| In recent years, the researches confirmed that the molecular modification of polysaccharide can enhance its bioactivity and produce new pharmaceutical value. In order to raise the bioactivity of polysaccharide, appropriate molecular modification can be applied. Siberian solomonseal rhizome polysaccharide (SRPS) and Achyranthes root polysaccharides (ARPS) are the main effective ingredient of Siberian solomonseal rhizome and Achyranthes bidentata, respectively, and have many biological activities such as immune-enhancing, anti-virus, anti-tumor, anti-stress, anti-oxidant and so on. In order to track their active site and the possibility of raising their activities by sulfated modification in antiviral and immune-enhancing aspect. SRPS and ARPS were selected as the research object. Two kinds of total and fractional SRPS and ARPS were extracted respectively by water-decocting and one-step or stepwise ethanol precipitation method. Their antiviral and immune-enhancing activities in vitro were compared. The polysaccharides active sites were choice out and further modified by sulfated method. The antiviral and immune-enhancing activities of sulfated SRPS (sSRPS) and ARPS (sARPS) were compared. The experiments included following nine sections:Experiment 1. Extraction of SRPS and ARPS The total SRPS (SRPSt) and four fractional SRPS (SRPS30, SRPS50, SRPS70 and SRPS80), total ARPS (ARPSt) and four fractional ARPS (ARPS30, ARPS50, ARPS60 and ARPS70) were extracted respectively by water-decocting and one-step or stepwise ethanol precipitation method, their carbohydrate and protein content wer determined by phenol-sulfuric and coomassie brilliant blue G-250 methods. The results showed that the extraction rate of SRPSt was highest reaching 13.84%, of SRPS50 was highest (4.88%) and of SRPS30 was lowest (0.38%) among fractional SRPSs. The carbohydrate content of SRPS50 was highest (77.43%). The protein content of SRPS30 was highest (4.8%), of SRPS50, lowest (0.83%). The extraction rate of ARPSt was highest (2.48%). ARPS70 had highest carbohydrate content (32.89%), ARPS30, lowest (10.55%). The protein content of ARPS70 was highest (1.84%), ARPS, and ARPS40, lowest (microcontent).Experiment II. Effect of SRPS and ARPS on cellular infectivity of NDV in vitro In order to screen out the antiviral active site of SRPS and ARPS, firstly the safe concentrations of five SRPSs and five ARPSs on Chicken embryo fibroblast (CEF) were compared by MTT method. Then ten polysaccharides at five concentrations within safe concentration and Newcastle disease virus (NDV) were added into the cultivating system of CEF respectively in three models, pre-, post-adding polysaccharide and simultaneous adding polysaccharides and NDV after mixed. The cellular infectivity of NDV were determined by MTT method. These results showed that during pre-adding polysaccharide, SRPS50, SRPS70, SRPSgo, SRPSt, SRPS30 and SRPS70 at 1.94-31.25 μg·mL-1, SRPS50 at 3.92-31.25 μg·mL-1, ARPS70 and ARPSt at 19.53-156.25 ng’ml"1 significantly inhibited NDV to infect CEF. During post-adding polysaccharide, SRPS30 and SRPS70 at 1.94-31.25 μg·mL-1, SRPS50 at 3.92-31.25 μg·mL-1, SPSP80 at 3.92-7.84 μg·mL-1, SRPSt at 31.25 μg·mL-1, ARPS30 at 19.53-39.06 μg·mL-1, ARPS60 at 19.53 μg·mL-1, ARPSt at 39.06 μg·mL-1 significantly inhibited NDV to infect CEF. During simultaneous adding polysaccharides and NDV after mixed, SRPS30, SRPS50, SRPS70 and SRPS80 at 1.94-31.25 μg·mL-1 significantly inhibited NDV to infect CEF. In general evaluation, SRPS50, SRPS70, SRPSt and ARPS, had better action and maybe was the antiviral active site of SRPS and ARPS.Experiment Ⅲ. Effect of SRPS and ARPS on chicken peripheral lymphocyte proliferation in vitro In order to screen out the immune-enhancing active sites of of SRPS and ARPS. The ten polysaccharides at five concentrations within safe concentration were added into the cultivating system of chicken peripheral lymphocytes singly or synergistically with PHA. The lymphocyte proliferations were measured by MTT method. The results show that in single adding SRPS, SRPS50, SRPS70 and SRPS80 at 1.953-31.25 μg·mL-1, SRPS30 at 31.25 μg·mL-1 significantly stimulated lymphocyte proliferation and the proliferation rate of SRPS70 group was highest. In synergistically adding SRPS with PHA, SRPS30 and SRPS70 at 31.25 μg·mL-1, SRPS50 at 1.953-31.25 μg·mL-1, SRPS80 at 1.953 μg·mL-1 significantly stimulated lymphocyte proliferation and the proliferation rate of SRPS50 group was highest, in single adding ARPS, ARPS30 and ARPS70 at 19.531-312.5 μg·mL-1, ARPSt at 78.12-312.5 μg·mL-1 significantly stimulated lymphocyte proliferation and the proliferation rate of ARPSt was highest. In synergistically adding ARPS with PHA, ARPS50 at 312.5 μg·mL-1, ARPS60 at 19.531 μg·mL-1, ARPS70 at 19.531-312.5 μg·mL-1, ARPSt at 39.0625-312.5 μg·mL-1 significantly stimulated lymphocyte proliferation and the proliferation rate of ARPSt was highest. In general evaluation, SRPS50, SRPS70 and ARPSt presented better action and maybe was the immune-enhance active site of SRPS and ARPS.Experiment IV. Purification and sulfated modification condition optimization of SRPS and ARPS SRPSt and ARPS, were purified respectively by Trichloroacetic-acid method to remove protein, H2O2 method removed pigment and chromatography through DEAE-Sepharose A-50 or Sephadex G-100 column in turn. The sulfated modification conditions of chlrulfonic-pyridine method, reagent formula, reaction temperature and reaction time, were optimized by orthogonal design taking the yield, degree of substitution (DS) and carbohydrate content as index. The DS and structure of sulfated polysaccharide (sPS) were determined respectively by Barium chloride-gelatin method infrared spectra. The results showed that SRPS appeared one polysaccharide peak through DEAE-Sephadex A-50 column and the yield was 10%. ARPS appeared two polysaccharide peaks through Sephadex G-100 column, the second peak with higher content and yield was 30%. The effect of reaction temperature on DS was greatest and of reagent formula on yield and carbohydrate content were greater. The optimum modification conditions for SRPS were reaction temperature of 70℃, the reaction time of 2 h and the ratio of chlrosulfonic acid to pyridine of 1:4. The optimum modification conditions for ARPS were reaction temperature of 85℃, the reaction time of 1 h and the ratio of chlrosulfonic acid to pyridine of 1:4. The infrared spectra of sPS presented two characteristic absorptive peak which proved that the sulfated group had linked with polysaccharide to form sulfate ester.Experiment V. Effect of two kinds of sPSs on cellular infectivity of NDV in vitro In order to compare the antiviral effect of the two kinds of sPSs,4 purified polysaccharides (SRPS50p, SRPS70p, SRPStp and ARPStp) and 4 sPSs (sSRPS50, sSRPS7o, sSRPSt and sARPSt) were obtained according to test IV. Firstly the safe concentrations of eight polysaccharides and two rude polysaccharides (SRPStc and ARPStc) to CEF were determined by MTT method. Then ten polysaccharides at five concentrations within safe concentration were added into the cultivating system of CEF in three models (pre-, post-adding polysaccharides and simultaneous adding polysaccharides and NDV after mixed), the cellular infectivity of NDV were observed by MTT assay. The results showed that sSRPS50, sSRPS70, sARPSt and sSRPSt at 0.9775 μg·mL-1 and other polysaccharides at 3.91 μg·mL-1 significantly inhabited NDV to infect CEF in pre-adding polysaccharides pattern. SSRPS50 and sSRPS70 at 0.2444-0.9775 μg·mL-1, ARPStp, SRPS50p and SRPS70p at 0.2444-1.955 μg·mL-1, sARPSt at 0.2444 μg·mL-1 significantly inhabited NDV to infect CEF in post-adding polysaccharides pattern. The polysaccharides except ARPStc significantly inhabited NDV to infect CEF in simultaneous adding polysaccharides and NDV after mixed pattern. In general comparison, SRPS50p and sARPSt possessed better action, which indicated that sulfated modification could increase antivirus activity of ARPS.Experiment Ⅵ. The effects of two kinds of sPSs on lymphocyte proliferation in vitro In order to compare the immune-enhancing action of the two kinds of sPSs, ten polysaccharides above-mentioned at five concentrations within safe concentration were added into the cultivating system of chicken peripheral lymphocytes singly or synergistically with PHA, the lymphocyte proliferation measured by MTT method. The results show that in single adding, sSRPS50, sSRPS70 and sARPSt at 0.2444-3.91 μg·mL-1, sSRPSt at 0.2444-0.9775 μg·mL-1, SRPS70P at 0.2444-1.955 μg·mL-1, ARPStp and SRPStp at 0.4887-1.955 μg·mL-1, SRPS50p at 0.4887-0.9775 μg·mL-1 and ARPStc at 0.2444 μg·mL-1 stimulated lymphocyte proliferation significantly. The proliferation rate in sARPSt group was highest (91.7%), the next were sSRPS50 (75.59%) and sSRPS70 (68.3%). In synergistically adding, sSRPS50 at 0.9775-1.955 μg·mL-1, sARPSt at 0.2444-3.91 μg·mL-1, SRPS70p at 0.2444 μg·mL-1, SRPS50p at 0.4887-0.9775 μg·mL-1 and ARPStp at 0.2444-0.4887 μg·mL-1 stimulated lymphocyte proliferation significantly synergistically with PHA. The proliferation rate in sARPSt group was highest and the following were sSRPS50 and sSRPS70. In comprehensive comparison, sSRPS50, sSRPS70 and sARPSt presented stronger action, which indicated that the sulfated modification could raise the immune-enhancing activity of SRPS and ARPS.Experiment Ⅶ. Effect of two kinds of sPSs on immune response of ND vaccine in chickens Ⅰ In order to compare the immune-enhancing action in vivo of the two kinds of sPSs,360 14-day-old chickens were randomly divided into 12 groups and vaccinated with Newcastle disease vaccine except blank control (BC) group, repeated vaccination at 28-day-old. At the same time of the first vaccination, the chickens in 10 polysaccharides groups were injected respectively with corresponding polysaccharides, in non-adjuvant control (NA) and BC group, physiological saline, once a day for three successive days. On days 7,14,21 and 28 after first vaccination, the blood samples were collected randomly from each group for determination of lymphocyte proliferation by MTT method and serum antibody titer by β-micromethod. The results show that the lymphocyte proliferation in sSRPS70, sSRPSt, sARPSt and SRPS50p group at D7, sSRPS50, sSRPS70, sARPSt, SRPS70p, SRPS5op, SRPStp and SRPStc group at D14, sSRPS50, sARPSt, SRPS70p, SRPStp, SRPS50P group at D21, sARPSt and SRPS50p group at D28 were significantly higher than those in NA group, the average proliferation rate of 4 time points in SRPS50p group was highest (86.63%), the following was sARPSt group (62.2%), in two groups was significantly higher than that of NA group. The antibody titers at all time points after immunization in all polysaccharide group were higher and at D14 and D21 were significantly higher than those in NA group. In general comparison, the action of sARPSt and SRPS50p were stronger in improving both cellular and humoral immunity, which indicated that sulfated modification could significantly raise the immune-enhancing activity of ARPS.Experiment Ⅷ. Curative comparison of two kinds of sPSs for ND in chickens In order to compare the antiviral effect in vivo of the two kinds of sPSs, 420 27-day-old chickens were divided randomly into 12 groups and challenged with NDV except for blank control (BC) group. After 6 hours of challenge, the chickens in ten polysaccharide groups were injected with corresponding polysaccharide, once a day for three successive days, in challenge control (CC) and BC groups, physiological saline. The clinical symptoms and death were observed daily, the died chickens were autopsied, and on day 1 before challenge and days 3,7 and 14 after challenge, the blood samples were collected for determination of serum antibody titer. The results showed in all polysaccharide groups the mortalities were lower and total effective rate were higher as compared with CC group, and in sARPSt and SSRPS50 group presented significant differences compared with CC groups. The antibody titers in all polysaccharide groups at all time points after challenge were higher than those of the CC groups, in sARPSt and sSRPS50 group were significantly higher than those in CC groups. The results indicated that all polysaccharides possessed determinate curative effect on artificially infected ND, and the efficacies of sARPSt and sSRPS50 were most significant.Experiment IX. Effect of two kinds of sPSs on expression of NDV NP gene mRNA infected CEF In order to verified the antiviral action and mechanism of two kinds of sPSs, the effects of sSRPSso, sARPSt and SRPSsop on expression of NP gene mRNA of NDV infected CEF were determined. The chick embryo fibroblasts (CEF) were cultivated, the MEMs were replaced with 2 mL of NDV when CEF grew into monolayer, and the virus solutions were replaced with 2 mL of sSRPS50, sARPSt and SRPS50p at two concentrations after incubation for 30 min at 4℃, respectively. after incubation for 72 h, the cells were collected and total RNA was extracted. According to Primer Premier software, a pair of special RT-PCR primers of NDV NP gene were designed to amplify NP gene protein fragment of 176 bp, and the relative expression quantities of NDV NP gene mRNA were determined by fluorescence quantitative real time RT-PCR assay. The results showed that the relative expression quantities of NDV NP gene mRNA in all sulfated polysaccharide group were significantly lower than that in virus control group, in high dose of sARPSt group was lowest and following was high dose of sSRPSso. These results indicated that three polysaccharides could significantly inhibit NDV proliferation in CEF and the action of sARPSt was strongest. |
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