Comparison Of Anti-inflammatory Activities Of Different Parts Of Chloranthus Serratus And Preliminary Separation And Purification Of Its Active Components

Objective:To compare the anti-inflammatory activities of the water and alcohol extracts from the roots of Chloranthus serratus（C.serratus）in vitro and in vivo,so as to select the appropriate extraction method in clinical use,and preliminary separation and purification of polysaccharides as anti-inflammatory active component.Methods:1)Fifty-six male SD rats were randomly divided into the blank（KB）,model（MX）,positive control（YX）,low dose of the water extract from the roots of C.serratus（GSL）,high dose of the water extract from the roots of C.serratus（GSH）,low dose of the alcohol extract from the roots of C.serratus（GCL）and high dose of the alcohol extract from the roots of C.serratus（GCH）groups.The positive drug aspirin（ASP）,the water and alcohol extracts from the roots of C.serratus were dispersed in 0.5%sodium carboxymethyl cellulose（CMC-Na）solution to form a suspension,respectively.In the GSL,GSH,GCL and GCH groups,the doses of the drugs of 0.189,0.378,0.108 and 0.216 g/kg/d（the amount of original medicinal materials was equal,obtained by conversion according to the extraction rates of the water and alcohol extracts from the roots of C.serratus）were given by intragastric administration,respectively.In the YX group,the doses of the drug of 0.1 g/kg/d was given by intragastric administration.The equal doses of CMC-Na solution were given by intragastric administration in the KB and MX groups.After continuous administration for 7 days,the feet of the right hind of the rats were subcutaneously injected with 0.1 m L of 1%the normal saline solution of carrageenan to establish inflammatory model.The change rates of body weight and the organ indexes of the thymus,adrenal and spleen of the rats in each group were calculated and compared.The contents of vitamin C（Vit C）and cholesterol（CHOL）in the adrenals were measured.The ankles of the rats were stained by hematoxylin-eosin（HE）and the histopathological changes were observed.The contents of nitric oxide（NO）in serum of the rats were measured by one-step method.The contents of oxidative stress indicators including malondialdehyde（MDA）,total superoxide dismutase（T-SOD）,glutathione（GSH）and catalase（CAT）were measured.The contents of interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）in serum of the rats were measured by ELISA method.The contents of prostaglandin E₂（PGE₂）in the inflammatory feet of the rats were measured by ultraviolet spectrophotometry.The expression levels of the inflammation-related pathway including the JAK/STAT,NF-κB and p-ERK/ERK in the inflammatory feet of the rats were detected by Western blot method.2)The effects of different drug doses on the viability of RAW264.7 macrophages were detected by CCK-8 method and the low and high doses of the water and alcohol extracts from the roots of C.serratus were screened.RAW264.7 macrophages induced by lipopolysaccharide（LPS）were used as inflammatory model,set the KB,MX,GSL,GSH,GCL and GCH groups.After the cells in the drug groups were pretreated with corresponding the doses of drugs for 0.5 h,the cells in each group（except the KB group）were stimulated with 1 mg/m L of the solution of LPS for 23.5h.The morphology of the cells in each group were observed under fluorescence inversion microscope.The contents of NO in cell culture supernatant were detected by one-step method.The contents of oxidative stress indicators such as T-SOD,GSH,MDA and CAT in cell lysate were measured.The contents of TNF-α,IL-6,IL-1βand PGE₂were measured by ELISA method.The contents of reactive oxygen species（ROS）in the cells were measured with DCFA-HA fluorescence probe.The expression levels of the inflammation related pathway including the Nrf2/HO-1,MAPK,NF-κB and JAK/STAT and the TLR4 proteins were detected by Western blot method.3)Using LPS as a modeling drug,inflammation induction of RAW264.7macrophages,set the KB,MX,Dexamethasone acetate positive（YX）and low,middle,high doses of C.serratus crude polysaccharides groups.The low,medium and high doses of C.serratus crude polysaccharides were 200,400 and 800μg/m L.After the cells in the drug groups were pretreated with corresponding doses of the drugs for 0.5h,the cells in each group（except the KB group）were stimulated with 1 mg/m L of the solution of LPS for 23.5 h.Took pictures to compare the morphological changes of the cells in each group and the cell viability changes in each group were observed by CCK-8 assay.The contents of NO were measured by one-step method.The contents of IL-1β,IL-6,PGE₂and TNF-αwere determined by ELISA method.The contents of GSH,T-SOD and MDA in cell lysate were measured and the contents of ROS in the cells were measured with DCFA-HA fluorescence probe.4)The C.serratus polysaccharides were extracted by the method of water extraction and alcohol precipitation.The contents of polysaccharides were determined by sulfuric acid-phenol method.The ratio of extraction material to liquid,extraction temperature,extraction time and extraction times of the polysaccharides were optimized by single factor experiments and orthogonal experiments.The feasibility of the determination method of the contents of C.serratus polysaccharides were verified by precision,repeatability,stability and sample recovery experiments.The alcohol settling time and alcohol concentration of C.serratus polysaccharides were optimized by single factor experiments.The C.serratus polysaccharides were decolorized by activated carbon adsorption,the decolorization rates,retention rates of polysaccharides and comprehensive scores were used as indicators to evaluate the decolorization effect and optimize the decolorization time,decolorization temperature and the amount of activated carbon in the decolorization process by response surface methodology.Results:1)The water and alcohol extracts from the roots of C.serratus could reduce the plantar swelling rates and inhibit the changes of the thymus and spleen organ indexes,but had no obvious effect on the adrenal organ indexes.They could extremely significantly reduce the contents of Vit C and CHOL in the adrenals and PGE₂in the inflammatory feet.They could significantly reduce the contents of NO,TNF-α,IL-1β,IL-6 and MDA.They could obviously increase the contents of CAT,T-SOD and GSH.They could significantly inhibit the expression of the inflammation-related pathway including the JAK/STAT,p-p65/p65 and p-ERK/ERK.They could reduce the infiltration of inflammatory cells and interstitial hemorrhage in the inflammatory feet of the rats in a dose-dependent manner.The water extract from the roots of C.serratus showed stronger anti-inflammatory activities in the investigation of several inflammatory indicators and inflammation-related pathway.2)The doses of the GSL,GSH,GCL and GCH groups were 20,200,11.4 and114μg/m L（the amount of original medicinal materials was equal,obtained by conversion according to the extraction rates of the water and alcohol extracts from the roots of C.serratus）,respectively.The water and alcohol extracts from the roots of C.serratus could inhibit the changes of cell morphology and improve cell viability after LPS stimulation in a dose-dependent manner.They could significantly reduce the production of NO,PGE₂,TNF-α,IL-1β,IL-6,MDA and ROS.They could obviously increase the contents of T-SOD,GSH and CAT.They could significantly inhibit the expression of inflammation-related pathway including the MAPK,NF-κB,JAK/STAT and TLR4 proteins.They could extremely significantly increase the expression of the Nrf2/HO-1 pathway.Among them,the water extract from the roots of C.serratus had a more significant effect on the inflammatory indicators and inflammation-related pathway than the alcohol extract.3)Each drug group of C.serratus crude polysaccharides could inhibit cell deformation and differentiation after LPS stimulation in different degrees.The cell viability of the MX group was significantly decreased in the treatment of LPS,however the drug groups of C.serratus crude polysaccharides could not only enhance cell viability,but also significantly reduce the production of NO,PGE₂,IL-6,IL-1β,TNF-α,MDA and ROS caused by inflammation in a dose-dependent manner.They could obviously elevate the contents of the protective indicators of GSH and T-SOD.4)The optimum water extraction conditions of C.serratus polysaccharides were determined as follows:the ratio of extraction material to liquid was 1:30,the extraction temperature was 100°C,extracted 3 times,the extraction time was 1.5 h.The optimum alcohol precipitation condition was 75%alcohol concentration and the alcohol settling time was 18 h.Through the response surface method,the optimum conditions for decolorization of C.serratus polysaccharides by activated carbon were obtained as follows:the decolorization temperature was 90°C,the decolorization time was 40 min,the activated carbon dosage was 1.8%.Conclusions:Both of the water and alcohol extracts from the roots of C.serratus have anti-inflammatory activities in vitro and in vivo.In vivo,the anti-inflammatory mechanism of the water and alcohol extracts from the roots of C.serratus may be related to the hypothalamus-pituitary-adrenocortical（HPA）axis,inhibition of JAK/STAT pathway and oxidative stress.In vitro,the anti-inflammatory mechanism may be related to inhibition of MAPK,NF-κB and JAK/STAT pathways,activation of Nrf2/HO-1 pathways and oxidative stress.The anti-inflammatory activities of the water extract from the roots of C.serratus is better than that of the alcohol extract.It shows that water extraction is a better method of extraction.The C.serratus crude polysaccharides have strong anti-inflammatory effects in vitro,which provides a powerful theoretical basis for further exploration of the anti-inflammatory activities of C.serratus.The extraction and decolorization process of C.serratus polysaccharides are optimized by single factor experiments and orthogonal experiments,which lay a theoretical foundation for the separation and purification of anti-inflammatory monomers contained in C.serratus in the future.