Anti- Malassezia globosa (MYA-4889, ATCC) activity of Thai propolis from the stingless bee Geniotrigona thoracica

Abstract

Malassezia globosa, a lipophilic pathogen, is known to be involved in various chronic skin diseases. Unfortunately, the available treatments have unwanted side effects and microbial drug resistance is evolving. As the antimicrobial activity of propolis is outstanding, this study aimed to examine the potential of propolis from the stingless bee Geniotrigona thoracica against the yeast. Anti-M. globosa growth activity was ascertained in agar well diffusion and broth microdilution assays and the inhibitory concentration value at 50 % (IC₅₀) was determined. Since the yeast cannot synthesize its own fatty acids, extracellular lipase is important for its survival. Here, anti-M. globosa extracellular lipase activity was additionally investigated by colorimetric and agar-based methods. Compared to the crude hexane and crude dichloromethane extracts, the crude methanol partitioned extract (CMPE) exhibited the best anti-M. globosa growth activity with an IC₅₀ of 1.22 mg/mL. After CMPE was further enriched by silica gel column chromatography, fraction CMPE1 (IC₅₀ of 0.98 mM or 184.93 μg/mL) presented the highest activity and was later identified as methyl gallate (MG) by nuclear magnetic resonance analysis. Subsequently, MG was successfully synthesized and shown to have a similar activity, and a minimal fungicidal concentration of 43.44 mM or 8.00 mg/mL. However, lipase assay analysis suggested that extracellular lipase might not be the main target mechanism of MG. This is the first report of MG as a new anti-Malassezia compound. It could be a good candidate for further developing alternative therapeutic agents.

Keywords: Antimicrobial drug; Bee product; Lipase; Natural product; Skin disease.

Fig. 1

Scheme of the bioassay-guided fractionation of the CME G. thoracica propolis extract. The box with color indicates the extract that exhibited anti-M. globosa growth activity in the agar well diffusion assay.

Fig. 2

Propolis collecting sites: (A) a man-made wooden hive used for meliponiculture, (B) the position of propolis at the entrance, (C) inside of a G. thoracica hive, and (D) raw propolis sample of G. thoracica.

Fig. 3

Average diameter of the inhibition zone (mm) from the (A) CME, CHPE, CDPE, and CMPE samples at concentrations of 6.25–400 mg/mL, and (B) CMPE1–3 at concentrations of 3.13–50 mg/mL. Note, KTZ at 2 or 4 μg/mL was used as the positive control within each treatment. Absenting area of bar graph plot (square) indicates the values were not applicable. The SD of each data is shown above the graph bar.

Fig. 4

Chemical structure of MG [47].

Fig. 5

Percentage growth inhibition of CME, CMPE, MG, and CMPE3 at 50 mg/mL in the agar well diffusion assay. Values were calculated relative to the zone of inhibition of KTZ from each treatment. Data are shown as the mean ± 1 SD. Means with a different superscript letter are significantly different (p ≤ 0.05; One-way ANOVA, Dunnett's T3).

Fig. 6

Representative results from the agar well diffusion assay for (A) CMPE and (B) MG. The symbols “+2”, “+4” and “–” represent the positive control (2 or 4 $\mu$ g/mL KTZ) and solvent control (DMSO), respectively. The numbers represent the extract concentrations (mg/mL).

Fig. 7

Percentage growth inhibition of M. globosa in the broth microdilution assay by (A) CME, CMPE, and MG along with (B) their representative results in the resazurin assay and colony pattern. For CME and CMPE, the positive control was KTZ at 0.80 mg/mL and the solvent control was 10 % (v/v) DMSO/5 % (v/v) Tween 60. For MG, the positive control was KTZ at 0.03 mg/mL and the solvent control was 1 % (v/v) DMSO were used. Data are shown as the mean ± 1 SEM.

Fig. 8

Representative results of the resazurin assay (upper panel) and colony growth pattern of M. globosa (lower panel) after treatment with MG at concentrations of 2.72–54.30 mM (0.50–10.00 mg/mL). Abbreviation: “rep.” represents technical replicate.

Fig. 9

Detection of the extracellular lipase activity from M. globosa on (A) agar medium supplemented with indicator dye (TW60-Vic B agar) and (B) agar medium without indicator dye (TW60 agar). Note, PPL was used as a positive control along with M. globosa at a cell density of 10⁶–10⁷ cells/mL, while Na₂PO₄ buffer and 1× PBS were used as negative solvent controls when examining PPL and the yeast cells, respectively.

Fig. 10

Percentage lipase inhibition of (A) CME at 0.20–0.50 mg/mL, (B) CME, CMPE, CDPE, and CHPE at 0.025–0.1 mg/mL, and (C) MG, CMPE2, and CMPE3 at 0.02–0.50 mg/mL. Data are shown as the mean ± 1 SEM. Means with a different superscript letter are significantly different (p ≤ 0.05; One-way ANOVA, Tukey HSD).

Fig. 11

The (A) percentage lipase inhibition and (B) M. globosa lipase activity on TW60-Vic B agar plate from yeast after treatment with MG at 0.06–1.00 mg/mL (0.33–5.43 mM). Data are shown as the mean ± 1 SEM. Abbreviations; “PPL” and “Rep.” refer to porcine pancreatic lipase (2 mg/mL) and replicate, respectively.

Supplement 1

Representative TLC images showing the compound profile of extracted MG (lane 1A), synthetic MG (lane 1B), and pure/commercial MG as control (lane 1A and 1B) under UV light. The mobile phase was 2:23 MeOH: CH₂Cl₂.

Supplement 2

¹H NMR (A) and ¹³C NMR (B) peak data of the CMPE1 fraction as methyl gallate.