Studies On Chemical Constituents And Anti-inflammatory Activity Of Scutellaria Barbata

Scutellaria barbata D.Don is a plant belonging to Scutellaria of Labiatae,which mainly contains neo-clerodane diterpenoids,flavonoids,polysaccharides,triterpenoids,sterols and other components.It has the functions of clearing heat and detoxification,disperses blood stasis and hemostasis,diuretic and detoxification,and is proverbially used in the treatment of various inflammatory diseases,snake bites and jaundice.Modern pharmaceutical research and clinical experiments have proved that the crude extract and monomer compounds of S.barbata have anti-inflammatory activity,but there are few reports concerning its related mechanism.In order to find the anti-inflammatory active components with novel structure,high efficiency and low toxicity in S.barbata,and further study its mechanism of action,this work systematically studied the chemical components and anti-inflammatory activity of the whole herb of S.barbata collected from Qinzhou City,Guangxi Province.By comprehensive utilization of reversed-phase column chromatography,MCI column chromatography,Sephadex LH-20 gel column chromatography,HPLC and modern spectrum analysis techniques such as NMR,HRESIMS,IR and UV,we isolated,purified and identified54 compounds from S.barbata,including 40 neo-clerodane diterpenoids,7 flavonoids,3steroids and 4 other compounds.Thirteen new compounds were found,all of which were neo-clerodane diterpenoids,respectively named Barbaolides（A-M）.Based on single-crystal X-ray diffractionanalysis and circular dichroism（ECD）comparison,the absolute configurations of the new compounds were assigned.Inflammation is an innate immune response to harmful stimuli.Excessive and persistent inflammatory response can cause many chronic diseases,such as stroke,tumor and Alzheimer’s disease.Macrophages are the primary cells involved in inflammatory response.At present,lipopolysaccharide stimulation of RAW264.7 macrophage is a usual in vitro model to test the anti-inflammatory activity of natural products.In this work,40 diterpenoids isolated from S.barbata were evaluated for anti-inflammatory activity by this activity model,and the effect of compound 36（Scutolide E）on NF-κB signaling pathway was further discussed.The results showed that all diterpenoids had no cytotoxicity to RAW264.7 at the concentration of 50μM,and compounds 26,27,32,34,36 and 37 could significantly inhibit the generation of NO in RAW264.7 macrophages,with IC₅₀values lower than 40μm;By analyzing the structure-activity relations of neo-clerodane diterpenoids,it was found that theα,β-unsaturated-γ-lactone moiety with an exocyclic conjugated double bond was necessary for maintaining and increasing its activity.Compound 36 showed the most active with an IC₅₀value of 10.6±1.5μM.The Western blot showed that it had a dose-dependent inhibitory effect on i NOS and p-IκBα（P<0.01）,thus revealing that the anti-inflammatory effect of compound 36 was related to the inhibition of NF-κB signaling pathway.