Introduction: Turpentine derivatives and eucalyptus oil are herbal substances traditionally used to treat various skin infections. Limited non-clinical data suggest they exert an immunological activity, but only scant information exists on their antibiotic effects. This in vitro study has been carried out to investigate the antibacterial and antifungal activity of a marketed skin ointment; its active pharmaceutical ingredients larch turpentine, eucalyptus oil, and turpentine oil; and their mixture, against bacteria and yeasts commonly present on the skin and causing skin infections. Methods: The antibiotic activity was tested using the drop dilution assay on the Gram-positive bacteria Staphylococcus aureus (wild type), a methicillin-resistant S. aureus strain, S. epidermidis, S. haemolyticus, Streptococcus pyogenes, the Gram-negative Pseudomonas aeruginosa, and the yeasts Candida albicans and C. tropicalis. Results: The ointment exerts a strong inhibitory effect on all Gram-positive bacteria at a concentration of 5 g/100 mL in the Müller-Hinton medium. It also has inhibiting effect on both Candida species but does not inhibit P. aeruginosa growth. As for the single active pharmaceutical ingredients, larch turpentine was the most active substance. The mixture of the three ingredients, in the concentrations used in the ointment, had a higher antibiotic effect than any of the individual ingredients studied, suggesting at least an additive activity. Conclusions: Our study has shown that the herbal ingredients and their combination exert antimicrobial activities, especially against Gram-positive bacteria, that justify their use in the treatment of skin infections.

The human skin is colonised by communities of microorganisms that are distinct according to the body site and organs, degree of moisture, pH, and temperature, as well as the chemical composition of the skin surface [1]. Several species of staphylococci, corynebacteria, propionibacteria, and yeasts colonise the skin in numbers that vary from a few hundred in exposed skin regions, such as the groin and axillae, to thousands per centimetre square [1]. They usually competitively inhibit other potentially pathogenic microorganisms, but some of them occasionally may progress from colonisers to pathogens [1‒3].

The most common skin infection agents are bacteria [4], mostly Gram-positive Staphylococcus species and Streptococcus pyogenes, as well as some Candida species [5]. Gram-negative bacteria such as Pseudomonas aeruginosa are no typical skin colonisers, but in rare cases, they also may cause cutaneous infections [2]. The diagnosis and management of bacterial skin and soft tissue infections are described in several guidelines [6], with some of them also addressing the treatment of multidrug-resistant microorganisms such as methicillin-resistant Staphylococcus aureus (MRSA) [7]. Topical treatment of skin infections includes mupirocin and fusidic acid creams and ointments, which are at least as effective as oral antibiotics in mild-to-moderate impetigo and are considered first-line empiric treatment of folliculitis [3].

Recently, herbal medicinal products have been proposed as adjuvants or even standalone treatments of skin infections caused by susceptible and multidrug-resistant bacteria [8]. Plant-derived secondary metabolites such as terpenes, flavones, flavonols, and some alkaloids and phenylpropanoids have shown antimicrobial activity in vitro [9], and essential oils have been suggested for the treatment of various types of acne [10]. Oregano oils, for instance, are apparently endowed with good antimicrobial properties [11].

Turpentine derivatives and eucalyptus oil are traditionally used to treat various skin infections [12]; for these substances, disinfectant, analgesic, antiparasitic [13], and antimicrobial activities [14‒16] have been described. Turpentine oil is derived from Pinus pinaster Ait. Its anti-inflammatory properties have been shown in animal studies [17].

Larch turpentine consists of the pure resin of the European larch (Larix decidua Mill.), and according to the (now superseded) Commission E official monograph, it can be used topically to treat furuncles based on its antiseptic and blood flow-enhancing effects [18]. The evidence for its efficacy, however, is scant and based only on naturopathic reports [19].

Eucalyptus oil is extracted from various Eucalyptus species and is described in the corresponding European Community Herbal Monograph as a traditional herbal medicinal product indicated for the relief of cough associated with cold [14]; however, some authors have also reported antimicrobial properties [15, 16]. Overall, however, the non-clinical and clinical data on the pharmacological activity of essential oils are scant at best, and little is known on their mode of action.

An ointment1 containing turpentine oil, larch turpentine, and eucalyptus oil as active pharmaceutical ingredients has been used for more than 90 years for the symptomatic treatment of mild, localised, purulent inflammations of the skin, and its efficacy has been demonstrated in clinical trials [20, 21]. Non-clinical data point to an immunological activity of the ointment [22], but no information exists on its antibacterial activity, and the evidence for antibiotic effects of its components is also scant and outdated [13‒16].

Turpentine oil, larch turpentine, and eucalyptus oil can be considered as safe at the dosages used in medicinal products. Turpentine’s main ingredients, α- and β-pinenes, have a low irritancy potential and are, therefore, included in the US Food and Drug Administration (FDA) inventory of compounds generally recognised as safe [13]. Genotoxic and mutagenic effects of turpentine after prolonged occupational exposure to turpentine fumes have been recently reported [23], but they hardly can be expected at the concentrations used in medicinal ointments. For eucalyptus oil, no undesirable effects are mentioned in the herbal monograph issued by the European Medicines Agency Committee on Herbal Medicinal Products [14]. While allergic contact dermatitis caused by repeated contact with Eucalyptus cinerea has been recently reported [24], the allergenic potential of eucalyptus oil is extremely uncommon, even if in rare cases it may present as severe cutaneous reactions [25]. At the dosages used in the ointment tested, toxic or irritative effects of the active ingredients are not to be expected; however, allergic reactions may occur in cases of predisposing hypersensitivity.

This in vitro study has been carried out to confirm in vitro the clinical data gathered by Fuchs-Algrim et al. [20] and Görne et al. [21] on the antibacterial and antifungal activity of a skin ointment and its individual components larch turpentine, eucalyptus oil, and turpentine oil, as well as their mixture in the concentrations used in the ointment, against bacterial and yeast microorganisms commonly present on the skin and sometimes causing skin infections.

Test Products

The ointment tested contains a lipophilic mixture of 7.2% pine turpentine oil (Gebrüder Unterweger GmbH, Assling, Austria), 5.4% larch turpentine (Gebrüder Unterweger GmbH, Assling, Austria), and 1.2% eucalyptus oil from E. globulus Labill (Düllberg Konzentra GmbH & Co. KG, Hamburg, Germany) as active pharmaceutical ingredients. The turpentine oil consists of approx. 95% monoterpenes such as α- and β-pinene. Larch turpentine contains up to 65% resin acids (mainly larixyl acetate) and essential oils, whereas eucalyptus oil includes ≥80% 1,8-cineole. Essential volatile oils (<0.3%) from Salvia rosmarinus Spenn. and Thymus vulgaris L., as well as thymol (all Frey + Lau GmbH, Henstedt-Ulzburg, Germany), are included in the ointment as excipients. The ointment basis contains 72% Vaseline (Hansen & Rosenthal KG, Hamburg, Germany), as well as beeswax (Kahl GmbH & Co. KG, Trittau, Germany), chlorophyll paste (Th. Geyer Ingredients GmbH & Co. KG, Höxter, Germany), stearin (Nordmann, Rassmann GmbH, Hamburg, Germany), oleic acid (VWR International GmbH, Darmstadt, Germany), and polysorbate 20 (Croda GmbH, Nettetal, Germany).

Strains and Antimicrobial Agents

The antibiotic activity of the ointment and its components was tested on the Gram-positive bacteria S. aureus wild type (ATCC 23923), MRSA (ATCC 700699), S. epidermidis (ATCC 12228), S. haemolyticus (SMIC 01), St. pyogenes (ATCC 19615), the Gram-negative bacterium P. aeruginosa (ATCC 27853), and the yeasts Candida albicans (ATCC 14053) and C. tropicalis (SMIC 02). Cultures were grown on Müller-Hinton agar (MHA, Oxoid) without supplementation at 37°C.

Study Design

We evaluated the effects of the marketed ointment, its individual active components, as well as of their mixture (in the ratio 5.4% larch turpentine, 1.2% eucalyptus oil, and 7.2% turpentine oil, as present in the ointment) (Table 1). The maximum concentration of the ointment in the growth medium (MHA) was chosen based on the solubility of the ointment in the medium. The concentrations of the pharmaceutical ingredients to be added to the medium were those present in the ointment. These amounts, as well as a lower and a higher concentration for each compound and the corresponding mixture, were tested to study any dose-dependent effects on microorganism growth.

Table 1.

Compounds tested and concentrations used

Test compounds and controlsg/100 mL MHA
Eucalyptus oil 0.6, 1.2, 20 
Larch turpentine 2.7, 5.4, 20 
Turpentine oil 3.6, 7.2, 20 
Mixture 6.9, 13.8 
Ointment 2.5, 5 
Vaseline (positive control) 2.5, 5 
Fusidic acid (negative control) 2.5, 5 
MHA (positive control) 
Test compounds and controlsg/100 mL MHA
Eucalyptus oil 0.6, 1.2, 20 
Larch turpentine 2.7, 5.4, 20 
Turpentine oil 3.6, 7.2, 20 
Mixture 6.9, 13.8 
Ointment 2.5, 5 
Vaseline (positive control) 2.5, 5 
Fusidic acid (negative control) 2.5, 5 
MHA (positive control) 

A negative control inhibits bacterial or yeast growth, whereas a positive control has no influence on growth. Bold: concentration of the active ingredient in the ointment.

MHA, Müller-Hinton agar.

Supplementation of Growth Media with the Test Products

After autoclaving, MHA was cooled to approximately 50°C, then the ointment, the active pharmaceutical ingredients, or the mixture, as appropriate, were added and thoroughly mixed with the medium. In addition to MHA, Vaseline (Bombastus, Freital, Germany) was also used as a positive control (i.e., no influence on bacterial or yeast growth). A cream containing fusidic acid (Fucidin 2% ointment, LEO Pharma A/S, Ballerup, Denmark), a topical antibiotic commonly used to treat bacterial skin infections [3], was used as a negative control (i.e., as a bacterial and yeast growth inhibitor).

Antibiotic Activity Testing

A drop dilution test [26, 27] was used to assess the concentration of viable bacterial and yeast cells in a liquid suspension. This assay involves preparing serial dilutions of the sample and depositing small volumes of each dilution onto a suitable growth medium. The plates are then incubated under appropriate conditions, and the number of colonies that grow from each drop is evaluated visually.

The microbial inoculum was prepared by culturing the microorganisms overnight and resuspending them in 0.85% NaCl. The suspensions were then adjusted to a final density of 1.5 × 108 colony-forming units (CFUs) per mL, at a final optical density of 0.5 McFarland for bacterial and 1 McFarland for yeast strains as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [28]. The microorganisms were diluted from 108 to 103 CFU/mL, and 10 µL of each dilution were spotted on MHA supplemented with the ointment, the individual APIs, or their mixture, at the concentrations listed in Table 1.

Plates were incubated at 37°C for 48 h and checked for growth at 24 h and 48 h. The results are based on the 48-h readings.

Measurement of Inhibition and Statistical Analysis

To determine the growth inhibition of the bacterial and yeast strains by the ointment and its active components, the growth on the positive controls (MHA and MHA supplemented with Vaseline) was used as the baseline value (100%). All experiments were performed in quadruplicates on two different days, thus yielding 8 replicates for each strain and condition tested.

No formal confirmative statistical analysis was carried out. Inhibition is presented graphically as the mean concentration at which inhibition was observed. As these data are only semi-quantitative and could be considered categorical, we did not compute any standard deviation or the corresponding non-parametrical counterparts (percentiles). Literature data estimate that the abundance of microorganisms on skin is 103–104/cm2, increasing to 106–107/cm2 in wet areas or during skin infections [29]. A biologically significant inhibition was arbitrarily defined as no or reduced growth (less than 75% of the control) at a concentration of 103 CFU/mL (log 10 value of 3, dashed line in the graphical displays) and no growth at higher concentration of the microorganisms studied. Figures were prepared using R version 4.3.2 [30], using the packages ggplot2 [31] and ggsci [32].

No growth inhibition was observed on MHA or MHA supplemented with Vaseline. As expected, most strains did not grow on fusidic acid already at a 2.5 g/100 mL concentration, and 5 g/100 mL fusidic acid completely inhibited growth of all strains tested (Fig. 1).

Fig. 1.

Antibacterial activity of Müller-Hinton agar (MHA), MHA supplemented with Vaseline, and Fucidin. Graphically, the more a mean of the 8 replicates (represented by a blue or red dot) is farther right, the larger is the inhibitory effect of the compound on the tested strains. Red dots indicate that the strains have been inhibited in their growth and blue dots indicate no inhibition. The scale from 0 to 6 refers to the log of the colony-forming unit (CFU) concentrations, thus indicating indirectly the level of inhibition, i.e., at which concentration the microorganism can grow, with 0: no inhibition (the microorganisms grew in all drops from 10 to 106 CFU/mL) and 6: maximum inhibition (no growth observed at any concentration). Biologically significant inhibition was arbitrarily defined as no or reduced growth at a concentration of 103 CFU/mL (log 10 value of 3, dashed line).

Fig. 1.

Antibacterial activity of Müller-Hinton agar (MHA), MHA supplemented with Vaseline, and Fucidin. Graphically, the more a mean of the 8 replicates (represented by a blue or red dot) is farther right, the larger is the inhibitory effect of the compound on the tested strains. Red dots indicate that the strains have been inhibited in their growth and blue dots indicate no inhibition. The scale from 0 to 6 refers to the log of the colony-forming unit (CFU) concentrations, thus indicating indirectly the level of inhibition, i.e., at which concentration the microorganism can grow, with 0: no inhibition (the microorganisms grew in all drops from 10 to 106 CFU/mL) and 6: maximum inhibition (no growth observed at any concentration). Biologically significant inhibition was arbitrarily defined as no or reduced growth at a concentration of 103 CFU/mL (log 10 value of 3, dashed line).

Close modal

A concentration of 5 g/100 mL of the ointment exerted a strong antibacterial activity against all Gram-positive bacteria tested (S. aureus WT and MRSA, S. epidermidis, S. haemolyticus, and St. pyogenes) but was ineffective against the Gram-negative P. aeruginosa. C. albicans growth was inhibited, and only a limited activity on C. tropicalis was observed. Decreasing the ointment concentration in MHA from 5 g/100 mL to 2.5 g/100 mL decreased the inhibitory activity of the ointment on all tested strains (Fig. 2). A 13.8 g/100 mL concentration of the mixture of the active ingredients suppressed Gram-positive bacteria and yeast growth. P. aeruginosa, however, grew at both mixture concentrations (Fig. 2), in accordance with the results from the experiments with the ointment.

Fig. 2.

Antibacterial and antifungal activity of the ointment (final concentration in MHA: 2.5% and 5%) and of the mixture of its three active pharmaceutical ingredients (larch turpentine, turpentine oil, and eucalyptus oil). Final concentration of the mixture in MHA: 6.9% and 13.8%). For additional details, see Fig. 1.

Fig. 2.

Antibacterial and antifungal activity of the ointment (final concentration in MHA: 2.5% and 5%) and of the mixture of its three active pharmaceutical ingredients (larch turpentine, turpentine oil, and eucalyptus oil). Final concentration of the mixture in MHA: 6.9% and 13.8%). For additional details, see Fig. 1.

Close modal

Turpentine oil inhibited only C. tropicalis and St. pyogenes both at 3.6 g/100 mL and 7.2 g/100 mL. An increase by a factor 2 of the turpentine oil concentration, therefore, did not improve its inhibitory effect in a dose-dependent manner (Fig. 3).

Fig. 3.

Individual antibacterial and antifungal activity of larch turpentine, turpentine oil, and eucalyptus oil. The second highest concentrations are those used in the formulation of the commercial ointment. For additional details, see Fig. 1.

Fig. 3.

Individual antibacterial and antifungal activity of larch turpentine, turpentine oil, and eucalyptus oil. The second highest concentrations are those used in the formulation of the commercial ointment. For additional details, see Fig. 1.

Close modal

The lowest larch turpentine concentration (2.7 g/100 mL) inhibited C. albicans, and 5.4 g/100 mL all Gram-positive bacteria and yeasts. No activity on P. aeruginosa was observed. Twenty g/100 mL larch turpentine completely suppressed the growth of S. haemolyticus, St. pyogenes, and C. tropicalis (Fig. 3). Concentrations of 0.6 and 1.2 g/100 mL eucalyptus oil had no effect on bacterial or yeast growth, and only 20 g/100 mL could inhibit the growth of all strains tested, except P. aeruginosa (Fig. 3).

Our study has shown that 5 g/100 mL of the ointment tested exerts a strong inhibitory effect on all Gram-positive bacteria. It has also a limited effect on C. albicans and C. tropicalis, but it does not inhibit P. aeruginosa growth. The results of the tests with the individual components suggest that larch turpentine accounts for the main share of the whole activity.

The mixture of the active pharmaceutical ingredients of the ointment, at a concentration of 6.9 g/100 mL, had a stronger inhibitory activity than any of the individual ingredients studied. This suggests at least an additive or possibly even a synergistic activity of the ingredients. A synergistic activity of these herbal extracts would fit previous observations made with other herbal medicines [33], showing that the combination of two or more herbal extracts may lead to enhanced efficacy, as already reported by other authors for other herbal medicinal products [34].

The marketed pull ointment investigated in this study is indicated for the treatment of mild, localised, purulent inflammations of the skin, such as nodules, inflamed hair follicles, and sweat gland inflammations2. Previous non-clinical studies have reported anti-inflammatory activity in vitro [22], and its clinical efficacy in the therapy of mild bacterial skin infections has been shown in double-blind and open, randomised clinical trials [20, 21].

We could confirm the antibacterial activity of eucalyptus oil as reported in previous publications [16, 35‒37] only at a 20 g/100 mL concentration; 0.6 g/mL and 1.2 g/100 mL eucalyptus oil were slightly active only against C. tropicalis. A systematic review [38] concluded that essential oils extracted from E. globulus are effective against MRSA and their efficacy can be enhanced by combining the oil with other essential oils and antibiotics. This agrees well with our hypothesis of an additive/synergistic activity of the ointment, and we suspect that only comparatively high eucalyptus oil concentrations are effective against bacteria and yeasts.

Study Limitations

In this study, we could not determine the minimum inhibitory concentration (MIC) of the individual components, the mixture, or the ointment. The drop test [26, 27, 39, 40] is not recommended as a routine assay by the EUCAST for MIC determination, but it provides valuable information on the viability of microorganisms in a sample. The drop dilution assay is a valuable tool in microbiology and has been used to quantify microbial populations [26], assess the efficacy of antimicrobial agents [41] and disinfectants [42], or to test environmental conditions on microbial growth [43]. In their guidance documents, the EUCAST recommends broth microdilution, disk diffusion, and gradient diffusion methods as the reference methods for antimicrobial susceptibility testing (AST) and provides a standardised framework for the performance of AST in microbiology laboratories, facilitating accurate and comparable susceptibility testing results across different settings. In our study, the hydrophobic nature of the ointments and APIs and the correspondingly low or absent diffusion in agar hindered the use of any of the methods recommended by the EUCAST. Diffusion is essential to create a gradient of antimicrobial concentration in the agar, which is necessary to determine the MIC of the antimicrobial agent against the microorganisms tested. Lipophilic antimicrobial agents do not diffuse evenly in the agar, leading to inconsistent results and difficulty in interpreting the inhibition zones. In such situations, the drop test is the only viable alternative to conduct AST.

This is the first time that the antimicrobial effects of the active ingredients of this ointment have been shown in a controlled in vitro model. The assay used has allowed determining the antimicrobial activity of the ointment and its components against a series of skin pathogens, thus confirming experimental studies in humans showing that the ointment studied is an effective and safe option for the treatment of skin infections [20], in particular folliculitis [21]. While these two studies have offered an empiric proof of efficacy in humans, our study has finally shown in a validated model that the herbal ingredients and their combination exert antimicrobial activities that justify their use in the treatment of skin infections.

The ointment and its components were kindly provided by Cesra Arzneimittel GmbH & Co. KG, Baden-Baden, Germany. SMIC strains were kindly supplied by the Clinical Microbiology Service of the local hospital (SMIC, cantonal hospital of Ticino, Switzerland).

An ethics statement was not required for this study type since no human or animal subjects or materials were used.

E.P. and C.F.-C. received a grant from Cesra Arzneimittel GmbH & Co. KG. O.P. is a consultant to Cesra. C.Z. and N.G. are employees of Cesra.

E.P. and C.F.-C. received a grant from Cesra Arzneimittel GmbH & Co. KG to carry out the laboratory experiments.

Conceptualisation: N.G. and C.Z.; methodology and writing – original draft: C.F.-C., E.P., and O.P.; investigation: C.F.-C. and E.P.; writing – review and editing: C.F.-C., E.P., O.P., C.Z., and N.G.; and visualisation: J.D.-M. and O.P. All authors gave final approval of the manuscript and are accountable for all aspects of the work.

1

ilon® Salbe classic, Cesra Arzneimittel GmbH & Co.KG, Germany.

2

Patient’s Information Leaflet, ilon® Salbe classic, Cesra Arzneimittel GmbH & Co. KG, Germany.

All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author or to the company.

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