3-Phenyllactic Acid and Polyphenols Are Substances Enhancing the Antibacterial Effect of Methylglyoxal in Manuka Honey

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TL;DR

This study demonstrates that 3-phenyllactic acid (3-PLA) and polyphenols (using gallic acid as a representative) enhance the antibacterial effect of methylglyoxal (MGO) in manuka honey against Bacillus subtilis, even though these compounds alone show no bacteriostatic effect at honey-relevant concentrations. The synergistic effect is partly explained by 3-PLA stabilizing MGO in the medium, resulting in higher apparent MGO concentrations over 24 hours. These findings contribute to understanding the antibacterial mechanism of manuka honey and support its medical applications in wound healing, though the exact quantitative contribution of these synergists requires further investigation.

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**Background:** Manuka honey (Leptospermum scoparium) is known for its pronounced antibacterial activity, primarily attributed to high concentrations of methylglyoxal (MGO), which can reach up to 1541 mg/kg. While MGO's bacteriostatic effects have been established, the potential synergistic contributions of other naturally occurring compounds in manuka honey—such as 3-phenyllactic acid (3-PLA), a marker substance present at up to 1400 mg/kg, and polyphenolic compounds—have not been systematically investigated. This study aimed to evaluate whether these substances enhance the antibacterial activity of MGO against Bacillus subtilis.

**Methods:** Four commercially available manuka honeys labeled for wound healing (MGO30+, MGO250+, MGO400+) and a cornflower honey were analyzed. Artificial honey (44 g fructose, 37 g glucose, 2 g sucrose per 17 g water) was used as a blank matrix to maintain constant osmotic pressure. MGO was quantified via HPLC-UV after derivatization with ortho-phenylenediamine. 3-PLA was quantified using HPLC-MS/MS with forchlorfenuron as internal standard. Total polyphenol content was estimated using the Folin-Ciocalteu method, expressed as gallic acid equivalents (GAE). Antibacterial activity was assessed by measuring growth delays of B. subtilis W168 in 96-well plates over 24 hours at 37°C, with OD600 measured every 5 minutes. The end of the bacterial lag phase was defined as OD600 reaching 5-fold above initial. Growth delay was calculated as the ratio of lag phase in test samples to lag phase in artificial honey control. To test synergistic effects, artificial honey was spiked with MGO (100–400 mg/kg) and 3-PLA (up to 2000 mg/kg) or gallic acid (up to 2000 mg/kg). MGO stability was assessed by measuring MGO concentrations at 0, 1, 3, 5, 8, and 24 hours in LB medium with and without 3-PLA.

**Key Results:** Cornflower honey showed a growth delay of 2.3 compared to artificial honey, while manuka honey MGO250+ showed a growth delay of 5.3. Higher MGO contents consistently led to longer lag phases. However, similar MGO contents in different honeys did not produce equivalent growth delays—for example, at 30 µg MGO/mL, honey 1 and honey 3 yielded growth delays of 4 and 6, respectively. 3-PLA alone (up to 2000 mg/kg) showed no bacteriostatic effect. When added to artificial honey containing 250 mg MGO/kg or more, 3-PLA dose-dependently increased growth delay; e.g., adding 2000 mg/kg 3-PLA to artificial honey with 400 mg/kg MGO increased growth delay from 4.06 to 5.05. In manuka honey MH30+ spiked with MGO and 3-PLA, adding 2000 mg/kg 3-PLA to honey containing 472 mg/kg MGO increased growth delay from 5.64 to 6.85. MGO stability testing showed that without 3-PLA, MGO concentration dropped from 120 mg/L to 7 mg/L over 24 hours, while with 600 mg/L 3-PLA, MGO decreased only to 35 mg/L. Gallic acid similarly enhanced MGO activity: at 400 mg/kg MGO, increasing gallic acid from 1500 mg/kg to 2000 mg/kg resulted in a bactericidal effect. Analysis of the four commercial honeys showed honey 1 (highest growth delay) contained 734 mg/kg 3-PLA and 636 mg/kg GAE, while honey 4 (lowest growth delay) contained 334 mg/kg 3-PLA and 386 mg/kg GAE. To achieve a growth delay of 5, honey 1 required 24 µg MGO/mL, while honey 4 required 31 µg MGO/mL. When comparing artificial honey spiked with 259 mg/kg MGO and 467 mg/kg 3-PLA to commercial manuka honey with identical MGO and 3-PLA levels, the commercial honey produced a growth delay of 6.2 at 27.3 µg MGO/mL versus 3.4 for the spiked artificial honey.

**Clinical Implications:** This study provides the first evidence that 3-PLA and polyphenols act as synergists enhancing MGO's antibacterial activity in manuka honey, partly through MGO stabilization. These findings have direct relevance for wound management applications, where manuka honey is used as a non-adherent dressing to maintain sterility and stimulate tissue regeneration. The results suggest that MGO content alone is insufficient to predict antibacterial potency; 3-PLA and polyphenol levels should also be considered in quality evaluation. However, the incomplete replication of commercial honey's antibacterial activity with MGO, 3-PLA, and gallic acid indicates additional unidentified compounds contribute to the overall effect. The authors recommend transferring this assay to other bacteria, particularly those lacking a proper glyoxalase system such as Staphylococcus aureus, and to other Gram-positive and Gram-negative bacteria.