Studies On Rapid Identification And Anti-inflammatory Of Prenylated Flavonoids From Artocarpus

Artocarpus is a well-known in the family Mulaceae.There are 52 species in the world,which are widely distributed distributed on some islands in Southeast Asia and the Pacific.The plants of Artocarpus have very important medicinal value,and many plants are often used as traditional folk medicine in the folk.In recent years,Artocarpus plants have attracted the attention of phytochemical workers because they are rich in phenolic components such as prenylated flavonoids with significant biological activity.Prenylated flavonoids（PFs）are a class of structurally distinct chromone derivatives characterized by several prenyl units linked to a flavone nucleus by C–C and/or C–O bonds.PFs possess high structural diversity due to various prenyl substitution patterns on the flavone skeleton,which correspond to their wide range of biological activities such as anti-inflammatory,tyrosinase inhibition,and cathepsin K inhibition.Due to their complex chemical diversity and wide range of biological activities,the search for structurally interesting and biologically active PFs from natural products has attracted the intense attention of natural product chemists and pharmacologists in the past few decades.A variety of bioactive PFs were previously identified from Artocarpus using the conventional process of isolation and purification on a preparative scale followed by structure elucidation via spectroscopic methods.However,the purification of PFs from the crude extract by this conventional method is usually time-consuming,tedious,and labor-intensive due to their thermal sensitivity and low abundance in Artocarpus.The combination of quadrupole time-of-flight mass spectrometry（Q-TOF-MS）and linear trap quadrupole orbitrap mass spectrometry（LTQ-Orbitrap-MS）is a powerful analytical strategy to rapidly screen and identify trace compounds.It is often used to identify the structure of traditional Chinese medicine extracts through the fragmentation behaviors of compounds.Despite there being vast numbers of known PFs,their fragmentation behaviors and corresponding mechanisms remain relatively unexplored.In view of this,this paper will systematically study the fragmentation pattern,structure identification and preliminary evaluation of the anti-inflammatory activity of prenylated flavonoids.Specific research contents are as follows:1.Studies on characterization and identification of PFs from Artocarpus.In this study,a combination of quadrupole time-of-flight mass spectrometry（Q-TOF-MS）and linear trap quadrupole orbitrap mass spectrometry（LTQ-Orbitrap-MS）was performed to investigate the fragmentation behaviors of prenylated flavonoids（PFs）from Artocarpus plants.Fifteen PFs were selected as the model molecules and divided into five types（groups A–E）according to their structural characteristics in terms of the position and existing form of prenyl substitution in the flavone skeleton.The LTQ-Orbitrap-MSⁿ spectra of the[M–H]^–ions for these compounds provided a wealth of structural information on the five different types of compounds.The main fragmentation pathways of group A were the ortho effect and retro Diels–Alder（RDA）,and common losses of C₄H₁₀,CO,and CO₂.The compounds in group B easily lose C₆H₁₂,forming a stable structure of a1,4-dienyl group,unlike those in group A.The fragmentation pathway for group C is characterized by obvious ^1,4A^-,^1,4B^-cracking of the C ring.The diagnostic fragmentation for group D is obvious RDA cracking of the C ring and the successive loss of CH₃ and H₂O in the LTQ-Orbitrap-MSⁿspectra.Fragmentation with successive loss of CO or CO₂,·CH₃,and CH₄ in the LTQ-Orbitrap-MSⁿspectra formed the characteristics of group E.The summarized fragmentation rules were successfully exploited to identify PFs from Artocarpus heterophyllus,a well-known Artocarpus plant,which led to the identification of a total of 47 PFs in this plant.2.Studies on Anti-inflammatory of PFs from ArtocarpusIn this study,lipopolysaccharide（LPS）-induced mouse peritoneal macrophages（RAW 264.7）were used as an inflammation model,and applied the CCK-8 method to determine the cell viability of 41 PFs isolated in the previous period on RAW264.7cells;31 PFs showed low toxicity to RAW264.7 cells viability range greater than or equal to 50μmol/L,and only PFs 5,7,9-10,12,15,27-28,33,and 36 showed relatively low cells viability.Within cell viability range,the anti-inflammatory activity of 41 PFs of the compound was determined.The results showed that 23 PFs had significant anti-inflammatory activity within their activity,and their IC50 ranged from 4μmol/L to 96μmol/L.Among them,PFs 10,12,17,27,28,33,and 36 total 7compounds.The IC₅₀ is lower than or close to 15μmol/L,and even the IC₅₀ of PF 10is only 4.75μmol/L.A total of 4 PFs of PF 2,17,26,and 41 showed low toxicity and good anti-inflammatory activity.Summarize the structure-activity relationship of PFs:the number of side chain prenyl substitution is different,three prenyl substitution>two prenyl substitution>geranyl substitution>single prenyl substitution.If a single prenyl substitution is in a different position,the C-8 prenyl substitution>C-3 prenyl substitution>C-6 prenyl substitution.In summary,this paper mainly studies the characterization,identification and anti-inflammatory of PFs from Artocarpus.These results indicate that the developed analytical method could be used as a rapid,effective technique for the qualitative characterization of PFs in Artocarpus plants and in other PF-accumulating medicinal herbs.At the same time,research on anti-inflammatory activity shows that PFs have significant anti-inflammatory activity,providing a basis for research and development of new anti-inflammatory drugs.