Sephadex g 50 fine

Before MALDI TOF analysis the gel filtration chromatography using the Sephadex G-50 (Fine, Sigma-Aldrich) column was made for separation of components of obtained AMP extract according to their molecular weights [7 (link), 12 (link)]. The fraction 1 (MW from 2500 to 17,500 Da) and the fraction 2 (MW from 2750 to 4750 Da) were collected, lyophilised, and stored at −20 °C for further analysis.
The mass analysis of the components of PRP was carried out by the MALDI method with sinapinic acid (3,5–dimethoxy-4-hydroxycinnamic acid, M = 224.2 Da, Sigma-Aldrich, Poland) used as a matrix, whereas antimicrobial peptides from the neutrophil crude extract were analyzed using the CCA matrix (α-cyano-4-hydroxycinnamic acid 189.2 Da, Sigma-Aldrich, Poland) [13 (link)].

Sephadex G-50 (fine) was obtained from Sigma-Aldrich. All reagents for biochemical separations were obtained from Acros Organics (silica gel 60) and Sigma-Aldrich (chloroform and methanol).

Bacterial strains and plasmids used in this study are listed in Table 1. The Lactobacillus salivarius IBB3154 strain used in this study was cultured in MRS-liquid or MRS-agar (solidified with 1.5% agar) medium (Difco Laboratories, Detroit, MI, USA) at 37°C (Kobierecka et al., 2015 (link)). The E. coli strain TG1 was used as a host for the construction of recombinant plasmids. The E. coli strain Rosetta (DE3) pLysS was used to overexpress pUWM1414, pUWM1293 and pUWM1313, and E. coli strain BL21 (DE3) was used to overexpress pUWM1282 (Łaniewski et al., 2014 (link)). C. jejuni and E. coli strains were grown under standard conditions (Łaniewski et al., 2014 (link)) unless otherwise indicated. When needed, media were supplemented with antibiotics at the following concentrations: 30 μg ml^-1 kanamycin and 15 μg ml^-1 chloramphenicol, Campylobacter Selective Supplement (Oxoid) and IPTG (3 mg ml^-1) in DMF (dimethyl-formamide).
The 1,2-Dipalmitoyl-sn-glycero-phosphatidylcholine (DPPC), 1,2-Distearoyl-sn-glycero-phosphoethanolamine-N-[poly(ethyl-ene glycol)2000] (DSPE-PEG 2000) and cholesterol (Chol) were purchased from Northern Lipids, Inc. (Vancouver, BC, Canada). Sephadex G-50 fine was obtained from Sigma–Aldrich Chemie GmbH (Steinheim, Germany). All the other reagents were of analytical grade.

Apigenin (AP) with 97.95% purity was purchased from TargetMol Chemicals Inc. (MA, USA). Soy lecithin (> 94% phosphatidylcholine), cholesterol (≥ 99%), chitosan (low molecular weight, ≥ 75% degree of deacetylation), Sephadex^® G-50-Fine, 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), Folin-Ciocalteu’s phenol reagent, Trolox, gallic acid, fluorescein sodium salt, pepsin (porcine, ≥ 500 U/mg), pancreatin (porcine, 4 X USP), and bile extract porcine were supplied by Sigma-Aldrich (Missouri, USA). Tween 80, maltodextrin (10–12 DE), iron (III) chloride, acetonitrile (HPLC grade), hydrochloric acid, and sodium chloride were purchased from Chemiz (M) Sdn. Bhd. (Malaysia). Methanol, sodium carbonate, trichloroacetic acid, and potassium persulfate were obtained from Fisher Chemicals (MA, USA). Other chemicals used include ethanol absolute (VWR International Ltd, UK), isopropanol (Systerm Chemicals, Malaysia), iron (II) sulfate, potassium chloride, and dipotassium hydrogen phosphate (R&M Chemicals, Malaysia). All chemicals used were of analytical grade unless specified. Water purified from Milli-Q system (Millipore, USA) was used in all analyses.

The freeze-dried sclerotial powder TM02® (reg no. MAL 11035004TC) was provided by LiGNO™ Biotech Sdn. Bhd. (Balakong Jaya, Selangor). Preclinical toxicological study determined that the product was not associated with any toxicity concerns. No-observed-adverse-effect level dose was more than 1000 mg/kg. The powder also did not cause detectable adverse effect on rats’ fertility, teratogenic and genotoxicity effects (22 (link)). Hot water, cold water and methanol extractions were carried out in a mass to volume ratio 1:20 (g/mL) as described earlier (23 (link)). Cold water extract of TM02® sclerotia was further fractionated by Sephadex® G-50 (fine) (Sigma-Aldrich, Merck, St. Louis, MO, USA) gel filtration chromatography column equilibrated with 0.05 M ammonium acetate (Sigma-Aldrich, Merck) buffer. Eluted fractions were subsequently grouped based on their molecular masses.

The hydrogenated soya phosphatidylcholine Phospholipon 100 H (HSPC) was donated by Phospholipid GmbH (Cologne, Germany). 1,2-Distearoyl-sn-glycero-phosphoethanolamine-N-[poly(ethylene glycol)2000] (DSPE-PEG 2000) and cholesterol (Chol) were purchased from Northern Lipids, Inc. (Vancouver, British Columbia, Canada). Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, EDTA diammonium salt, HEPES and Sephadex G-50 fine were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Epirubicin hydrochloride (EPI) was donated by the Pharmaceutical Research Institute (Warsaw, Poland). All the other reagents were of analytical grade.

The asolectin-based carriers (LIP) were prepared using our previously published preparation protocol [25 (link),26 (link)], which was slightly modified for successful encapsulation of the present neuroactive drug molecule. Namely, 100 mg of asolectin was dissolved in 10 mL CHCl₃:CH₃OH/9:1 mixture (c_lipid = 10 mg cm⁻³). The organic solvent was removed by vacuum evaporation for 8–10 min at 50 °C to form a lipid film on the flask wall. The solid lipid film was hydrated in 20 mL of aqueous solution containing KYNA (cKYNA = 0.01 M) by continuous magnetic stirring at 800 rpm for 20 min. To achieve optimal size and maximum encapsulation efficiency, the sample was further treated by a half-hour sonication (37 kHz) and continuous stirring at 800 rpm for 20 min. Finally, non-encapsulated KYNA excess was removed by gel chromatography. The scheme of the preparation is presented in Figure S5. The water-bloated hydrophilic matrix structured dextran gel (Sephadex^® G50 fine, Sigma) as the stationary phase was added into an Eppendorf-filter (0.5 mL capacity) and centrifuged for 5 min at 13,000 rpm for compression. The separation of the excess KYNA was performed using ultracentrifugation at 7000 rpm for 5 min.