell membranes,affecting the function of membrane surface enzymes and receptors.We need more studies about that whether linoleic acid plays the role of endogenous HNE inhibiting effects in vivo.3-oxo-18β-glycyrrhetinic acid（16）had84.45%HNE inhibition rate.Besides,Glycyrrhetinic acid is the aglycone of glycyrrhizic acid,the inhibition rate of glycyrrhetinic acid was better than glycyrrhizic acid in vitro,however,with lower content in Glycyrrhizae Radix et Rhizoma.So saponins maybe were hydrolyzed into sapogenins by enzymes then absorbed into the body through the intestinal metabolism.The total sapogenins possessed better HNE inhibitory activities than the total saponins of Platycodonis Radix.However,the total sapogenin was separated and purified to obtain five pentacyclic triterpenoids,with no effective HNE inhibitory activities.Comparing the structure of oleanolic acid,ursolic acid,glycyrrhetinic acid（standard）,3-oxo-18β-glycyrrhetinic acid（compound 16）and saponin（compound19-21）isolated from platycodon grandiflorum.There were structural differences mainly between the rings A/C and E.It was speculated that the increase of hydroxyl substitution on the A ring might reduce the activity of inhibiting HNE,and the carbonyl structure at the C-11 position and the ring E might also affect the HNE activity.Based on the above research,we concluded that although there are many pentacyclic triterpenoids in the Jie Geng Gan Cao decoction,inhibition of HNE activity It might not be the main mechanism of anti-inflammatory effect of pentacyclic triterpenoids through inhibiting HNE activity.