Evaluation Of Preparation Technology And Hepatoprotective Effect Of Total Flavonoids From Carthamus Tinctorius L.Leaf.

Objective:Carthamus tinctorius L.leaves,a waste product of Carthami flos production,are rich in flavonoids.In this study,flavonoids from Carthamus tinctorius L.leaf（TFCTLL）were used as the study object,aiming to optimize the extraction and purification process of Total flavonoids from Carthamus tinctorius L.Leaf（TFCTLL）,and Combined with network pharmacology and in vitro and in vivo experiments to study the liver protective pharmacological activity and mechanism of TFCTLL..Methods:1.HPLC chromatogram of Carthami flos and its non-medicinal parts（bud,leaf and stem）was performed at first,and chemical constituents of Carthamus tinctorius L.leaves were further identified to determine the subject of the experiment.2.Single factor test and Box-Behnken response surface method were used to optimize the extraction and purification process of TFCTLL.Taking the extraction rate of total flavonoids as the evaluation index,the optimal levels of four extraction factors including solid-liquid ratio,ethanol concentration,extraction time and extraction times were investigated.Single factor experiment was used to investigate the optimal enrichment conditions of different macroporous resin,elution solvent,loading amount and p H.3.ultra-performance liquid chromatography-high resolution mass spectrometry（UPLC-Q-TOF/MS）was used to collect mass spectrometry data.Combined with literature reports and standard natural products high-resolution mass spectrometry database,the chemical constituents of TFCTLL and TFCTLL into blood were identified and analyzed.Combined with network pharmacology,the important active ingredients,main therapeutic targets and pathways of TFCTLL in the treatment of liver injury were screened out.4.The effect of TFCTLL on H2O2-induced oxidative damage of L02 cells and the anti-inflammatory effect of TFCTLL on LPS-induced RAW264.7 inflammatory cells were studied in vitro,providing some theoretical basis for the subsequent animal model study on the liver protection effect of TFCTLL in mice.5.The therapeutic effect and mechanism of TFCTLL on acute and CLI models were investigated by ELISA,tissue section observation,RT-PCR,immunofluorescence and molecular docking using CCl4-induced acute and CLI models.Results:1.The HPLC chromatographic characteristics of the non-medicinal parts（bud,leaf,stem）and Carthami flos were different,and the chemical components in Carthamus tinctorius L.leaves were more and mainly flavonoids.Therefore,TFCTLL was further determined as the research object.2.Through single factor test and Box-Behnken response surface method,the optimal extraction process of TFCTLL was as follows:solid-liquid ratio of 1∶27,ethanol concentration of 80%,extraction time of 65 min,and the actual extraction rate was 4.921%.The extraction solution of Carthamus tinctorius L.leaves under the optimal extraction process was adsorbed on the pretreated macroporous resin column AB-8 and eluted with 95%ethanol.The total flavonoids purity reached61.42%.3.Eighteen compounds,including luteolin,vicenin-1,vicenin-2,roseoside and Quercetin-7-O-β-D-glucopyranoside were identified in TFCTLL samples.Main components were identified in serum samples of TFCTLL mice after administration,including luteolin,vicenin-1,vicenin-2 and roseoside,etc.101 potential anti-liver injury targets were obtained through network pharmacological screening,and the enrichment of KEGG pathway was found.Intersection targets are significantly enriched in the PI3K-AKT signaling pathway,which is involved in the body’s response to oxidative stress and inflammation,and may be a potential key pathway for anti-liver injury.4.TFCTLL pretreatment could significantly improve the cell viability of H₂O₂-induced L02 cells and LPS-induced RAW 264.7 cells.It can inhibit the production of NO and the content of IL-1βand IL-6 in cells of LPS-induced RAW 264.7.In vitro cell experiments have proved that TFCTLL has anti-inflammatory and antioxidant effects,which provides a theoretical basis for the subsequent study of the liver protection effect of TFCTLL.5.CCl4 was used to induce acute liver injury model in mice,and studies showed that TFCTLL could reduce the contents of ALT,AST and TBA in model mice.It can improve the degeneration,necrosis and inflammatory cell infiltration of liver cells in model group.CCl4 was further used to induce the mouse CLI model,and the study showed that TFCTLL could significantly improve the levels of ALT,AST,TBA,MDA,SOD,CAT,GSH-Px,IL-6,IL-1βand TNF-αin vivo.The liver surface and cell necrosis,inflammatory infiltration,collagen fiber proliferation and fibrosis were significantly reduced in CLI mice.The relative gene expressions of TLR2,TLR3,TLR4,NF-κB p65,IRF3,AKT,TRIF,PI3K,My D88,TNF-αand IL-1βwere significantly down-regulated in CLI mice.Further immunofluorescence results showed that TFCTLL could inhibit the protein expression of NF-κB p65 and cytoplasmic translocation to the kernel in CLI mice.Molecular docking results showed that luteolin,vicenin-1,vicenin-2 and roseoside,had good binding effect with TLR4,PI3K and AKT1.Conclusions:1.The optimal extraction and purification process of TFCTLL was determined.The method was simple,efficient and feasible,and could be used for the extraction of total flavonoids from Carthamus tinctorius L.leaves.2.Luteolin are the main chemical and blood components of TFCTLL,The PI3K-AKT signaling pathway may be a potential key pathway for anti-liver injury.3.TFCTLL has anti-inflammatory and antioxidant pharmacological activities,and has protective effects on both acute and CLI.The mechanism of its anti-liver injury may be related to the anti-inflammatory and immunomodulatory effects of TLRs/NF-κB and PI3K/AKT signaling pathways.In conclusion,this study can provide support for the resource utilization of non-medicinal parts of Carthamus tinctorius L.and the development of efficient and safe natural drugs for the treatment and anti-liver injury.