| Semen cuscutae is the dry and ripe seeds of convolvulaceous plant Cuscuta australis R.Br.or Cuscuta chinensis Lam,and it is a well-known traditional Chinese medicine first recorded in the famous Shen Nong’s Herbal as an upper grade drug.Tropism of taste:Gan Wen.Owned by liver,kidney and spleen meridian.It has been used to nourish liver and kidney,prevent miscarriage and improve eyesight for thousands of years.The salt-processed product(SPP)of the semen cuscutae is first documented in Comprehensive Recording of Sage-Like Benifit(Sheng Ji Zong Lu)in the North Song Dynasty of China.Herbs processed with salt are mainly to be manifested their effects on kidney meridian.The SPP is better in warming and tonifying kidney.This study is a part of the national natural fund project—the material foundation and principle of the research which Cuscuta australis processed by salt enhance the efficacy of protecting kidney.This study aimed at comparing the effects of tonifying kidney between the crude product(CP)of semen cuscutae and the SPP and then determine their material basis,studying the difference of chemical components and biological effects of the CP and SPP by multivariable statistical analysis and to explain potential mechanisms of processing in enhancing the nourishing kidney.To unveil which is the key factor to lead the phytochemical changes during the processing procedure by multivariable statistical analysis,and compare the biological activity on nourishing kidney of CP and other 4 kinds of processed products.Furthermore,to preliminary study the potential mechanisms of processing.To obtain the new polysaccharides isolated from semen cuscutae and studying their structures.Objective:1.To establish the continuous method for extracting the total flavonoids and total polysaccharides in semen cuscutae.2.To establish the purification technology of total flavonoids in semen cuscutae.3.To study the difference of chemical components and biological effects of the CP and SPP by multivariable statistical analysis,compare the biological activity of nourishing kidney between CP and SPP,and then to explain potential mechanisms of processing in enhancing the nourishing kidney.4.To unveil which is the key factor to lead the phytochemical changes during the processing procedure by multivariable statistical analysis,and compare the biological activity on nourishing kidney of CP and other 4 kinds of processed products.Furthermore,to preliminary study the potential mechanisms of processing.5.To obtain the new polysaccharides isolated from semen cuscutae,and study their structures of molecular weights,homogeneity and monosaccharide composition.Methods:1.Optimize the process parameters of continuous extraction technology by the orthogonal design and the single factor design on the basis of the transfer rates of hyperoside,astragalin,total flavonoids,the content of total polysaccharide,and extract-acquiring rate.2.Compare the purification effect of different types of macroporous resin from the form of adding sample,removing impurity,elution,etc,and optimize the purification process parameters of total flavonoids on the basis of the elution rate of hyperoside,astragalin,total flavonoids.3.Multivariable statistical analysis was used to analysis the difference of chemical components and to find out the potential chemical markers for the discrimination between CP and SPP.And then detected the contents of 5 flavonoids(hyperoside,astragalin,quercetin,kaemferol and isorhamnetin),total flavonoids and total polysaccharide.4.Based on hydrocortisone induced kidney-yang deficiency rat model,to evaluate the biological activity of CP and SPP(the CP flavonoids extract,CP polysaccharide extract,SPP flavonoids extract,SPP polysaccharide extract,CP crude flavonoids extract)on nourishing kidney.And then combine with the change of its chemical profile,to explain potential mechanism of processing in enhancing the nourishing kidney.5.The CP,SPP,WPP,WPP+S and SSP were obtained according to the key factors in salt-processing and then to unveil which is the key factor to lead the phytochemical changes during the processing procedure by multivariable statistical analysis,and then determine the contents of 5 flavonoids,total flavonoids and total polysaccharide.6.Based on hydrocortisone induced kidney-yang deficiency rat model,to evaluate the biological activity of CP SPP,WPP,WPP+S and SSP.And then combine with the change of its chemical profile,to explain the potential mechanisms of processing.7.The new polysaccharide were obtained first by using different extraction methods,grading alcohol sink for preliminary separation and then separated by DEAE-cellulose and S-400 repeatedly.The physicochemical properties of new polysaccharide were studied by HPGPC,UV,IR.Results:1.The best process parameters of continuous extraction technology were refluxed twice for lh each time with 8 times of 60%ethanol.The residue after extracting by 60%ethanol was extracted with 8 times water and refluxed twice for 1h each time.2.The best process parameters of purification of total flavonoids were by HPD-100 macroporous resin with the column diameter ratio was 1:6,the sample amount was 0.8BV,the suitable concentration of 0.35 g/ml,the adding sample rate was 3 BV/h,the first water and 10%ethanol for removing impurity,with 40%and 80%ethanol elution 6 BV and 4BV respectively,the elution rate of 4 BV/h.3.The result of PCA indicated that there were 8 peaks(2,8,3,6,7,9,10,1)changed and these peaks are the most important components to distinguish CP and SPP.Then the contents of 3,6,9,10 and total flavonoids,total polysaccharidethe were determined.The results showed that quercetin,total flavonoids and total polysaccharide had increased while hyperoside,astragalin,kaemferol and isorhamnetin decreased significantly after salt-processing.4.The CPF,CPT,CPCF,SPPF and SPPT groups all could improve the kidney-yang deficiency syndrome.The CP and SPP had different advantages in different ways of improving the kidney-yang deficiency syndrome.On the whole,the SPP showed better.The flavonoids and the polysaccharide were both the effective components.5.The results of PCA indicated that the samples were classified into 4 groups which were CP,WPP and WPP+S,SPP,SSP.The products which were processed with heat could be separated from the products that processed without heat;The products which were processed with salt couldn’t be separated from the products that processed without salt.However,the products which added salt before heating and which added salt after heating was separated obviously.The results of contents determination showed that compared with the CP,hyperoside,astragalin,astragalin,isorhamnetin in SPP had decreased,while quercetin,total flavonoids and total polysaccharide had increased.The contents of astragalin,kaemferol,isorhamnetin in WPP,WPP+S had decreased,while quercetin,total flavonoids and total polysaccharide had increased.In SSP,the contents of quercetin and isorhamnetin had decreased.6.The CP and 4 kinds of processed products groups all could improve the kidney-yang deficiency syndrome.The wight result had indicated that all groups could increase the wight of the kidney-yang deficiency rats.The viscera index result had indicated that SPP could increase 3 viscera indexes,and was better than other groups.As shown as in the results of the kidney function,the WPP and WPP+S groups could decreased the level of BUN.As shown as in the results of the level of T,all groups could increase the level of T,especially the WPP+S and SSP groups.7.There were 3 polysaccharide obtained,C-7WR1,C-7WR2,C-7WR3.Their structures were also identification.The molecular weight of C-7WR1 was 7.59×104,it was composed of 2 monosaccharides with molar ratios as Fru:Man=0.02:1.The molecular weight of C-7WR2 was 3.23×104,it was composed of 4 monosaccharides with molar ratios as Fru:Man:Xyl:Ara=0.01:1:0.14:0.33.The molecular weight of C-7WR3 was 2.25×104,it was composed of 4 monosaccharides with molar ratios as Fru:Man:Xyl:Ara=0.01:1:0.10:0.47.They all were without protein and nucleic acid.Conclusions:1.The process parameters of continuous extraction technology were stable,feasible and suitable for the flavonoids and polysaccharide of semen cuscutae.2.The process parameters of purification of total flavonoids were reasonable,stable,feasible and suitable for purificating the flavonoids of Semen cuscutae in large-scale production.3.Salt-processing changed the chemical constituents,which in turn affect its bioactivity profile.The potential mechanisms of processing in enhancing the nourishing kidney were clear.4.Heat and the way of adding salt are both important factors in processing for the change of chemical constituents in salt-processing.The heat and salt were both the key factors in processing for their biological activities.So the heat and salt are are both important factors in processing.The mechanisms of salt-processing were clear.5.Three new polysaccharides were first obtained.Their molecular weights were 7.59×104,3.23×104,2.25×104.Their monosaccharide compositions were different,however,they all had Man.They were without protein and nucleic acid and their IR spectra were similar. |
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