The Antiviral And Immune-Enhancing Activities Comparison Of Garlic Poly-Saccharides And Their Sulfated Derivants

In recent years, it has been discovered by medical researches that the biological activity of components of traditional Chinese medicine can be improved through molecular modification. Some researches aslo confirmed that the biological activity or new one of natural polysaccharide can be enhanced or generated by sulfated modification. Garlic polysaccharide (GPS) has many biological activities such as anti-virus, enhancing immunity, antioxidant, and so on. In order to trace the active site of GPS and to investigate the possibility of sulfated modification in improving the antiviral and immune-enhanced activity of GPS, this study firstly used column chromatography to classify GPS and surveyed their antiviral and immune-enhanced activity in vitro, then selected out more effective polysaccharides to carry out further sulfated modification, at last compare the antiviral activity and immune-enhanced of polysaccharide pre-and post-modification. The tests are divided into the following five sections:Experiment1. Extraction of Garlic polysaccharides The crude garlic polysaccharide (GPSc) was extracted from garlic by hot water extraction, and the deproteinized garlic polysaccharides (GPSp) was obtained from GPSc by Sevag deproteiniczation method. Garlic neutral polysaccharide (GPSpl) and two acidic polysaccharides (GPSp2and GPSp3) were fractionated from the GPSp by DEAE-52cellulose column chromatography. Their polysaccharide contents were determinated by phenol-sulfuric acid method and protein content were determinated by Coomassie brilliant blue method. The results showed that extraction rate of the GPSc was the highest, up to7.24%, the polysaccharide contents and protein content were68.35%and2.7%, respectively; The extraction rate and protein content of the GPSp lowered to3.31%and1.2%, polysaccharide content increased to91.2%; The extraction rate of the GPSpl, which was neutral polysaccharide, further decreased to2.2%, while polysaccharide contents was the highest, up to95.83%; The extraction rate of GPSp2and GPSp3were the lowest,0.17%and0.16%, and polysaccharide contents were86.03%and33.7%, protein content were0.85%and4.9%, respectively.Experiment2. The selection of antiviral activity site of GPS in vitro Firstly, the safe concentrations of GPSc、GPSp as well as three kinds of hierarchical polysaccharides GPSpl. GPSp2、and GPSp3were determined on chicken’s embryo fibroblast (CEF). Then, five points within the safe concentration range of each kind of GPS were selected and added) with Newcastle disease virus (NDV) into CEF in three manners:pre-, post-adding polysaccharide or simultaneous adding polysaccharides and NDV. The effect of NDV on the CEF was determined by MTT method. The results differed according to the addition manner:when the polysaccharide was added first, with GPSc at0.78～1.56μg·mL-1, GPSp at3.125～12.5μg·mL-1were able to prevent the infection of CEF remarkably (P<0.05); when the polysaccharide was added later, with GPSc at1.56ug·mL-1, GPSp at0.78～1.56μg·mL-1, GPSp1and GPSp2at0.78-3.12μg·mL-1were able to prevent the infection of CEF evidently (P<0.05); when the polysaccharide and the NDV were added simultaneously, with GPSc at0.78-3.12μg·mL-1, GPSp and GPSp at0.78～6.25μg·mL-1, and GPSps at1.56μg·mL-1were able to prevent the infection of CEF dramatically (P<0.05), furthermore, the effective concentration range of GPSp is the most and the maximal virus inhibition rate of it is higher than any other kinds. In conclusion, the effect of GPSp is the best, and it may be the optimal antiviral activity site of GPS.Experiment3. The selection of immune-enhanced activity site of GPS in vitro Five polysaccharides at five concentrations within safe concentration range were added into cultural chicken peripheral blood lymphocytes, and the lymphocyte proliferation was measured by MTT method (the value A570of cells and lymphocytes proliferation rate). The results showed that, with GPSc and GPSp at0.78～6.25μg·mL-1, GPSpl at0.78～1.56μg·mL-1, the value A570of cells are significantly higher than those of cells control group when used only (P<0.05); with GPSc at1.56～6.25μg·mL-1, GPSp at0.78～3.12μg·mL-1, GPSpl at1.56～3.12μg·mL-1, the value A570of cells is much more than those of PHA control group, in combination with PHA; The general evaluation indicated that lymphocyte proliferation rates of GPSp by two types of action were relatively higher. The results showed that garlic polysaccharide could stimulate lymphocyte proliferation at certain concentrations by used only or in combination with PHA. GPSp has the best effect, so may be the optimal immune-enhanced activity site of GPS.Experiment4. The sulfated modification conditions of GPS L9(34) orthogonal test was designed with three factors of reaction temperature, reaction time and reagent ratio. The results showed that the reagent ratio has great influence on yield, when chlorosulfonic acid and Pyridine was1:4, the yield of product was the highest; The effects of reaction temperature on polysaccharide content and DS were obvious, polysaccharide content was the highest at45℃, decreaseing with the ascension of temperature; While DS was the lowest at45℃, increasing with the ascension of temperature.Experiment5. The comparison of antiviral activity of sulfated GPS in vitro Firstly, the safe concentrations of9sGPSs were determined on chicken embryo fibroblast (CEF). Then compare GPSp with sGPSs.9sGPSs and GPSp at five concentrations within safe concentration range were added with NDV into CEF monolayers by three models, and the effect of NDV on the CEF was determined by MTT method. The results indicated that viral would be significantly inhibited by GPSp, sGPS5and sGPS6at3.125ug·mL-1during pre-adding polysaccharide (P<0.05), and the sGPS5has the highest maximal virus inhibition rate; During post-adding, all sGPSs were able to prevent the infection of CEF evidently at certain concentrations except sGPS1and sGPS2(P<0.05), sGPS5and sGPS6have the highest maximal virus inhibition rate, and higher than that of GPSp significantly (P<0.05); when simultaneous adding polysaccharides and NDV,9sGPSs were able to prevent the infection of CEF evidently at certain concentrations (P<0.05), and virus inhibition rate of sGPS5and sGPS8are significantly higher than GPSp and other sGPSs (P<0.05). These manifested that sulfated modification could enhance the antiviral activity of GPS. Especially sGPS5has the best effect which modified by chlorosulfonic acid and Pyridine was1:8, reacting for6h at the temperature of65℃.Experiment6. The comparison of immune-enhanced of sulfated GPS in vitro9sGPSs and GPSp at five concentrations within safe concentration range were added into cultural chicken peripheral blood lymphocytes, and the lymphocyte proliferation was measured by MTT method. The results showed that when used only, all sulfated GPS groups were significantly higher than cells control group at certain concentrations (P<0.05), and the lymphocyte proliferation rates of sGPS5and sGPS3were significantly higher than GPSp (P<0.05); when combine with PHA, sGPS5、sGPS6、sGPS8、sGPS9were significantly higher than PHA control group at certain concentrations (P<0.05), the lymphocyte proliferation rate of sGPS5and sGPS9were much more than other sGPSs (P<0.05), and more than GPSp (P>0.05). It was synthetically indicated that sulfated modification could enhance the antiviral activity of GPS. Especially sGPS5has the best effect which modified by chlorosulfonic acid and Pyridine was1:8, reacting for6h at the temperature of65℃.