Preliminary Study On The Extraction Of TAR And Antioxidant And Anti-Inflammatory Activity Of Rubia Cordifolia L.

Objective:To find the main components of antioxidant and anti-inflammatory effects of Rubia cordifolia L.,the extraction process of the total anthraquinone of Rubia cordifolia L.(TAR)product was optimized and purification process were studied with macroporous resin to obtain TAR product with high purity.And FRAP,DPPH,ABTS method was applied to detect the antioxidant activities.Macrophage RAW264.7 inflammation model and the freund’s complete adjuvant-induced arthritis model in mice were duplicated to evaluate anti-inflamnatory activities in this study.Methods:1.A spectrophotometric method for the determination of TAR was introduced to analyze Rubia cordifolia L.samples from different sources.The extraction process of the TAR was optimized by controlling the variable method to increase its yield.The macroporous resin with the best performance were selected by static and dynamic adsorption and dissociation experiments.And the purification process of TAR by D101 was optimized.2.The free radical scavenging ability and total antioxidant capacity of different batches of madder herbs were determined by chemical detection of FRAP,DPPH and ABTS,and the difference of antioxidant activity between different batches of medicinal materials was determinated.The fingerprints of the low polarity of Rubiaceae were established and correlated with the fingerprints using PLS,OPLS and other statistical methods to find potential substances related to antioxidant activity.3.MTT assay was used to detect the cytotoxicity of the purified TAR product and the different chemical sites extract of Rubia cordifolia L.on macrophages RAW264.7 inflammation model.The macrophages RAW264.7 were treated with different concentrations of TAR and purpurin.And then,LPS was added to the mixture that TAR and purpurin were preintervention on murine macrophages RAW264.7 with a final concentration of 0.1μg/mL and cultured 18 h.The concentration of IL-β,IL-6,IL-10 and TNF-α in the culture supernatant was evaluated by ELISA..4.The freund’s complete adjuvant-induced arthritis model in mice were duplicated to evaluate anti-inflammatory activities.The experimented mice were divided into six groups(blank group,model group,positive control group,low,medium and high concentration group).The weight and right ankle diameter was measured and the inhibition rate was evaluated every 3 days.After the end of the experiment,the serum and liver was collected.And the kidney,liver,spleen and other major organs were collected for the histopathological evaluation.The concentration level of IL-1β,IL-6,IL-10 and TNF-α,IFN-γ in serum were determined by ELISA,and the activities of SOD and MDA in 10%liver homogenate were measured by the chemical kits.Results:1.A stable and reproducible spectrophotometer method for the determination of TAR was established.The RSD of precision,stability and repeatability were 0.21%,0.20%,L83%,respectively.And average of recovery was 105.3%with the RSD was 1.57%.And optimized the extraction method of total anthraquinone was obtained.D101 macroporous resin was selected to purify total anthraquinone.And the optimized process was as follows:the column was packed by wet packing method.10mL wet pretreated D101 resin,and added 40mL sample and 60mL water were mixed by uninterrrupted shak.After 12 hours of adsorption,the column was wet packed and the liquid was collected to calculate the column volume.After 6BV pure water was uesd for elution,colunmn was eluted by 75%ethanol in 15 BV.75%ethanol eluatlon was collected.2.The results of antioxidant activities showed that the good batches of free radicals were QC-14,QC-1 and QC-3,and the poor was QC-6.From ABTS results,the good batches of free radicals were QC-14,QC-1,QC-2,the worst is the QC-13;FRAP results show that the good batches of free radicals were QC-14,QC-1,QC-3,the worst is QC-13.3.IL-1β,IL-6 and TNF-α(p<0.001,p<0.01)were significantly higher in the model group than in the blank group.Compared to model group,the different concentrations of EER,TAR,CFR and hydroxyglycine(1μg/mL,0.5μg/mL)were able to significantly reduce the expression of INF-α,IL-1β,IL-6 with a dose-dependent manner.EAER can only slightly decrease the level of proinflammatory factors.All extracts can improve the expression of IL-10.In addition,TAR and CFR significantly improved the expression of IL-10(p<0.01).4.The results of ELISA showed that IL-1β,IL-6 and TNF-α concentrations in the model group were significantly higher than those in the blank group(p<0.001).Compared with the model group,EER and EAER,CFR decreased IL-1β,IL-6,TNF-α,and showed a dose-dependent.EER can significantly reduce the concentration of IL-1β and TNF-α(p<0.05).The concentrations of IL-1β,IL-6 and TNF-α were significantly decreased in the high and medium concentrations of CFR and the high concentration of EAER(p<0.05).While the low concentration of CFR and EAER low,medium concentration group compared with the model group was no significant effect.Compared with the blank group,the IL-10 of the model group increased.Compared with the model group,the concentration of IL-10 was significantly increased in middle and high concentrations of EER(p<0.05).5.Compared with the blank group,the levels of IL-1β,IL-6 and TNF-α in the model group were significantly higher than those in the blank group(p<0.05).EER,TAR and Purpurin could significantly decrease the levels of IL-1β,IL-6 and TNF-α(p<0.05)in comparison with the model group.The efficacy of TAR and Purpurin was superior to that of positive control group and EER group.Compared with the blank group,the IL-10 concentration in the model group was significantly improved(p<0.01).Compared with the model group,EER,TAR and Purpurin in the high concentration group can significantly improve the concentration of IL-10.6.The levels of SOD,NO and GSH-Px in liver homogenate were significantly increased(p<0.01).POD.Superoxide anion radical and MDA were decre,ased with the increase of the concentration of the extract.Compared with the model group,the POD was significantly improved in the low,medium and high concentration groups(p<0.01).Superoxide anion radical,MDA significantly decreased(p<0.01).Compared with the-model group,low and medium concentration group(p<0.05,p<0.01).The levels of T-GSH,T-GSH-2GSSG were significantly increased in low and medium concentrations,and the levels of T－GSH,T-GSH-2GSSG,GSSG concentration(p<0.05,p<0.01).Conclusion:The experimental results indicate that D101-type macroporous resin is suitable for the purification of TAR.EER contains antioxidant activity compounds.TAR and extracts of different chemical parts and purpurin have good effects on anti-inflammatory in vitro which indicates that the TAR could be a potential reagent for anti-inflammatory.Both the high and medium concentration dose of EER has good therapeutic effect on adjuvant arthritis mice.