Whatman no 3 paper

The fungal specimens were dried and then crushed into powder form and sequentially extracted into four extracts as previously described [11 (link),12 (link)]. For large-scale extraction, powdered O. tomentosa CL312 (87 g) was first extracted with 80% ethanol for 3 h at 65 °C. The solution, filtered through Whatman paper No. 3, was then referred to as E1, whereas the residue was subjected to the second step: 50% methanol extraction for 3 h at 65 °C. This filtrate, designated as E2, was retained while the residue was subjected to the third step: water extraction for 6 h at 65 °C. The filtrate from the water extraction step was referred to as E3. Finally, this residue was subjected to the final step: 5% sodium hydroxide (NaOH) extraction at 65 °C for 6 h. The filtered solution from this step was referred to as E4. Both E1 and E2 liquid extracts were concentrated using a rotary evaporator before being subjected to lyophilization to yield solid extracts of 13.2 g (15.2%) and 5.3 g (6.1%), respectively. All lyophilized extracts were reconstituted in water at 20 mg/mL and filter-sterilized using a 0.2 μm filter (Sarstedt, Quebec), before they were assessed for antiproliferative and immunomodulatory activities as described below.

The in vitro antibacterial activity of the synthesized compounds was investigated by using the disc diffusion technique. Bacteria samples from one over-night grown colony were suspended in a test tube containing physiological water. The turbidity (expressed as optical density OD) of the microorganism suspensions was measured with an optical spectrophotometer (wavelength = 625 nm) and adjusted to 0.6 to 0.8. A sterilized cotton swab was immersed in the resulting suspension, and a lawn of bacteria was applied onto the surface of the plates containing Tryptic Soy Agar for bacteria and Sabouraud dextrose agar for the fungal sample. Then, sterile filter paper disks (diameter 6 mm, Whatman paper n^o3) were first impregnated with 30 μl of each dimethylsulfoxide (DMSO) extract (6 mg/ml). The side containing the particles was facing downwards to ensure direct interaction with the agar and microorganisms. The bacterial plates were incubated at 37 °C for 24 h, and the fungal plates were incubated at 25 °C for 48 h. Finally, the diameter of the growth inhibition zones was measured using a zone inhibition reader (Fisher Lilly Antibiotic Zone Reader). Disks with 30 μl of distilled water were used as negative controls, and antibiotic discs were used as positive controls such as Nystatine (10 mg/ml), Gectapen (40 mg/ml), Azimycine (30 mg/ml). Each experiment was carried out twice.

One hundred grams of powder was measured using an electric balance (SF-400) and introduced into 1 L of 95% ethanol. The concentration is 10% g/ml. The mixture was homogenised for 72 h (every 4 hours, the extract was stirred) and filtered using Whatman paper no. 3. The resulting filtrate was dried in an oven at 45°C until the dry extract was obtained.

Powder B. micrantha (100 grams) using an electrical balance (SF-400) was weighed into a 2-litre container, with an addition of 95% ethanol (one litre) followed by agitation for five minutes and maceration for 72 hours. Whatman paper no. 3 was used to filter the homogenate with the resulting filtrate evaporated at 45°C in an oven [15 ]. The aqueous extract was prepared by infusion in distilled water at 100°C for three hours, followed by filtration to obtain the filtrate.

The fungal specimens were dried and then crushed into powder, then sequentially processed into four extracts as previously described [2 (link),3 (link)]. For large extraction, 350 g of powdered specimen was processed with 3.5 L of 80% ethanol for 3 h at 65 °C. The solution, referred to as 1A, was filtered through Whatman paper No. 3. The residue was subjected to a second step of 50% methanol extraction for 3 h at 65 °C. This filtrate was designated as 1B. The residue was subjected to a third step which involved water extraction for 6 h at 65 °C. The filtrate from the water extraction was referred to as 1C. The residue was subjected to the final step of 5% sodium hydroxide (NaOH) extraction at 65 °C for 6 h. The filtered solution from this step was referred to as 1D. All extracts were subjected to roto-evaporation followed by lyophilization. All lyophilized extracts were reconstituted in water at 20 mg/mL and were filter-sterilized using a 0.2 μm filter (Sarstedt, Quebec) before they were assessed for immunomodulatory and antiproliferative activity as described below.

Bridelia atroviridis barks were harvested at Mbalmayo in December 2018. The plant was authenticated by Mr. Ngansop Eric, a botanist at the National Herbarium, Yaoundé (Cameroon), in comparison with the specimen voucher N35241/HNC Cam. The barks were air-dried in the shade and pulverized into powder. A kilogram of powder from B. atroviridis was soaked in 5 L of hydroethanolic solvent mixture (30% water and 70% ethanol; v/v) for 72 hours with regular agitation. Then, the mixture was filtered with Whatman paper No. 3, and the filtrate was evaporated to dryness using a rotatory evaporator (Buchi Rota vapor, Switzerland) at a temperature of 45°C. The residual water was removed by ventilation under a hood (Burdinola ST 1800) giving a powdered crude extract.