The Antiviral And Immune-Enhancing Activities Comparison Of Common Yam Rhizome Polysaccharides And Their Sulfated Derivants

Some researches confirmed that original biological activity of natural polysaccharide could be enhanced and probably new biological activity be obtained by sulfated modification. More sulfated polysaccharides can be obtained by construction reform to enhance the biological activity of Chinese herbal polysaccharides. Common Yam Rhizome Polysaccharide (CYRP) are important bioactive substances of Common Yam Rhizome, and have many biological activities such as enhancing immunity, antivirus, anti-tumor, anti-aging, antioxidant, hypoglycemic and so on. In order to track the optimal active site of enhancing immunity and antivirus of CYRP, and investigate the possibility of sulfated modification to improve the antiviral and immune-enhancing activitiesof CYRP, CYRPs and CYRPt were extracted fractionally by stepwise and one-step alcohol precipitation, and the best active sites were selected by determination of their antiviral and immune-enhancing activity in vitro, then further sulfated modification was performed to compare antiviral and immune-enhancing activities of polysaccharides pre-modified with post-modified. The tests are divided into six sections as follows:Experiment1Extraction of Common Yam Rhizome polysaccharide The total (CYRPt) and five fractional polysaccharides CYRPs, CYRP40, CYRP550, CYRP560, CYRP70and CYRPgo were extracted respectively by one-step and stepwise alcohol precipitation after water decoction, and their polysaccharides contents were determined by phenol-sulfuric acid method. The results showed that the extraction rate of CYRP, was the highest up to0.8%. The extraction rate of CYRP40was the highest (0.13%) in five fraction polysaccharides; and the polysaccharides content of CYRP40was the highest up to72.7%.Experiment2The selection of antiviral activity site of CYRP in vitro In order to selected the strongest antiviral active site of Common Yam Rhizome polysaccharides (CYRP), total CYRP (CYRPt) and5fractional polysaccharides (CYRP40, CYRP550, CYRP56o, CYRP70and CYRP80) were assayed by MTT method to determine the maximum safe concentrations of CYRP to chick embryo fibroblast. Then6polysaccharides within each safe concentration range were added with NDV by three means of pre-adding, post-adding CYRP and simultaneously adding CYRP mixed with virus, to survey the infectivity of.NDV to cells. The results showed that CYRP40, CYRP80and CYRPt could significantly inhibit the infectivity of NDV to CEF (P＜0.05); CYRP70, CYRP80and CYRPt could significantly inhibit the infectivity of NDV to CEF when per-added (P<0.05), and the virus inhibition rate of CYRPt was the highest,79.618%; CYRP60, CYRP70, CYRP80and CYRPt all could significantly inhibit the infectivity of NDV to CEF when added with NDV simultaneously, the virus inhibition rate of CYRPt was the highest,up to181.6%(P<0.05). Therefore, CYRPt was the strongest antiviral active site of CYRP.Experiment3The selection of immune-enhancing active site of CYRP in vitro CYRPt and5fractional polysaccharides (CYRP40, CYRP50, CYRP60, CYRP70, CYRP8o) at five concentrations within safety concentration ranges, alone or simultaneously with PHA respectively, were added into cultural chicken peripheral blood lymphocytes, to measure the lymphocyte proliferation by MTT method. The results showed that, CYRP40, CYRP50, CYRPgo and CYRPt could significantly stimulate lymphocyte proliferation when added alone; these six polysaccharides could all significantly stimulate lymphocyte proliferation in coordination with PHA. The lymphocytes proliferation rates of CYRPt were both the largest among all the CYRP no matter added alone or mixed with PHA, indicating that CYRPt had the best effect on enhancing immune and was the optimal immune-enhancing active site of CYRP.Experiment4The sulfated modification of CYRP Purified CYRPt was sulfated by chlorosulfonic acid-pyridine method. L9(34) orthogonal test was designed with reaction temperature, time and reagent ratio as three factors, yield and Sulphuric acidyl substitution degree (DS) as index. DS was determinated by barium chloride-gelatin method. The result showed that when the modification was performed at80℃, with ratio of chlorosulfonic acid to pyridine at1:4for3hours, the yield of sulfated CYRP was the highest up to753.5mg. The influence rank of each factor on yield was as follows:ratio of chlorosulfonic acid to pyridine, reaction time, temperature; When the modification was conducted at70℃, with ratio of chlorosulfonic acid to pyridine at1:6for3hours, the substitution degree was the highest up to1.237. The influence rank of each factor on DS was as follows:ratio of chlorosulfonic acid to pyridine, reaction time, temperature. All above_indicated that reagent ratio had the greatest effects on both yield and DS, while reaction temperature had the minimum effects.Experiment5The comparison of antiviral activity of sulfated CYRP in vitro Nine sCYRP (obtained by sulfation in test4) were assayed by MTT method to determine the maximum safe concentration of sCYRP to chick embryo fibroblast. Then9sCYRP and CYRPt at five concentrations within safety concentration ranges were added with NDV by three means of pre-adding, post-adding CYRP and simultaneously adding CYRP mixed with virus, to survey the infectivity of NDV to cells. The results showed that sCYRP1, sCYRP2, sCYRP3, sCYRP4, sCYRP5, sCYRP6, sCYRPy and CYRPt could significantly inhibit the infectivity of NDV to CEF when per-added (P<0.05), and the virus inhibition rate of sCYRP3at3.906μg·mL-1was the highest among all the CYRP; these ten CYRP could all significantly inhibit the infectivity of NDV to CEF when post-added (P<0.05), and the virus inhibition rate of sCYRP8at0.488μg.mL-1was the highest; sCYRP1, sCYRP2sCYRP3, sCYRP5, sCYRP6, sCYRP7, sCYRP8, sCYRP9and CYRPt could significantly inhibit the infectivity of NDV to CEF when added with NDV (P＜0.05), and the virus inhibition rate of sCYRP5at1.953μg·mL-1was the highest. Compared synthetically, sCYRP1was the strongest antiviral activity part of all the CYRP under the modification conditions of reaction temperature:60℃, ratio of chlorosulfonic acid to pyridine:1:4, and reaction time:1h; proving that sulfated modification could enhance the antiviral activity of CYRP.Experiment6The comparison of immune-enhancing activity of sulfated CYRP in vitro Nine sCYRP and CYRPt at five concentrations within safety concentration ranges, alone or simultaneously with PHA respectively, were added into cultural chicken peripheral blood lymphocytes, to measure the lymphocyte proliferation by MTT method. The results showed that, the A570values of sCYRP,, sCYRP3, sCYRP4, sCYRP5, sCYRP6, sCYRP8and CYRPt were significantly larger than cell control group when added alone; the A570values of sCYRP1, sCYRP3, sCYRP4, sCYRP5, sCYRP6, sCYRP7and sCYRP9were larger than PHA control group when added with PHA, and the A570value of CYRPt was significantly larger than PHA control group. The lymphocytes proliferation rates of sCYRP6were both the largest among all the CYRP no matter added alone or mixed with PHA, illustrating that sCYRP6had the best immune-enhancing effect under the modification conditions of reaction temperature:70℃, ratio of chlorosulfonic acid to pyridine:1:8, and reaction time: lh. Therefore, sulfated modification could enhance the immune-enhancing activity of CYRP.