Cytotoxicity and antiviral test

Using the Vero cell/HSV-1 model, cytotoxicity was evaluated by incubating 100 µl of cellular suspension (3.5 × 10⁵ cells/ml) with various dilutions (concentration 5–1,000 µg/ml) of potential extract (NP31-01 and KJB-07) in 96 well plates in Eagle’s MEM with a total volume of 200 µl. Vero cells were placed in Eagle’s MEM with a final volume of 200 µl in each well and were used as positive controls. The well plates were incubated at 37 °C for 72 h. Cytotoxic activity was observed using microscope and cells were tested using the neutral red dye method. Optical density was measured at 540 nm using a spectrophotometer (SpectraCount™; Packard, Paris, France). The 50% cytotoxic concentration (CC₅₀) of the test compound was defined as the concentration that reduced the absorbance of mock-infected cells to 50% of that of controls. CC₅₀ values were determined as the percentage of destruction (%D): ((OD_c)C − OD_c)MOCK/(OD_c)C) × 100. (OD_c)C − (OD_c)MOCK were the OD values of the untreated cells and treated cells (Langlois et al., 1986).

Using the Vero cell/HSV-1 model, 100 µl of cellular suspension (3.5 × 10⁵ cells/ml) in Eagles’s MEM containing 8% FCS were incubated with 50 µl of a dilution of extracts (concentration 5–1,000 µg/ml) in 96 well-plates (48 h, 37 °C, 5% CO₂) Three replicates were infected using 50 µl of medium and a HSV-1 suspension at a multiplicity of infection (MOI) of 0.001 ID₅₀/cells. Acyclovir (9-(2-hydroxyethoxymethyl) was used as a control positive against HSV-1 ranging from 0.05–5 µg/ml. Cultures were grown in incubation at 37 °C for 72 h in a humidified CO₂ atmosphere (5% CO₂). Antiviral activity was observed using Olympus model CX23LEDRFS1 Optical Microcope and cells were tested using the neutral red dye method. The protection of the extract from virus-infected cells was expressed by 50% effective antiviral extract concentration (EC₅₀). OD was measured at 540 nm. The OD was related directly to the percentage of viable cells, which was inversely related to the cytopathic effect. EC₅₀ values were determined as the percentage of cell protection (%P): ((OD_t virus − OD_c virus)/(OD_c MOCK − OD_c virus)) × 100. OD_c and OD_t were the OD values of the virus control and test sample, OD_c MOCK was the OD of mock-infected control (Langlois et al., 1986). Interpretation of the data was presented using a system of linear regression equations.