The bioactivity of proteins in Southern African Fynbos honey

Abstract

Medical grade honey aids in wound debridement, reducing inflammation and infection, and promoting cellular infiltration due to its sugar content, acidic pH, glyoxal, hydrogen peroxide, polyphenols, and peptides. The contribution of the protein fraction of honey to these activities is not well understood. Investigating the bioactivity of proteins isolated from honey could lead to the discovery of novel antimicrobial, anti-inflammatory, and wound-healing agents with potential applications in medicine, functional foods, and pharmaceuticals. This research fills critical knowledge gaps by characterizing honey proteins, elucidating their mechanisms of action, and assessing their stability, bioavailability, and therapeutic potential, offering new insights beyond the well-studied carbohydrate and polyphenol components of honey. This study evaluated the bioactivity of whole honey and corresponding isolated protein fractions from Southern African Fynbos (FB) honeys which were compared to the medically approved Manuka (MA) honey and a Buckwheat (BU) honey as controls.

The protein fractions of five FB honeys and the control honeys were isolated using gel filtration chromatography. Analyses included protein content, total polyphenol content (TPC), sugar content, and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis. At tested non-cytotoxic concentrations, the effect of the whole honey and the protein fractions on cellular migration was evaluated with the scratch assay using the HaCaT keratinocyte and the SC-1 fibroblast cell lines. Antioxidant activity was evaluated with the in vitro oxygen radical absorbance capacity (ORAC) assay and the Trolox equivalent antioxidant capacity (TEAC) assay. The cellular antioxidant activity (CAA) was also determined using HaCaT and SC-1 cell lines. Nitric oxide (NO) mediated effects on inflammation were evaluated in the in vitro lipopolysaccharide (LPS) induced NO scavenging/RAW 264.7 cell model. Antibacterial activity in terms of minimum inhibitory concentrations (MIC’s) against Escherichia coli (E. coli ATCC 700928) and Staphylococcus aureus (S. aureus DSM 2569) were determined.

The protein content varied across honey samples, with BU (1.33 ± 0.15 mg/g) and FB6 (1.19 ± 0.1 mg/g) showing significantly higher levels than MA (0.95 ± 0.07 mg/g, p < 0.0001), while FB2 (0.78 ± 0.08 mg/g) and FB3 (0.72 ± 0.06 mg/g) had significantly lower levels (p < 0.0001). The protein fractions showed a similar trend, with MF1 (0.05 ± 0.01 mg/g), FB1 (0.05 ± 0.01 mg/g), FB2 (0.07 ± 0.02 mg/g), and FB3 (0.04 ± 0.01 mg/g) exhibiting significantly lower concentrations than MA (0.12 ± 0.01 mg/g, p < 0.0001).The sugar/fructose content in honey ranged from 25.18 ± 0.10 mg/100 g (MF1) to 38.99 ± 0.18 mg/100 g, with MF1 showing significantly lower values than MA (p = 0.0212). In protein fractions, sugar content was highest in MA (15.95 ± 3.15 mg/100 g) and lowest in BU (11.07 ± 0.63 mg/100 g). Total phenolic content (TPC) was highest in BU (1.05 ± 0.03 mg GAE/g, p = 0.0273) and lowest in MF1 (0.47 ± 0.04 mg GAE/g, p = 0.0002), with FB2 and FB3 also significantly lower than MA (p < 0.0001). Molecular mass analysis confirmed the presence of distinct protein bands between 45-70 kDa.

Cell viability in HaCaT and SC-1 cells exceeded 80% at tested concentrations. Wound closure rates in HaCaT cells for whole honey (1.25% v/v) ranged from 71 ± 8% to 90 ± 3%, and for protein fractions (0.0044-0.0146 mg/g) from 75 ± 8% to 89 ± 3%. In SC-1 cells (0.625% v/v), rates ranged from 46 ± 15% to 85 ± 3% for whole honey and 66 ± 11% to 86 ± 6% for protein fractions. Antioxidant capacity (TEAC) was highest in BU honey (8.66 ± 0.14 µM TE/g) and lowest in FB2 (2.05 ± 0.35 µM TE/g). ORAC values ranged from 1.55 ± 0.40 to 2.67 ± 0.73 µM TE/g. Protein fractions showed minimal antioxidant activity. Oxidative damage in HaCaT cells ranged from 82.2 ± 4.4% to 100.0 ± 8.2% (honey) and 107.9 ± 5.3% to 117.1 ± 7.3% (protein). In SC-1 cells, damage was 79.1 ± 3.1% to 95.1 ± 5.6% (honey) and 87.8 ± 4.1% to 95.0 ± 8.5% (protein). NO levels were similar across samples. Anti-inflammatory activity was highest in BU honey (1.60 ± 0.25 µM), while FB9 (8.78 ± 2.90 µM) which had significantly lower activity than MA (p = 0.0353) and BU (p = 0.0002). Protein fractions exhibited lower NO scavenging, particularly BU (10.45 ± 0.47 µM, p = 0.0265).

Antibacterial activity (MIC50) against E. coli was strongest in MF1 (26.8 ± 6.4 mg/mL) and BU (29.9 ± 6.4 mg/mL). Against S. aureus, MF1 (0.01 ± 0.01 mg/mL) and FB1 (0.05 ± 0.04 mg/mL) had the highest efficacy among protein fractions, while BU and MF1 honeys showed no activity.

Tentatively, the proteins in the honey samples and protein fractions could be assigned to the major royal jelly protein (MRJP) family, glucose oxidase and bee defensin-1 (DF-1) in the based on molecular mass. The protein fraction did not contribute significantly to the antioxidant activity of honey nor to the NO scavenging activity in vitro. For several honeys and protein fractions the MIC50 was determined and generally, the honey and protein fractions showed increased targeting of E. coli than S. aureus similar to Manuka honey. FB honeys exhibited anti-inflammatory, antioxidant, and antimicrobial properties similar to the MA honey control. The findings highlight the potential of Southern African Fynbos honeys to exhibit bioactivities comparable to medically approved honeys, suggesting their promising role in wound care and antimicrobial applications.