Antibiotic antimycotic mixture

Vero E6 cells were cultured in Dulbecco’s modified Eagle’s medium (Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (Gibco, New York, NY, USA), and 1% antibiotic–antimycotic mixture (Lonza). The cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂. An hCoV-19/Egypt/NRC-3/2020 SARS-CoV-2 virus (Accession Number on GSAID: EPI_ISL_430820) was propagated in Vero E6 cells and harvested after cytopathic effects (CPE) appearance. Viral stock was titred titrated using plaque infectivity assay and stored at −80 °C.
Curcumin (Bio Basic, Canada INC.), hesperidin (Carl Roth, Karlsruhe, Germany), and quercetin (Molekula, Molekula, United Kingdom) compounds were evaluated for their potential antiviral activity against SARS-CoV-2 virus. Hydroxychloroquine (Sanofi, Paris, France) was used as a positive control antiviral drug throughout our study. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was used at 10% in water as a proper solvent of the three tested compounds as well as Hydroxychloroquine. The prepared stock solutions were sterilized by syringe filter with 0.2 micron pore size (Millipore, Billerica, MA, USA) and stored at −20 °C.

Human hepatocellular carcinoma cell line HUH-7 was purchased from Vacsera (Giza, Egypt), sorafenib p- Toluenesulfonate salt was purchased from LC Laboratories (Woburn, MA, United States), Avastin^® (bevacizumab, Genentech, United States) was purchased, in its formulated commercial preparation, from a community Pharmacy (Cairo, Egypt) and fucoidan extracted from Laminaria Japonica was purchased as a crude extract from Buchem BV (Holland). Dulbecco’s modified Eagle’s medium (DMEM) medium and fetal bovine serum (FBS) were purchased from Gibco^®, Thermo Fisher Scientific, United States. Antibiotic-antimycotic mixture (100 U/ml of penicillin, 0.1 U/ml streptomycin and 0.25 μg/ml of Amphotericin B) was purchased from Lonza^®, Walkersville, MD, United States. All other chemicals were purchased from Sigma Aldrich, United States unless otherwise specified.

The antiproliferative effects of the prepared extracts were determined on a panel of human adherent cancer cell lines of gynecological origin. MCF7 and MDA-MB-231 cells were isolated from breast cancers, while A2780 and SiHa cells were isolated from ovarian and cervical malignancies, respectively. Cells were cultivated in minimal essential medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and an antibiotic-antimycotic mixture (Lonza Ltd., Basel, Switzerland). Cancer cells were seeded in a 96-well microplate at the density of 5000/well and allowed to adhere overnight and 200 μL new medium containing the tested extract was added. After incubation for 72 h at 37°C in humidified air containing 5% CO₂, the percentage of living cells was assessed after addition of 20 μL MTT solution. The medium was removed and the precipitated formazan crystals were dissolved in 100 μL DMSO during a 60 min period of shaking at 37°C. The absorbance values were read at 545 nm, using a microplate reader; wells with untreated cells were utilized as control and inhibition % values were calculated. All in vitro experiments were carried out on two microplates with at least five parallel wells. Cisplatin was used as positive control. Stock solutions of the tested substances (50 mM) were prepared in DMSO.