Preparation, Purification And Characterization Of Anti-complementary Polysaccharides From Bupleurum

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 efficient, low toxicity complement inhibitor with specificity is needed very much. China has abundant resources of traditional drugs; from which natural, low cost and non-toxicity complement inhibitors are isolated, and most of them can be directly digested and absorbed by human body. This research mainly focused on anti-complementary polysaccharides isolated from Bupleurum. Two homogeneous polysaccharides BC-PS1and BC-PS2were isolated from the roots of B. chinense DC. Their anti-complementary activities and preliminary mechanism of anti-complementary effect were determined. The optimized preparation procedures of crude polysaccharides from B. smithii Wolff var. parvifolium Shan et Y. Li. were demonstrated. The methods to determin and eliminate LPS from crude polysaccharides were also established. And for the first time, we evaluated the quality of crude drugs from Bupleurum with the function of clearing heat and detoxicating, by comparing their anti-complementary activities. The main results of this research were as follows:1. Comfirmation of anticomplementary constituents of B. chinenseThe anti-complementary activities of10commercial crude drugs of radix bupleuri were determined. Ethanol extracts showed none anti-complementary activity, while hot water extracts indicated varying degrees of anti-complementary activities. Bupleurum chinense from Chengde (CP50value of hot water extract:0.236±0.03mg/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 activities with the CP50and AP50values of0.135±0.03mg/ml and0.418±0.02mg/ml. Thus the crude polysaccharide was the anti-complementary component of B. chinense.2. Preparation procedure of crude polysaccharides from BupleurumTook B. smithii var. parvifolium for example, orthogonal design experiments were established to investigate process parameters for hot water extraction and alcohol precipitation of polysaccharides. And three deproteinization methods, including Sevage method, Trichloroacetic Acid Method and Papain Method, were evaluated. The optimal process was as follows:the dried roots were ground into fine particles and defatted using95%EtOH, then the residues were dried in the shade. The residues were soaked with16-time water for1.5hours, decocting2times and2hours every time. The hot water extraction was concentrated to the equivalent of0.12g crude drug per ml, then ethanol was added until the concentration of80%. After standing overnight, the supernatant was discarded after centrifugation, and the precipitation was dissolved with water. The water solution was added with trichloroacetic acid until the concentration of7%(volume percentage) at4℃, and then kept in refrigerator for2hours at4℃. The solution was taken out and added with10%NaOH until the PH value of7.0, then centrifuged, and dialyzed against running water for three days. The supernatant was concentrated and lyophilized to produce the crude polysaccharide. Repeated experiments showed that the yield of crude polysaccharides was about5%. The process was stable with higher yield of polysaccharides.3. Determination and elimination of LPS in crude polysaccharidesAn LAL-based method for determination of LPS in crude polysaccharides was established. The contents of LPS in crude polysaccharides of Arnebia euchroma, B. smithii var. parvifolium, B. chinense and Houttuynia cordata were:29.58±3.27ng/mg,28.39±0.73ng/mg,66.77±1.90ng/mg and59.33±1.79ng/mg. All of the crude polysaccharides contained very low contents of LPS (ng level).The LPS in A. euchroma and B. smithii var. parvifolium were 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 A. euchroma and B. smithii var. parvifolium crude polysaccharides were44.51%and39.77%, and their CP50values were0.096±0.013mg/ml and0.288±0.037mg/ml. Results 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 B. chinense and study on their preliminary mechanism of anti-complementary effectBioactivity-guided fractionation of a hot-water extract from the roots of B. chinense DC. led to the isolation of two polysaccharides BC-PS1and BC-PS2. Determination results of high performance gel permeation chromatography (HPGPC) and high performance capillary electrophoresis (HPCE) showed that BC-PS1and BC-PS2were homogeneous polysaccharides. Using NMR, GC, GC-MS, IR and methylation, the structure features of BC-PS1and BC-PS2were identified. BC-PS1, which was identified as a branched polysaccharide with average molecular weight about2,000KDa, composed of Gal, GalA, Glc, Ara and Man in the ratio of1.6:1.1:1.8:1.7:1.0, along with trace of Rha, Fuc and Xyl,36.95%of uronic acid and only1.21%of protein. The main linkages of the residues of BC-PS1include terminal,1,4-linked and1,3-linked Galp, terminal,1,4-linked,1,6-linked, and1,4,6-linked Glcp, terminal and1,5-linked Araf, terminal,1,6-linked, and1,4,6-linked Manp. BC-PS2, which was identified as a higher branched polysaccharide with average molecular weight about1,600KDa, composed of Glc, Ara, Gal and Man in the ratio of3.5:2.4:2.0:1.0, along with trace of Rha and Xyl, and only1.11%of protein. The main linkages of the residues of BC-PS2include terminal,1,6-linked,1,3-linked and1,3,6-linked Glcp, terminal and1,5-linked Araf, terminal,1,4-linked,1,6-linked, and1,4,6-linked Galp, terminal,1,4-linked, and1,4,6-linked Manp. Human serum was used as complement source to evaluate anti-complementary activities of BC-PS1and BC-PS2. Results showed that BC-PS1and BC-PS2not only inhibited the complement from guinea pig serum, but also antagonized the complement from human serum. The CP50values of BC-PS1and BC-PS2were0.234±0.025mg/ml and0.264±0.013mg/ml, respectively. The AP50values of BC-PS1and BC-PS2were0.369±0.017mg/ml and0.362±0.032mg/ml, respectively. The CP50and AP50values of heparin (the positive control drug) were0.226±0.014mg/ml and0.329±0.026mg/ml, respectively.The targets of BC-PS1, BC-PS2and the crude polysaccharide of Bupleurum chinense in the complement activation cascade were identified by self-made complement components depleted sera. Results showed that BC-PS1selectively interacting with C1q, C2, C5and C9, BC-PS2selectively interacting with C1q, C2and C9, and the crude polysaccharide interacted with all components, which suggested that BC-PS1and BC-PS2showed more selectivity on the complement system, and both of them inhibited the key components of complement activation cascade.Due to the anticoagulant effect, heparin played a rather limited role for complement inhibition in vivo. In this research, effects of BC-PS1and BC-PS2on the coagulation system were also investigated with the thrombin reagent. BC-PS1and BC-PS2showed potent anti-complementary activities without anticoagulant properties, which indicated that they will have good prospects.5. Anti-complementary activities of Bupleurum polysaccharide derivativesUsing crude polysaccharides of B. chinense and B. smithii van parvifolium as the substrates, the sulfation, hydroethylation and carboxymethylation products were obtained. The IR spectra of sulfation products indicated a narrower OH stretching vibration peak at about3400cm-1compared with the crude polysaccharide, and the818cm"1and1232cm-1stretching vibration peaks were attributed to the C-S-O and S=O bonds. The IR spectra of hydroethylation products showed a wider OH stretching vibration peak from3200cm-1to3600cm-1, and a stronger CH stretching vibration at about2900cm-1compared 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 CP50values of sulfation, hydroethylation and carboxymethylation products of Bupleurum chinense and Bupleurum smithii var. parvifolium were:0.107±0.03mg/ml,0.145±0.02mg/ml,0.099±0.02mg/ml, and0.159±0.013mg/ml,0.327±0.022mg/ml,0.059±0.006mg/ml, respectively. The AP50values were0.102±0.04mg/ml,0.164±0.03mg/ml,0.188±0.04mg/ml, and0.22±0.03mg/ml,0.144±0.02mg/ml,0.164±0.04mg/ml, respectively. 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 crude polysaccharides, and they also interacted with the key components of complement system.6. Quality evaluation of radix bupleuri by determining anti-complementary activities of crude polysaccharides in vitroThe hemolysis assay was further optimized, and a systematic complement inhibition activity determination method was completely established for the quality evaluation of clearing heat and detoxicating Traditional Chinese Medicines. Twenty-two batches of radix bupleuri were collected, and we prepared Twenty-two batches of crude polysaccharides. The anti-complementary activities of the crude polysaccharides on both classical and alternative pathways were also determined. Results showed that crude polysaccharide from B. smithii had the strongest anti-complementary activities than the others. The limited values of anti-complementary activities were also provided according to the average CP50and AP50values of22batches of crude polysaccharides. For the drugs of radix bupleuri, the CP50and AP50values should not exceed0.426mg/ml and0.694mg/ml.