The antiviral activities on HSV-1 and CVB₃ in vitro of polysaccharide from alga eucheuma striatu

Cen Yingzhou¹, Khoo Gaikming¹, Ye Shaoming¹, Wang Yifei²
(¹Department of Chemistry, Faculty of Life Science and Technology, ²Guangzhou Jinan Biomedical Research and Development Center, Jinan University, Guangzhou, Guangdong, China 510632)

Tropical red alga Eucheuma striatum is widely cultivated in Hainan Island, China. The major constituent, polysaccharide, takes up 60% total weight of its dry mass. Hitherto，researches on Eucheuma striatum mainly concentrate in food processing and daily necessities chemicals processing, but lack of mentioning its biological activities. This paper describes the antiviral activities of the polysaccharide samples from Eucheuma striatum (ESP) and its preliminary research on antiviral mechanism.

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 4^oC.
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 37^oC 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 37^oC 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 37^oC. 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 37^oC, 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 37^oC 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 2 Antiviral activity of ESP against HSV-1 mg/mL

Table 3 Antiviral activity of ESP against CVB₃ mg/mL

2.3 Discussion
Monoherpes virus belongs to the a-herpes virus family. Herpes infections are ubiquitous. HSV-1 is normally associated with orofacial infections, keratitis and encephalitis. Acyclovir(ACV) is a common drugs used to treat herpes-infection at present but acyclovir-resistant HSV infections have emerged because of the increase in drug use frequency. Coxsackie virus is a member of the enterovirus genus of the picornavirus family. Coxsackie virus group B especially CVB₃ is regarded as the most common agent of viral myocarditis and are also capable to induce a chronic myocarditis associated with a persistent type of heart muscle infection. Coxsackie virus infects humans year-round, especially causing high mortality among the newborns. No compounds have yet been found that are clinically effective against the Coxsackie virus diseases. Thus, there is an urgent need for novel anti-Coxsackie agents.
    In this study, the antiviral activity in vitro of the ESP was described. The results showed that ESP could inhibit the CPE of cells infected by HSV-1 and CVB3 in certain concentration range. Furthermore, it showed low cytotoxicity to the normal Vero cells. Its antiviral activities were better than Acyclovir and Ribavirin which were run in parallel as positive control samples. The results suggested that the polysaccharide from Eucheuma striatum may be a potential anti-HSV and anti-Coxsackie virus drug candidates.
    The antiviral mechanism was studied through three methods. Firstly, the effects of pre-treatment by ESP on Vero cells, to observe the ability of ESP protecting Vero cells from attacking by the virus. Secondly, the inhibition effects of ESP to virus-infected cells, to observe whether this polysaccharide was able to inhibit the biosynthesis or release of viruses. Thirdly, the direct inactivation effects of polysaccharide to the virus. The results showed that the antiviral activities of the ESP are prominent in all methods, especially the first and the third method mentioned above. Thus, we presume that this polysaccharide not only could extinguish the virus in Vero cells, protect the Vero cells from attachment of virus, but also could execute the virus effectively at a low concentration of 0.16~3.12mg/mL. In conclusion, the polysaccharide from Eucheuma striatum may be able to use as preventive medicine or direct treatment drugs for HSV or CVB infected diseases.