Determination of antiviral activity

The Vero cell line (ATCC: CCL-81) and herpes simplex virus (HSV-1), Coxsackie B4 virus (CoxB4), and hepatitis A virus (HAV) were used in this study. All viruses were obtained from the Microbiology Department, Faculty of Medicine for Girls, Al-Azhar University, Cairo.

The cytotoxic activity of the different concentrations (15.62, 31.25, 62.5, 125, 250, and 500 μg/mL) of NMF6 extract on viral host cells (Vero cells, ATCC CCL-81) was evaluated using MTT assay as described earlier. The maximum tolerated concentration, which did not cause toxicity or morphological alteration toward Vero cells, was determined, and the percentage of cytotoxicity was calculated as [(A − B)/A × 100], where A and B are the absorbance values of control and treated cells, respectively. The CC₅₀ value (the concentration that caused 50% toxicity) was determined from the cell viability standard curve using the GraphPad Prism 5 software program [37, 38].

Antiviral activity was evaluated using MTT assay as described previously [38]. Briefly, the Vero cell culture (10,000 cells/200 μL of media) was dispensed into each well of a 96-well plate (three wells were left empty as blank control). The plate was incubated overnight at 37 °C under 5% CO₂ to permit adherence of cells to the wells. Next, 100 μL of virus/sample suspension, prepared by mixing equal volumes of nonlethal concentrations (15.62, 7.8, and 3.9 μL/mL) of NMF6 extract and virus suspension and incubated for 1 h, was added to each well, shaken for 5 min at 150 rpm, and incubated at 37 °C for 24 h under 5% CO₂ to permit the activity of the virus. Then, 20 μL of MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well, mixed thoroughly by shaking for 5 min at 150 rpm, and incubated for 1–5 h to allow metabolism of MTT. The medium was poured off, and the plate was dried to remove any residue. The MTT metabolic product (formazan) was resuspended in 200 μL of DMSO and shaken well to ensure complete solubility. The optical density was determined at 560 nm, and the background was subtracted at 620 nm. The IC₅₀ value (the concentration required to reduce the viral cytopathic effect to 50%) was estimated using the GraphPad Prism 5 software program. The SI value was calculated as the ratio of CC₅₀ to IC₅₀, and the antiviral activity was calculated as follows:

where A, B, and C are the absorbance values of sample, virus, and cell control, respectively.

GC-MS analysis was conducted according to previously described methods [39]. Briefly, the Thermo Scientific Trace GC1310 gas chromatograph attached with a mass spectrometer was used to identify the individual components present in the EtOAc of strain NMF6. The instrument was coupled with the Agilent J&W DB-5 column, and the temperature was set at 40–280 °C. The sample was injected at 300 °C and eluted under low-pressure helium gas (1 mL/min). The mass spectra of the compounds were compared with Wiley Registry8e library.

The FT-IR spectra of NMF6 extract were recorded using a Bruker Tensor 27 FTIR spectrophotometer and the conventional KBr disk method. A total of 32 scans were collected at a spectral resolution of 4 cm⁻¹.