Antibacterial Effect of Lemongrass (Cymbopogoncitratus) against the Aetiological Agents of Pitted Keratolyis

Abstract

Pitted keratolysis (PK) is a bacterial skin infection mostly affecting the pressure-bearing areas of the soles, causing unpleasant symptoms. Antibiotics are used for therapy, but the emergence of antiobiotic resistance, makes the application of novel topical therapeutic agents necessary. The antibacterial effects of 12 EOs were compared in the first part of this study against the three known aetiological agents of PK (Kytococcus sedentarius, Dermatophilus congolensis and Bacillus thuringiensis). The results of the minimal inhibitory concentration, minimal bactericidal concentration and spore-formation inhibition tests revealed that lemongrass was the most effective EO against all three bacterium species and was therefore chosen for further analysis. Seventeen compounds were identified with solid-phase microextraction followed by gas chromatography-mass spectrometry (HS-SPME/GC-MS) analysis while thin-layer chromatography combined with direct bioautography (TLC-BD) was used to detect the presence of antibacterially active compounds. Citral showed a characteristic spot at the Rf value of 0.47, while the HS-SPME/GC-MS analysis of an unknown spot with strong antibacterial activity revealed the presence of α-terpineol, γ-cadinene and calamenene. Of these, α-terpineol was confirmed to possess an antimicrobial effect on all three bacterium species associated with PK. Our study supports the hypothesis that, based on their spectrum, EO-based formulations have potent antibacterial effects against PK and warrant further investigation as topical therapeutics.

Keywords: antibacterial; bioautography; compound identification; gas chromatography; lemongrass; pitted keratolysis; solid-phase microextraction.

Figure 1

Drop plate antibacterial test results of the 12 essential oils on the three bacterium species associated with pitted keratolysis. Values represent the inhibition zones in diameters, determined around the drops. Clindamycin and erythromycin were used as positive standards (applied concentrations were 2 mg/mL).

Figure 2

Chromatogram of the HS-SPME/GC-MS analysis of the West Indian lemongrass essential oil (Cymbopogon citratus) used in this study obtained from Aromax Ltd. Each compound can be identified based on the data in Table 2 (tR (min)).

Figure 3

Compounds with antibacterial activities of lemongrass essential oil revealed on B. thuringiensis by TLC-DB. Order and quantity of the volatile test materials on the plate was the following: 1-lemongrass EO (0.2 mg); 2-citral (0.02 mg); 3-citral (0.04 mg); 4-citral (0.08 mg).

Figure 4

HS-SPME/GC-MS chromatogram of the white cut-out spot from TLC-BD silica plate.

Figure 5

Antibacterial components in the lemongrass EO used in this study after TLC-DB. (A) Plate under UV 254 nm, (B) TLC plate after treatment with vanillin–sulfuric acid reagent and documented in visible light, (C) TLC-DB assay: bioautograms using B. thuringiensis, (D) TLC-DB assay: bioautograms using K. sedentarius (bright zones indicate antibacterial effects). Mobile phases: dichloromethane and toluene–ethyl acetate 93:7 (v/v). The applied volumes of compounds used were as follows: lemongrass EO—6 µL, citral—4.5 and 6 µL, α-terpineol—1.5 µL. The stock solutions had concentrations as follows: lemongrass EO—200 mg/mL, citral—20 mg/mL, α-terpineol—100 mg/mL. Order and quantity of the volatile test materials on each plate were as follows: 1-lemongrass EO (1.2 mg); 2-citral (0.09 mg); 3-citral (0.12 mg); 4-α-terpineol (0.15 mg).

Figure 6

Antibacterial components in the lemongrass EO used in this study after TLC-DB. (A) plate under UV 254 nm, (B) TLC plate after treatment with vanillin–sulfuric acid reagent and documented in visible light, (C) TLC-DB assay: bioautograms using D. congolensis (bright zones indicate antibacterial effects). Mobile phases: dichloromethane and toluene–ethyl acetate 93:7 (v/v); 1, 4.5 and 6 µL indicated the applied volumes of the EO and the standards (lemongrass essential oil: 1µL, citral: 4.5 and 6 µL, α-terpineol: 4.5 µL). The lemongrass essential oil was applied without diluton and the stock solution in the cases of citral and α-terpineol were 100 mg/mL. Order and quantity of the volatile test materials on each plate were as follows: 1-lemongrass essential oil (undiluted); 2-citral (0.45 mg); 3-citral (0.6 mg); 4-α-terpineol (0.45 mg).