The antiviral activities on HSV-1 and CVB3 in vitro of polysaccharide from alga eucheuma striatu Cen Yingzhou1, Khoo Gaikming1, Ye Shaoming1, Wang Yifei 2(1Department of Chemistry, Faculty of Life Science and Technology, 2Guangzhou Jinan Biomedical Research and Development Center, Jinan University, Guangzhou, Guangdong, China 510632) Received Dec.31, 2003; Supported by the National Natural Science Foundation of China (No. 20172020), Foundation of Important Technological Items of Guangdong Province, China (2002A3050503), Natural Science Foundation of Guangdong Province, China (2001-10-010401, 2003£11) Abstract To assay the antiviral
activities on HSV-1 and CVB3 in vitro of the polysaccharide from Eucheuma
striatum, and its antiviral mechanism was explored. Vero cells were infected by HSV-1
and CVB3, and they were cultured with serial dilutions of polysaccharide. The
cells cytotoxicity of polysaccharide was evaluated by the MTT method. The inhibitory
effects were evaluated by the cytopathic effect (CPE). Its antiviral mechanism was studied
by the method of giving samples in different time. The polysaccharide could inhibit the
CPE of cells infected by HSV-1 and CVB3. It showed low cytotoxicity on Vero
cells. Its antiviral activities were better than Acyclovir and ribavirin which were run in
parallel as the positive control samples. The polysaccharide from Eucheuma striatum
has potent antiviral activities. Its antiviral mechanism is that it can prevent the virus
from absorbing to the cell surface. Such research work has not ever been reported before. 1 EXPERIMENTAL 1.1 Preparation of polysaccharide The Eucheuma striatum sample was obtained from Qionghai seaweed farm, Hainan, China. Dry ground material was mixed with 10% KCl and stirred for 2h in a boiling water bath. Filtered, the filtrate was then concentrated to 1/3 of its former volume and dialysis was used to extract the Cl-. The remainder was mixed with 3 volume ethanol and left over night. After filtration, the residue was rinsed thoroughly with ethanol and acetone, lyophilized and given the crude E. striatum polysaccharide . The crude polysaccharide sample was purified by using DEAE-cellulose column chromatography and obtained the E. striatum polysaccharide (ESP), and its chemical structure was identified by IR and GC-MS. Finally , the ESP sample was diluted in distilled water to form a solution concentration of 20mg/mL, after filtration sterilization, stored in 4oC. 1.2 Cytotoxicity assays Vero cells were diluted with DMEM medium to a density of 2.5x105/mL and were added to 96-well plates 100m L/well at 37 oC . Medium was aspirated to the plates overlaid with serial dilution of ESP 100m L/well, 4 wells per dilution. Plates that overlaid with maintenance medium in absence of ESP were used as controls. After culturing at 37oC for 48h, the fluid was discarded and 5mg/mL MTT 10m L/well was added. After 4h, 10% SDS 100m L/well was added and was placed at 37oC for 8h. The optical densities were determined with ELISA reader at a test wavelength of 570nm and a reference wavelength of 630nm. Data were calculated as percentage of viability by the following formula: Cellular viability % = (ODt/ODc) x 100% ODt and ODc indicated the optical density of the test substances and the control respectively. The 50% cytotoxic concentration (CC50) and maximal non-cytotoxic concentration (MNCC) were calculated. 1.3 Antiviral Assays Parallel experiments of normal cells group, viral-infected cells group were carried out for assays below and were used as controls for comparison. ACV and Ribavirin groups were used as the positive control groups for HSV-1 and CVB3, respectively. 1.4 Effects of pre-treatment by ESP on Vero cells Vero cells monolayers were overlaid with serial dilution of ESP 50m L/well for 2h at 37oC. 100 TCID50 of virus was inoculated to the monolayers 50m L/well. The virus-induced CPE were scored every 24h under an inverted microscope (score 0, 0% CPE; score 1, 0-25% CPE; score 2, 25-50% CPE; score 3, 50-75% CPE; score 4, 75-100% CPE). When the controls appeared 75-100% CPE, CPE of each well was recorded at the same time, 50% inhibited concentration (IC50, m g/mL) and selectivity index (SI=CC50/IC50) were calculated. 1.5 Effects of ESP on virus-infected cells The Virus was inoculated separately to the Vero cells monolayer in 96-well plates 50m L/well for 1h at 37oC, and serial dilution of ESP were added, incubation was carried on. Observation method was the same as above. 1.6 Direct effects of ESP on virus The same volume of virus was mixed with serial dilution of ESP and incubated at 37oC for 1h. The mixtures were immediately inoculated onto Vero cells cultures 100m L/well. Incubation was carried on and observation method was same as above. 1.7 Viral binding assay 100 TCID50 of virus was inoculated to the Vero cells monolayers 50m L/well. Serial dilution of ESP was added at the same time 50m L/well. Incubation was carried on and observation method was the same as above. 2 RESULTS AND DISCUSSION 2.1 Cytotoxicity of ESP The cytotoxic effect of ESP against Vero cells was tested with MTT method. As shown in Table 1, the cytotoxicity effect of ESP to the normal Vero cells was very low. The viability of cells was more than 50% at ESP concentration of 4000mg/mL. Vero cells in culture incubated in the presence or absence of ESP showed a similar number at the ESP concentration of 31.25mg/mL.Thus, suggesting that cytotoxicity of ESP against normal Vero cells were very low. Table 1 Viability of Vero cells in presence of serial concentration of ESP after 48h incubation
2.2 Antiviral assays of ESP against HSV-1 and
CVB3
Table 3 Antiviral activity of ESP against CVB3 mg/mL
2.3 Discussion REFERENCES |