Whatman filter paper

Blood samples of patients infected with malaria falciparum were collected on Whatman™ filter paper (GE Healthcare Bio-Sciences Corp, NJ, USA) for routine chloroquine resistance surveillance [18 (link), 19 (link)]. DNA was extracted through a Chelex-100 based method [20 (link)]. A PCR method was used to confirm parasite species [3 (link)].

Human C1q was purified, as previously described [20 (link)]. Briefly, fresh frozen human plasma (100 mL) was centrifuged at 5000× g for 10 min, then passed through a Whatman filter paper (GE Healthcare, Hatfield, UK) to remove lipids. The plasma was then incubated with IgG-Sepharose (GE Healthcare, Hatfield, UK) for 2 h at room temperature (RT). The column was washed with a wash buffer (10 mM HEPES) and C1q was eluted using the elution buffer (100 mM CAPS, 1 M NaCl, 0.5 mM EDTA, pH 11). The eluted C1q was applied to a Hi-Trap Protein G column to remove residual IgG, and the flowthrough was collected. Finally, the purified C1q was dialysed against a 0.1 M HEPES buffer, pH 7.5, quantified (yield ~ 2 mg), and examined via SDS-PAGE (Supplementary Figure S1A).

The production of OLE was carried out as described in Conte et al. [26 (link)]. Briefly, after washing, the olive leaves were dried at 120 °C for 8 min in a ventilated oven (Argolab, Carpi, Italy) to reach a moisture content <1%, and then grounded with a blender (Waring-Commercial, Torrington, CT, USA). Milli-Q water was used as extraction solvent in a ratio 1/20 (w/v). The extraction process was ultrasound-assisted (CEIA, Viciomaggio, Italy) and the extract was filtered through Whatman filter paper (GE Healthcare, Milan, Italy), freeze-dried (BUCHI, Flawil, Switzerland, Lyovapor^TM L-200), and stored at −20 °C.
The total phenol content was determined on the extract using the Folin-Ciocalteu method, the antioxidant activity was assessed by ABTS and DPPH assays as reported in Difonzo et al. [19 (link)]. The content in oleuropein was determined by HPLC-DAD and external calibration curve with the relative standard, as reported in Centrone et al. [27 (link)]. All determinations were performed in triplicate. After the extraction process, the OLE was freeze-dried and stored at −20 °C.

The olive leaf extract was produced according to Difonzo et al. [23 (link)]. The olive leaves were dried at 120 °C for 8 min in a ventilated oven (Argolab, Carpi, Italy) and grounded with a blender (Waring-Commercial, Torrington, CT, USA). Milli-Q water was used as extraction solvent in a ratio 1/20 (w/v). The extraction process was ultrasound-assisted (CEIA, Viciomaggio, Italy) and the extract was filtered through Whatman filter paper (GE Healthcare, Milan, Italy), freeze-dried (BUCHI, Flawil, Switzerland, LyovaporTM L-200), and stored at −20 °C.
The OLE was characterized for total phenol content and antioxidant activity by Folin–Ciocalteu and ABTS assays (119 mg GAE/g and 687 µmol TE/g). The phenolic profile was determined according to Difonzo et al. [24 (link)] and the oleuropein content (85 mg/g) was determined by HPLC-DAD and external calibration curve with the relative standard.

The roots powder was soaked in methanol (25 g/250 ml) for 24 h withheld in a gently shaking apparatus at room temperature for extraction. The extract was filtered with a Whatman filter paper (125 mm; 1441–125, GE Healthcare Life Sciences, UK) while excess solvent was allowed to evaporate under reduced pressure at 40 °C using a rotary vacuum evaporator. Following to determining the mean yield as a mass of the obtained extract per 100 g of W. somnifera roots, the filtrate was stored in a refrigerator at 4 °C and used throughout the study.

Three methods were used in the formulation of the corn Starch-Neusilin UFL2 conjugate: physical, chemical, and microwave methods. The physical method involved simple mixing of corn starch and Neusilin UFL2 in the ratio 1 : 1. To ensure proper mixing tumbling method was used and the weighed starch and Neusilin were transferred into a beaker and the mouth of the beaker was closed ensuring that there is no spillage of the contents from the beaker while the mixing is carried out. The mixing was carried out for 15 minutes. In chemical method corn starch and Neusilin UFL2 were incorporated in the ratio 1 : 1. Corn starch was suspended in distilled water (q.s) and kept on magnetic stirrer at 270 rpm and temperature not more than 40°C. To this, solution of Neusilin UFL2 in NaOH (q.s) was gradually added with constant stirring. After 15–20 minutes the precipitated conjugate was filtered using Whatman filter paper (GE Healthcare UK Limited) having a pore size of 125 mm and dried in oven at a temperature not more than 50 ± 2°C. In microwave method physical mixture of corn starch and Neusilin UFL2 was subjected to microwave radiation at 590 watt for not more than 5 minutes.

Bark of U. parvifolia was obtained from National Institute of Horticultural and Herbal Science (Jeollabuk-do, Republic of Korea). U. parvifolia bark was grounded and extracted with 70% ethanol at 80°C for 3 h, filtered with Whatman™ filter paper (GE Healthcare, PA, USA), condensed in a rotary evaporator (Rotavapor^® R-100; B.U.CHI Labortechnik, Switzerland), and lyophilized to obtain powdered extract. The powder was stored at −30°C for further use in experiments.