Preparation, Purification And Characterization Of Anti-complementary Polysaccharides From Houttuynia Cordata Thunb

The complement system can effectively translated identification of pathogens to defend function of the initial infection for host. The three major results of complement activation included:opsonization effect on:pathogen, recruitment of inflammatory cells and the formation of membrane attack complex (MAC) to kill pathogens directly. However, the excessive activation of complement may cause abnormal reaction of the immune system, resulting in damage to normal tissue and the pathological process involved in many diseases. There is no ideal treatment in clinic, and effective, and low toxicity complement inhibitor with specificity is thus needed very much. Some natural complement inhibitors have been isolated from traditional Chinese medicines, and most of them can be directly digested and absorbed by human body. This research is mainly focused on anti-complementary polysaccharides obtained from Houttuynia cordata Thunb.. Three homogeneous polysaccharides HC-PS1、HC-PS2and HC-PS3were isolated from the herb of Houttuynia cordata Thunb.. Their anti-complementary activities and preliminary mechanism of anti-complementary effect were determined. The optimized procedures for preparation of crude polysaccharides from Houttuynia cordata Thunb. were established. The methods to determin and eliminate LPS from crude polysaccharides were also investigated. A series of derivatives of three homogeneous polysaccharides were obtained and their anti-complementary activities and preliminary mechanism of anti-complementary effect were determined comparatively.1. Comfirmation of anticomplementary fraction of H. cordataThe anti-complementary activities of11commercial crude drugs were determined. Both ethanol extract and hot water extract exhibited anti-complementary activity. H. cordata from Sichuan (CH50value of hot water extract:0.136±0.033mg/ml) were selected as the research object. The crude polysaccharide, which was purified by removing the free protein and small molecules, showed strong anti-complementary activity with the CH50and AP50values of0.089±0.008mg/ml and 0.405±0.023mg/ml. Thus, the crude polysaccharide was one of the anti-complementary components of H. cordata.2. Preparation procedure of crude polysaccharides from H. cordataFour kinds of extraction methods were compared and the extraction conditions were optimized by orthogonal experiment. Using the same markers, the conditions of alcohol precipitating process were also optimized by orthogonal test. Three kinds of deproteinization methods were compared and the optimal condition was determined according to the result of protein removed and polysaccharide maintained. The effects of three decoloration methods were compared in terms of pigment removing and polysacchardide losing, and the best decoloring method was optimized by orthogonal experimental design. The optimized process to prepare the anti-complementary polysaccharides in H. cordata was given as follows:extract the coarse powder3times with50times volume of water at90℃,2hours every time, combine the extracts and concentrate to form a thick extract correspond to0.12g of H. cordata per ml. Add4times volume of90%ethanol to the extract, allow to stand for24hours to precipitate totally, filter, evaporate the ethanol in the precipitate. Resolve the residue with water, add TCA to a concentration of20%to remove protein.Activated carbon added to a concentration of3%, pH at3.0, temperature at50℃and decoloration lasting50min. The established and optimized procedures are repeatable and reliable to prepare the anti-complementary polysaccharides with high quality and activity from H. cordata.3. Determination and elimination of LPS in crude polysaccharides of H. cordataAn LAL-based method for determination of LPS in crude polysaccharides was established. The content of LPS in crude polysaccharides of H. cordata was:58.09±1.67ng/mg. The crude polysaccharides contained very low contents of LPS (ng level).The LPS in H. cordata was eliminated by column chromatography technology. The coupling technique of hydrophobic interaction chromatography and affinity chromatography showed better elimination effect on LPS. The clearance rates of LPS in H. cordata crude polysaccharides was42.58%, and its CH50values were0.080±0.014mg/ml. Result indicated that the LPS elimination method was workable and would not weaken the anticomplementary effects of crude polysaccharides.4. Purification and characterization of homogeneous anti-complememntary polysaccharides from H. cordata Bioactivity-guided fractionation of a hot-water extract from H. cordata led to the isolation of two polysaccharides HC-PS1、HC-PS2and HC-PS3. Determination results of high performance gel permeation chromatography (HPGPC) and high performance capillary electrophoresis (HPCE) showed that HC-PS1、HC-PS2and HC-PS3were homogeneous polysaccharides. Using NMR, GC, GC-MS, IR and methylation, the structure features of HC-PS1、HC-PS2and HC-PS3were identified.HC-PS1,which was identified as a branched polysaccharide with average molecular weight about274530Da, composed of Rha, Ara, Man, Glc, GlcA, Gal, GalA in the ratio of1.0:3.0:3.9:2.5:2.4:6.1:3.0, along with trace of Xyl,24.08%of uronic acid and only1.41%of protein. The main linkages of the residues of HC-PS1include terminal,1,5-linked Araf;1,3,6-linked and1,4,6-linked Manp; terminal,1,4-linked,1,3-linked,1,3,6-linked and1,4,6-linked Glcp; terminal,1,4-linked and1,6-linked Galp;1,4-linked GalpA.HC-PS2, which was identified as a branched polysaccharide with average molecular weight about7928Da, composed of Man, Glc, GlcA, Gal, GalA in the ratio of1.0:0.3:1.7:0.7:4.6, along with a trace of Rha, Ara and Xyl,65.64%of uronic acid and only1.89%of protein. The main linkages of the residues of HC-PS2include terminal linked Araf; terminal,1,6-linked and1,4,6-linked Manp;1,4-linked,1,3-linked and1,6-linked Glcp;1,4-linked GlcpA; terminal,1,4-linked,1,3,4-linked,1,4,6-linked and1,3,6-linked Galp; terminal,1,4-linked and1,3,4-linked GalpA.HC-PS3, which was identified as a branched polysaccharide with average molecular weight about216384Da, composed of Man, Glc, GlcA, Gal, GalA in the ratio of1.0:0.5:0.4:1.2:0.4, along with trace of Rha, Ara and Xyl,20.19%of uronic acid and only1.53%of protein. The main linkages of the residues of HC-PS3include terminal and1,5-linked Ara/;1,3,4-linked,1,4,6-linked and1,3,6-linked Manp; terminal,1,4-linked,1,3-linked and1,4,6-linked Glcp; terminal,1,4-linked and1,6-linked Galp.Human sera was used as complement source to evaluate anti-complementary activities of HC-PS1、HC-PS2and HC-PS3. Results showed that HC-PS1、HC-PS2and HC-PS3not only inhibited the complement from guinea pig sera, but also anta-gonized the complement from human sera. The CH50values of HC-PS1%HC-PS2and HC-PS3were0.295±0.032mg/ml、0.183±0.017mg/ml and0.271±0.026mg/ml, respectively. The AP50values of HC-PS1、HC-PS2and HC-PS3were0.318±0.023mg/ml、0.219±0.018mg/ml and0.314±0.025mg/ml, respectively. The CH50and AP50 values of heparin (the positive control drug) were0.158±0.019mg/ml and0.205±0.021mg/ml, respectively.The targets of HC-PS1、HC-PS2and HC-PS3and the crude polysaccharide of H. cordata in the complement activation cascade were identified by complement components depleted sera. Results showed that HC-PS1selectively interacted with C2、 C4and C5, HC-PS2selectively interacted with Clq、C2、C4、C5and C9, HC-PS3selectively interacted with C2、4and C5.Due to the anticoagulant effect, heparin played a rather limited role for complement inhibition in vivo. In this research, effects of HC-PS1、HC-PS2and HC-PS3on the coagulation system were also investigated with the thrombin reagent. HC-PS1、HC-PS2and HC-PS3showed potent anti-complementary activities without anticoagulant properties, which indicated that they will have good prospects.Detection of antioxidative capacity of H. cordata homogeneous polysaccharides by scavenging organic free radical DPPH, the determining IC50values were as follows:0.89mg/mL,0.95mg/mL,0.94mg/mL.5. Anti-complementary activities of H. cordata homogeneous polysaccharides derivativesUsing homogeneous polysaccharides of H. cordata as the substrates, the sulfation, hydroxyethylation and carboxymethylation products were obtained. The IR spectra of sulfation products indicated a narrower O-H stretching vibration peak at about3400cm-compared with the crude polysaccharide, and the818cm-1and1232cm-1stretching vibration peaks were attributed to the C-S-0and S=O bonds. The IR spectra of hydroethylation products showed a wider O-H stretching vibration peak from3200cm-1to3600cm-1, and a stronger C-H stretching vibration at about2900cm-compared with the crude polysaccharide, proving that the hydroxyethyl had replaced some of the hydroxy on sugar ring. The IR spectra of carboxymethylation products showed the characteristic signal of carboxylic group at1736cm-1.The CH50values of sulfation, hydroxyethylation and carboxymethylation products of HC-PS1were:0.128±0.024mg/ml,0.243±0.044mg/ml and0.241±0.038mg/ml, respectively. The AP50values were0.084±0.012mg/ml,0.186±0.013mg/ml and0.085±0.006mg/ml, respectively. The CH50values of sulfation, hydroxyethylation and carboxymethylation products of HC-PS2were:0.091±0.020mg/ml,0.248±0.034mg/ml and0.202±0.022mg/ml respectively. The AP50values were0.068±0.008mg/ml,0.118±0.011mg/ml and0.075±0.008mg/ml, respectively. The CH50values of sulfation, hydroxyethylation and carboxymethylation products of HC-PS3were:0.137±0.029mg/ml,0.299±0.040mg/ml and0.260±0.014mg/ml, respectively. The AP50values were0.080±0.007mg/ml,0.213±0.010mg/ml and0.073±0.002mg/ml, respectively.Results showed that sulfation product of HC-PS1was selectively interacted with C2、C3、C4、C5and C9, hydroxyethylation product of HC-PS1was selectively interacted with C2、C3、C4、C5and C9, carboxymethylation product of HC-PS1was selectively interacted with C2、C3、C4、C5and C9; sulfation product of HC-PS2was selectively interacted with Clq、C2、C3、C4、C5and C9, hydroxyethylation product of HC-PS2was selectively interacted with C2、C3、C4、C5and C9, carboxymethylation product of HC-PS2was selectively interacted with Clq、C2、C3、 C4、C5and C9; sulfation product of HC-PS3was selectively interacted with C2、C3、 C4、C5and C9, hydroxyethylation product of HC-PS3was selectively interacted with C2、C3、C4、C5and C9, carboxymethylation product of HC-PS3was selectively interacted with Clq、C2、C3、C4、C5and C9;Results showed that the derivative products possessed themselves of stronger anti-complementary activities than crude polysaccharides. The targets in complement activation cascade of the derivative products were different from the original polysaccharides, and they also interacted with the key components of complement system.