Studies On The Immuno-active Constituents Of The Ethanolsoluble Fraction From Liuwei Dihuang Decoction Preliminary Studies On The Structure-Activity Relationship Of Polysaccharides

By activity-guided fractionation ,using PFC(plaque forming cell) test as immunoactivity-evaluating parameter, stepwise fractionation and isolation were carried out on the ethanol-soluble fraction of Liuwei Dihuang Decoction,a famous classical ancient prescription of traditional Chinese medicine(TCM). Frl-3A and Frl-4C18-A, two major active fractions,were obtained,from which four compounds were isolated,namely Frl-3A(3)a, Frl-3A(3)b , Frl-3A(4) and Frl-4C18-A(2).Bioassay results showed as follows: 1 .Among all the fractions, Fr1-3A and Frl-4C18-A showed the strongest activity as well as the comparatively high yield,accounting for the major active fractions. 2. Frl-4C18-A(2) significantly promoted the declined antibody production response in Cy-treated mice . Frl-3A(4) enhanced the splenocyte proliferation in SAMP8 . Frl-3A(3)b enhanced the splenocyte proliferation induced by Con A in normal mice. The above results suggested that the three compounds be the major immuno-active constituents.All the obtained compounds were identified by tandem MSⁿ ,together with ¹³C-NMR and ¹H-NMR.They were sweroside,morroniside,loganin and manninotriose respectively.The trace constituent in Frl-3A(3)b was also identified as loganin with the same technique.From the pulps of Cornus officinalis Sieb et Zucc, six acidic pectic polysaccharides were obtained, among which four polysaccharides' structural characteristics were elucidated on the basis of chemical and spectral evidences . HPLC analysis showed that the four polysaccharides , Cop-2c , Cop-3a , Cop-3b , and Cop-3c were homogenous, and their molecular weights were estimated to be 20000 , 24000 , 10000 and 17000Dal, respectively .The component sugar analysis with TMS method showed that Cop-2c , Cop-3a and Cop-3c consisted mainly of GalA , Gal and Rha in the molar proportions 4.2 : 1 : 0.7 , 1.4 : 1 : 0.81 and 7 : 1 : 1.6, respectively . whereas Cop-3b consisted mainly of GalA and Rha in molar proportion 99 : 1 . Only trace of protein in them were detected by Bradford method (bovine IgG ) .With methylation analysis, their glycosyl linkages were elucidated that they consisted mainly of l-*4 linked GalA , some branched in 2 or 3 position and 1—2 linked Rha or 1—2 linked Rha with branch in 4 position as side chain . Cop-2c ,Cop-3a , and Cop-3c had Gal residue , mainly in terminal position .The 13C-NMR and 'H-NMR spectra of Cop-2c, Cop-3b, Cop-3c, Cop-4b, Cop-4c, mainly composed of GalA , showed that their glycosyl linkages were a configurations.Taking all the mentioned above into consideration , we can conclude that the four polysaccharide are all pectin-like polysaccharides. Another two polysaccharides , Cop-4b and Cop-4c , were eluted as single peaks on HPLC, showing that they are homogenous, and their molecular weights were estimated to be 32000 and 22500Dal, respectively. The activity-evaluation in vitro on the six polysaccharides were carried out , showing that all of them promoted the splenocyte proliferation in LACA mice, among which Cop-4b , obtained from CTAB-Ppt , exhibited the strongest activity .Two polysaccharides,named SI and S2,were isolated from the root of Dioscorea opposita. HPLC analysis showed that they were homogenous,and their molecular weights were 63000 and 7400Dal respectively. TLC analysis on their acidic hydrolysates exhibited that they were consisted of glucose. On the basis of I3C-NMR and 'H-NMR, their glycosyl linkages were elucidated to be 1—4 and the anomeric carbons be a configuration.They were all glucans with such type as [ a -Glc ( 1 -*4 ) -]nCA4-3,an active polysaccharide component from Liuwei Dihuang Decoction, was subjected to hydrolysis with 2M TFA and pectinase respectively. Nine hydrolyzed fragments were isolated from their hydrolysates.The molecular weight and structural characteristic of the major fragment were determined on the basis of HPLC analysis, 13C-NMR and 'H-NMR.The activity-evaluation in vitro on mice-splenocyte proliferation were carried out on the fragments.The results showed that the fragments,whose molecular weights were large,had better activity than that of the original active polysaccharide, suggesting that the activity of acidic polysaccharides be stronger as the increase of proportion of their uronic acids and relate to their molecular weights as well...