Studies On Anti-inflammatory Activities And Chemical Constituents Of Clerodendron Trichotomum Tunberg Leaves

Chou wu tong,also named as Clerodendron trichotomum Thunb,belongs to the Verbenaceae family,the twigs and leaves have been used for centuries as Chinese folk medicine for high medicinal value and broad application prospect.They can be used for the treatment of rheumatoid arthritis,hypertension,dysentery,malaria for the diverse pharmacological activities such as antihypertensive,anti-inflammatory,analgesic.The leaves can also be external used to treat eczema,tinea manus,etc.In recent years,the domestic and foreign scholars have confirmed that the methanol extract from the leaves of Clerodendron trichotomum Thunb possess obvious anti-inflammatory activity.However,the active material of anti-inflammatory has not been reported.To further determine the active constitution of anti-inflammatory activity from Clerodendron trichotomum Thunb leaves and to find the natural anti-inflammatory active compounds,in this study,the anti-inflammatory activity of different polar solvent extracts from the 90%ethanol extract of the dried leaves of the Clerodendron trichotomum Thunb was investigated and the chemical constituents of the anti-inflammatory active fraction were systematic studied.In this experiment,the dried leaves of Clerodendron trichotomum Thunb were macerated in 90%ethanol at room temperature for 5 times.The residue,which was obtained by rotary evaporator under reduced pressure,was re-suspended in water and then partitioned successively with an equivalent volume of petroleum ether?PE?,ethyl acetate?EtOAc?,and n-butanol?n-BuOH?,respectively.The cytotoxicity of PE,EtOAc,n-BuOH and Water extract against mouse monocyte-macrophage RAW 264.7 was evaluated in vitro using the MTT assay.The RAW 264.7 cells induced by lipopolysaccharide?LPS?were used as the model of inflammation and the anti-inflammatory activity of different polar fraction was screened by determining the inhibition of nitric oxide?NO?production and Interleukin-1??IL-1??production,Indomethacin?Indo 100?g/mL?was used as a positive control.The results showed that the PE,n-BuOH and Water extract at a concentration of not more than 100?g/mL,had no obvious cytotoxic effect.EtOAc extract showed significant cytotoxicity to RAW 264.7 at the concentration of 100?g/mL and the cell viability was only 55.3%.PE extract and EtOAc extract could significantly inhibit the release of NO and IL-1?in RAW264.7 cells induced by LPS in a concentration-dependent manner.When the concentration of was PE extract 50?g/m L,the inhibition of NO release rate was 46%(^**P<0.01),and he inhibition of IL-1?release rate was 42%(^**P<0.01);and when the concentration of EtOAc extract was 50?g/m L,the inhibition of NO release rate was 84%(^**P<0.01),and he inhibition of IL-1?release rate was 65%(^**P<0.01).Therefore,PE extract and EtOAc extract were the main anti-inflammatory active fraction,and the anti-inflammatory activity of EtOAc extract is stronger than PE extract.The PE extract and EtOAc extract were further isolated and purified by silica gel column chromatography,MPLC,HPLC,ODS,Sephadex LH20 and other chromatographic methods,and the structures of the compounds were identified by TLC,EI-MS,¹H-NMR,¹³C-NMR and other spectroscophic methods.Nine compounds were isolated from PE extract and EtOAc extract of the leaves of Clerodendron trichotomum Thunb,and they were identified as:22-dehydroclerosterol?1?,hexadecanoicacid?2?,1-Heptadecanol?3?,apigenin?4?,luteolin?5?,n-Pentatriacontane?6?,Chrysoeriol?7?,3-O-?-D-galactopyranosyl-?24??-ethylcholesta-5,22,25-trien?8?,?2S,3S,4R,E?-2-[?2'R?-2'-hydroxyl-octacosane amino]-23-hexacosane-1,3,4-triol?9?.Among them,compound 3,5,6,7,8,9 were isolated from the leaves of Clerodendron trichotomum Thunb for the first time,and compound 3,9 were isolated from the Clerodendrum Linn for the first time.To sum up,this paper studied the anti-inflammatory activity of the different polar parts of the Clerodendron trichotomum Thunb leaves,and studied the chemical constituents of the anti-inflammatory active fraction,which laid a solid foundation for the further study of the anti-inflammatory activity of Clerodendron trichotomum Thunb.