Polysorbate 20

100 microlitres of the secreted fractions and 100 µl of 1:20 diluted (in PBS) lysates from the secretion assays were loaded into a bio-blot microfiltration blotting system (BioRad. Cat. No.: 1706545). Samples were left to flow through a 0.45 µm nitrocellulose blotting membrane (Amersham Protran. Cat. No.: 10600002) by gravity ^{3 (link),55 (link)}. Membranes were then blocked with a Tris-Buffered Saline (TBS) solution supplemented with 0.1% vol/vol polysorbate 20 (Sigma-Aldrich. Cat. No.: P1379) (TBST) and 2.5% weight/vol non-fat dry milk for 20–40 min at room temperature (RT) on a laboratory rocker. Primary antibodies were diluted in TBST/BSA 5% (weight/vol) and membranes were incubated with this solution overnight at 4 °C on a laboratory rocker. Fluorescent secondary antibodies (donkey anti rabbit IgG – Alexa Fluor 680 – Life technologies. Cat. No.: A10043 or donkey anti mouse IgG – Alexa Fluor Plus 800 – Invitrogen. Cat. No.: A32789) were diluted to 2 µg/µl in TBST/non-fat dry milk 2.5% and membranes were incubated with this solution for 60 min at RT on a laboratory rocker. Fluorescent signal was detected using the iBright imaging system (ThermoFisher Scientific) equipped with a high resolution (9.1 mega pixels) CCD camera and multiplexed laser excitation/emission filters.

Beauveria bassiana strain GHA was purchased in the form of Mycotrol ESO (LAM International Corporation, Butte, Montana, USA). Stock Mycotrol ESO was halved with distilled water at a concentration of 1.06 × 10¹⁰ conidia/ml (rounded to 1 × 10¹⁰). From the stock solution, serial dilutions were prepared using distilled water. Five concentrations of B. bassiana were tested, in addition to a control without fungus. Furthermore, 0.05% of polysorbate 20 (Sigma–Aldrich, St. Louis, Missouri, USA) was added to each treatment and control to reduce formula separation (Dong et al. 2012 (link)). Experimental fungus concentrations were based on previous studies and WHO guidelines (WHO 2005 ; Deng et al. 2017 (link), 2019a (link)).

Macadamia integrifolia (macadamia) seed oil, Olea europaea (olive) oil, Triticum vulgare (wheat) germ oil, Persea americana Mill. (avocado) oil, Oenothera biennis (evening primrose) oil, and Prunus dulcis Mill. (almond) oil were of cosmetic grade, and were purchased from Namsiang (Chiang Mai, Thailand). Polysorbate 20 (Tween^® 20), polysorbate 80 (Tween^® 80), polysorbate 85 (Tween^® 85), sorbitan oleate 80 (Span^® 80), butylene glycol, and hyaluronidase from bovine testes (EC 3.2.1.35) were of analytical grade, and were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was purified using a Milli-Q water system (EMD Millipore, Burlington, MA, USA). Bovine serum albumin (BSA) was purchased from Merck (Darmstadt, Germany). Sodium acetate (CH₃COONa), acetic acid (CH₃COOH), hydrochloric acid (HCl), potassium persulfate (K₂S₂O₈), sodium chloride (NaCl), monosodium phosphate (NaH₂PO₄), and disodium phosphate (Na₂HPO₄) were of analytical grade, purchased from RCI Labscan Co., Ltd. (Bangkok, Thailand).

Mangiferin, Mangifera indica, 1,3,6,7-Tetrahydroxyxanthone C2-β-D-glucoside (MG), polyoxyethylenesorbitan monolaurate, polysorbate 20 (TW₂₀), polyoxyethylenesorbitan monooleate, polysorbate 80 (TW₈₀), sorbitan monolaurate (SP₂₀), sorbitane monooleate (SP₈₀), dimethyldidodecylammonium bromide (DDAB), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), nylon, mixed cellulose esters (MCE), and polytetrafluoroethylene (PTFE) membranes (diam. 25 mm, pore size 0.2 μm) were purchased from Merck, Sigma-Aldrich (Santa Louis, MO, USA). The soybean lecithin (90% phosphatidylcholine) (PC) was Epikuron 200 from Lucas Meyer, Hamburg, Germany. Solvents were HPLC grade and all other chemicals were analytical grade.