Rubini elderberry liquid extract

The extract used in the trials is a proprietary product known as Rubini and was supplied by BerryPharma AG (Solinger Strasse 7, D-42799 Leichlingen, Germany). This particular elderberry extract was chosen for our studies because it is standardized by HPLC and always produced from the same "Haschberg" variety of S. nigra L., which is grown under cultivation in the Steiermark region of Austria. The elderberry-to-extract ratio of the product is 18:1. The extract is concentrated and standardized using membrane filtration to achieve a minimum anthocyanin concentration of 3.2%. The concentration of anthocyanins is achieved using a mechanical filtration procedure in which semipermeable membranes separate substances according to their different molecular sizes. The HPLC assay is based on the IFU N° 71, 1998 method, measured at pH 1, 510 nm using cyanidin chloride (Sigma Aldrich) for the reference standard.

All references to elderberry liquid extract in this study refer to the same proprietary, standardized extract.

Bacterial strains

Strains of S. pyogenes, group C and G Streptococci, and B. catarrhalis were directly isolated from patient samples and cultivated on sheep blood agar plates (37°C, 5% CO 2 ) and refreshed twice-weekly. Patient isolates were characterized using different reference antibiotics (see Additional file 1, Table S1). For the experiments, bacteria were grown in an appropriate broth overnight at 37°C with shaking at 180 rpm (Unitron, Infors). Overnight cultures were diluted 1:50 in 20 ml fresh BHI (Brain Heart Infusion) broth using a 100-ml Erlenmeyer flask and were incubated at the conditions noted above until they reached an optical density of OD 600 nm 1.0.

Cell line & viruses

Madin Darbin canine kidney cells (MDCK) were grown in DMEM (1x DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin). The following influenza virus strains were used: The human HPAIV isolate A/Thailand/KAN-1/2004 (KAN-1, H5N1) was supplied to S. Pleschka by P. Puthavathana, Thailand. The human strain B/Massachusetts/71 (B/Mass) was obtained from the IV strain collection in Giessen, Germany. KAN-1 and B/Mass were propagated on MDCK cells with low serum without trypsin for KAN-1 and with trypsin (2 μg/ml) for B/Mass. Strains were titrated by focus assay (see below).

Testing of antimicrobial activity of elderberry liquid extract in liquid cultures

The strains were grown at 37°C at 180 rpm for 12-16 h. The optical density was measured at OD 600 nm and differences were adjusted by taking different volumes to obtain the same amounts of bacteria.

Volumes of one milliliter fresh media were inoculated with 50 μl of bacteria overnight-cultured in 1.5-ml Eppendorf tubes. Elderberry liquid extract was added in amounts of 5%, 10%, 15%, or 20%. The prepared tubes were kept at 37°C and 180 rpm for another 16 h. Out of the tubes, 100 μl were diluted, plated on blood agar (Oxoid), and incubated at 37°C in broth for 24 h. Colony forming units (CFU) were counted and the counts were recalculated by factoring in the formerly made dilutions. Plated bacteria without any elderberry extract were set as 100% of possible growth. Growth figures from isolates exposed to elderberry liquid extract were set in relation to the strains not exposed to the extract. Every biological experiment was independently repeated at least three times with two replicates per trial.

MTT assay

MDCK cells grown in 96-well microplates were incubated with DMEM/BA media (1x DMEM, 0.2% BA, 100 U ml-1 penicillin and 0.1 mg ml-1 streptomycin) with different concentrations of elderberry extract (as described under Materials and Methods, page 6) at 37°C, 5% CO 2 for 12, 24, 36 and 48 h (16 wells per concentration and time point). The media was then aspirated and the cells were left to recover for 60 min in DMEM which was then replaced by 200 μl of MTT-mix (DMEM supplemented with 10% FCS and antibiotics containing 175 μg/ml MTT = 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan; Sigma). The cells were further incubated for 90 min at 37°C and subsequently fixed with 4% PFA (in PBS) for 30 min at RT. The cells were dried and the tetrazolium crystals were dissolved by adding 200 μl of isopropanol to each well. The plates were shaken for 10 min and analyzed photometrically at 560 nm excitation in an enzyme-linked immunosorbent assay (ELISA) reader.

Focus assay

MDCK cells grown in 96-well plates in DMEM to about 90% confluency were washed once with PBS++ and infected with 50 μl virus of a dilution resulting in about 100 foci/well in PBS/BA, for 1 h RT. The inoculum was aspirated and 150 μl MC-media (1x DMEM, 1.5% Methyl cellulose; Methocel MC, Fluka) for KAN-1 and Avicel-media (1x DMEM, 1.25% Avicel; FMC, Belgium) for B/Mass were added and the cells were incubated at 37°C, 5% CO 2 , for 36 h and 48 h. To detect foci of infected cells, the cells were fixed and permeabilized using 100 μl fixing solution (4% PFA, 1% Triton X-100 in PBS++) at 4°C for 60 min. The solution was then discarded and the cells were washed 3x with PBS++/0.05% Tween20 and further incubated with 50 μl 1st antibody (mouse-anti-Influenza A Nucleoprotein mAb, BIOZOL BZL 10908) diluted in PBS++, 3% BSA, for 1 h at RT. The cells were then were washed 3x with PBS++, 0.05% Tween20 and incubated with 50 μl of 2nd antibody (anti-mouse HRP-antibody) diluted in PBS++, 3% BSA, for 1 h at RT. The cells were then washed 3x with PBS++, 0.05% Tween20 and incubated in 40 μl/well "AEC" staining solution (Sigma) for 45 min at RT. After sufficient staining, the substrate was removed and the cells were washed 2x with dH 2 O to remove salts. To detect the size of foci (indicating a productive replication), the 96-well plates were scanned and analyzed using Photoshop software package.

Statistical data analysis of experiments

All experiments were performed a minimum of three times. Significant differences between two values were compared with a paired Student's t-test. Values were considered significantly different when the p value was less than 0.05 (p < 0.05).

Biosafety

All experiments using infectious virus were performed in accordance with German regulations applicable to the propagation of influenza viruses. All experiments involving highly pathogenic influenza A virus were performed at a biosafety level 3 (BSL3) containment laboratory approved for such use by the local authorities (RP, Giessen, Germany).