Studies On Lipophilic Constituents Of Lu Dangshen And Their Anti-inflammatory Activities

Objective:Lu Dangshen is derived from the dried roots of Codonopsis pilosula(Franch.)Nannf.,Which is a famous medicine in Shanxi.Radix Codonopsis,with indications for tonifying qi and strengthening the spleen and lung,is one of the important traditional Chinese medicines for tonics.It has a wide range of clinical applications.Modern researches show that Radix Codonopsis has a variety of chemical components and pharmacological effects,but the material basis and mechanism of traditional functions of Lu Dangshen are not clear.In this paper,we systematically study the chemical constituents and anti-inflammatory activities of the Lipophilic parts of Lu Dangshen,in order to enrich the research on the chemical composition and bioactivity of Codonopsis pilosula,and to provide a certain theoretical basis for the quality control,development and utilization of Lu Dangshen.Methods:1.Silica gel column chromatography and thin-layer chromatography(TLC)separation technology were used to systematically isolate and purify the chemical constituents of the petroleum ether fraction of Lu Dangshen methanol extract.For the obtained compounds,one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy(1D-NMR,2D-NMR),mass spectrometry(MS),ultraviolet(UV),infrared(IR)and circular dichroism(CD)spectroscopy techniques were used to identify the chemical structures.2.The anti-inflammatory activities were evaluated by establishing a lipopolysaccharide(LPS)-induced RAW264.7 inflammatory cell model in vitro.Griess method was used to measure the released level of nitric oxide(NO)in the cell supernatant,and enzyme-linked immunoassay(ELISA)was used to detect the contents of tumor necrosis factor(TNF-α)and interleukin-6(IL-6).By comparing the effects of compounds on LPS-induced mouse macrophage RAW264.7 cells,the inhibition rate of TNF-α and IL-6 contents was evaluated for the anti-inflammatory activity of each compound.Results:1.Eighteen compounds were isolated from the petroleum ether fraction of Lu Dangshen methanol extract.Fifteen compounds have been identified,which are Codonopiliate D(1),24-methylenecycloartanyl linolate(2),24-methylenecycloartanyl linolate(3),and Codonoplipliate B(4),taraxeryl acetate(5),friedelin(6),3β-hydroxy-glutin-5-ene(7),α-spinone(8),22,23-dihydrospinone(9),α-spinasterol(10),22,23-dihydrospinasterol(11),1-[(12E,16E)-12,16-eicosadienoyl]-2-[(E,E)-7,11-octadecadienoyl]-3-stearyl glycerol(12),linoleic acid(13),n-hexadecanoic acid(14),taraxerol(15).Compound 1 is a new compound.2.Compounds 1 ~ 4 all belong to cycloartane triterpenes.The anti-inflammatory activities on LPS-induced RAW264.7 inflammatory cell model showed that compounds 1 ~ 4 can significantly inhibit the contents of NO in the cell supernatants at concentrations of 3.75 μmol / L,7.50 μmol / L and 15.00 μmol / L.At the above three concentrations,the NO inhibition rates of Compounds 1 ~ 4: Compound 1 were 25.60%,46.91%,and 58.59%;Compound 2 were 10.73%,20.62%,and 30.07%;Compound 3 were 23.37%,29.56% and 46.74%;Compound 4 were 32.52%,48.51% and 65.20%.In general,the NO inhibitory activity of compounds 1 ~ 4 on LPS-induced mouse macrophages RAW264.7 was: 4 > 1> 3 > 2.3.Compounds 8 and 9 are a mixture of spinasterone,and compounds 10 and 11 are a mixture of spinosterol.The NO inhibition rates of the spinosterone mixture(8 and 9)and the spinosterol mixture(10 and 11)at concentrations of 1 μg / mL,2 μg / mL and 4 μg / mL were 22.69%,37.23% and 46.87%,11.17%,18.00% and 20.31%,respectively;the inhibition rates of TNF-α content reached 34.79%,50.61% and 82.48%,19.43%,44.67% and 72.79%;the inhibition rates of IL-6 content were 43.59%,62.23% and 88.37%,36.82%,49.28% and 83.10%.Combining the results of the three indicators,the anti-inflammatory activities of the spinosterone mixture(8 and 9)is better than the spinosterol mixture(10 and 11).Conclusion:1.Among the 15 compounds which have been isolated and identified,compounds 1 ~ 7 are triterpenoids,among which compounds 1 ~ 4 belong to cycloartane type triterpenes,compound 5 and 15 are dandelion type triterpenes,and compound 6 is oxane type triterpenes,compound 7 is oleanolane triterpene;compounds 8 ~ 11 are steroidal compounds.compounds 12 ~ 14 are aliphatic compounds.Compound 1 is a new compound,and compounds 7 and 12 are isolated for the first time in Codonopsis.2.This paper systematically compares the anti-inflammatory activity of cycloartane triterpenes and steroids in Codonopsis pilosula for the first time.Compounds 1 ~ 4 and 8 ~ 11 have significant NO inhibitory activities on LPS-induced RAW264.7 inflammation model,and compounds 8 ~ 11 have significant TNF-α and IL-6 inhibitory activity on LPS-induced RAW264.7 inflammation model,which indicate that these compounds may be potential active substances for the traditional efficacy of Lu Dangshen.