Analysis Of Anti-inflammatory Components In Two Medicinal Plants

Objective:The fragmentation pathways of the biflavonoids and the grayanotoxin diterpenes were concluded by establishing the UPLC-Q-TOF-MS/MS method,and the biflavonoids from the active parts of Selaginella uncinata,as well as the grayanotoxin diterpenes from Rhododendron amesiae were identified.The PDE4 inhibitory activities of different polar parts and biflavonoid monomer compounds in S.uncinata were studied,and the anti-inflammatory activities of biflavonoids were also studied at the cellular level.The objective of this research was to clarified the anti-inflammatory substances of two medicinal plants,and to provide a basis for the structural identification and rapid discovery of biflavonoids and grayanotoxin diterpenes in plant samples.Methods:1.Using the biflavonoids isolated from S.uncinata as the standard,the mass spectrometry rules of the biflavonoids was studied,and the biflavonoids of the active parts of S.uncinata were analyzed by UPLC-Q-TOF-MS/MS technology.The PDE4B2 inhibitory activity of different polarity extraction parts of petroleum ether,ethyl acetate and n-butanol as well as biflavonoids were determined by in vitro enzyme activity assay.The binding effect of monomer compounds with PDE4B was studied by using SYBYL molecular docking software.In addition,LPS-induced RAW 264.7 cell model was used to detect the inhibitory activity of the biflavones for the release of NO.2.By establishing the UPLC-Q-TOF-MS/MS method,the mass spectrometry rules of grayanotoxin diterpenes isolated from R.amesiae were summarized,and the components of the ethyl acetate fraction of R.amesiae were analyzed.Results:1.The main fragmentations were focused on a series of ions such as ^1,3IIB⁺ion、^0,2IIB⁺ion、^1,3IA⁺ion、^1,3IIB⁺ion、^0,4IIB⁺ion,which was generated by the cracking of C ring in the two parts of biflavonoids and the loss of some neutral fragments such as CO、CO₂、C₂H₂O.Based on the mass spectrometry pathways,21 biflavonoids were identified from the ethyl acetate layer crude extract of S.uncinata.2.The different polarity extraction parts of petroleum ether,ethyl acetate and n-butanol of S.uncinata all exhibited PDE4B2 inhibitory activity with IC₅₀ values of 513.0μg/mL,310.9μg/mL and 460.0μg/mL,respectively.Among them,the ethyl acetate extraction part was the best one.All the nine biflavonoids could inhibit the activity of PDE4B2 enzyme with the IC₅₀ range of 1.3～17.7μM.The molecular docking results showed that the biflavonoids mainly hydrogen-bonded with the amino acid residues Asn395 and Gln443 of PDE4B2 enzyme and the bound water.Raw 264.7cell model was induced by LPS,and seven compounds of diflavones were detected to inhibit NO release in the model.3.The grayanotoxin diterpenes had similar fragmentation pathways and three common characteristic ions were found.The typical fragmentation pathways of the diterpenes were the loss of H₂O and AcOH and the loss of ring A and ring D by the ring-opening reaction followed by the Mc Lafferty rearrangement.Based on the mass spectrometry analysis,8 grayanotoxin diterpenes were identified from the ethyl acetate extraction part of the R.amesiae.Conclusion:The main pathways and characteristic ions of biflavonoids and grayanotoxin diterpenes were summarized,which provided a reference for the structural identification and rapid discovery of these compounds in natural plants.By establishing the UPLC-Q-TOF-MS/MS method,21 biflavonoids and 8 grayanotoxin diterpenes were identified from the ethyl acetate layer crude extract of S.uncinate and R.amesiae,respectively.The anti-inflammatory activities of biflavones were investigated at the molecular and cellular levels.It is meaningful to elucidate the basis of anti-inflammatory active substances of these two plants and to further explore their medicinal value.