Effects of Natural Borneol on Germ Tube Formation and Preformed Biofilm Activity in Candida albicans

Abstract

Candida albicans infection mainly occurs in patients with suppressed immune function and is the main pathogen of nosocomial infections. The use of natural products aimed at controlling fungal diseases is considered an interesting alternative to synthetic fungicides due to their lower adverse reactions and the lower cost of plant preparations compared to modern conventional pharmaceuticals. Natural borneol has a long history of treating ulcers and local infections in traditional Chinese medicine. In this study, we present an analysis of the in vitro effects of natural borneol on planktonic cells of C albicans in the liquid and vapor phases. We also investigated the effects of natural borneol on germ tube formation and mature biofilm activity of C albicans. We found that vapor-phase borneol (minimum inhibitory concentration [MIC] 0.4 mg/cm³) inhibited C albicans more effectively than in the liquid phase (MIC 2 mg/mL). The C albicans germ tube decreased by 99% to 60% at sub-MICs of 0.5 to 0.125 mg/mL. The inhibitory effects of 0.25, 0.5, 1, 2, and 4 mg/mL borneol on the biofilm activity were 33.7%, 48.6%, 49.9%, 52.9%, and 58.2%, respectively. Natural borneol may have potential in the treatment and prevention of C albicans infections.

Introduction

Candida albicans is a fungus that usually colonizes the skin and mucous membranes without causing infection in healthy individuals. ¹ However, in immunocompromised patients, its overgrowth can cause a wide range of infections, ranging from superficial to septicemic candidiasis. ² Candidiasis and disseminated candidiasis together have a mortality rate of 40% and are the second leading cause of death from nosocomial infections in the United States. ³

Biofilms of Candida spp. are involved in the persistence or worsening of a number of chronic inflammatory diseases as well as acute deep systemic Candida infections. C albicans remains the most important causative pathogen of fungal biofilm diseases.^4,5 Most of the available antifungals are either ineffective against Candida biofilms, or are only effective at high concentrations that are associated with significant adverse side effects. ⁶ Therefore, new therapeutic strategies are urgently needed to treat Candida biofilm infections.

Natural borneol (Chinese name Tianranbingpian), an almost pure chemical substance with a chemical composition of (+)-borneol, has long been used in traditional Chinese medicine for the treatment of pain, burns, sprains and ulcers. It is a component of many essential oils (EOs) with known antifungal activity.^7–9 Recent studies have also shown that borneol can promote wound healing,^10,11 and has topical analgesic effects. ¹²

In this study, we used natural borneol extracted from fresh branches and leaves of Cinnamomum camphora trees. ¹³ We present an analysis of the in vitro effects of both liquid and vaporized natural borneol on planktonic cells of C albicans. We also investigated the effects of natural borneol on the germ tube formation and mature biofilm activity of C albicans.

Results

Minimum inhibitory concentration (MIC) and 50% inhibitory concentration (IC₅₀) Determination

The MIC and IC₅₀ values of borneol were determined against the various C albicans strains. The reference strain ATCC 10231 and 10 clinical isolates had a MIC of 0.4 mg/cm³ in the vapor phase and 2 mg/mL by agar and broth dilution methods. The IC₅₀ values varied from 0.77 mg/mL to 0.89 mg/mL. The results clearly demonstrate that vaporized borneol inhibited C albicans more effectively than liquid-phase borneol.

Effects of sub-MIC of Natural Borneol on C albicans Germ Tube Formation

Using liquid-phase natural borneol at sub-MICs of 1, 0.5, 0.25, and 0.13 mg/mL, we found that C albicans germ tube formation was significantly decreased by 99%, 97%, 78%, and 60%, respectively (Figure 1).

Figure 1.

The effect of sub-minimum inhibitory concentration (MIC) of natural borneol on germ tube formation in Candida albicans.

The Effect of Borneol on Preformed Biofilms of C albicans

Compared with the control group, we found that concentrations of 0.25, 0.5, 1, 2, and 4 mg/mL borneol significantly decreased C albicans biofilm activity by 33.7%, 48.6%, 49.9%, 52.9%, and 58.2%, respectively (Figure 2).

Figure 2.

The effect of borneol on the preformed biofilms of Candida albicans.

Discussion

To be effective, most of the available antifungals are either ineffective against Candida biofilms or require extremely high concentrations, often associated with adverse side effects. These limitations have led to the search for new molecules with anti-biofilm potential. New clinical approaches using natural compounds have been the subject of intense research. ⁶ Many studies have reported the effect of EOs on inhibiting fungal and bacterial growth and inhibiting biofilm activity in liquid and vapor phases.^7,9,14–18 These EOs are complex mixtures of compounds extracted from various plant materials, with most of them characterized by high concentrations of borneol, camphene, terpinene, and cis-sabinene hydrate.^7,9,15

In this study, we found that the anti-C albicans activity of natural borneol in the vapor phase (0.4 mg/cm³) was significantly higher than that in the liquid phase (2 mg/mL). This phenomenon has been observed in studies of the antibacterial properties of EOs. Researchers have reported that vapor generated by EOs has a greater antimicrobial effect compared to EOs in liquid form applied by direct contact.^19–22 The anti-Candida activity of natural borneol depends on its diffusibility and solubility in the liquid phase, while its activity in the vapor assay depends on its volatility. The fact that the biological activity of molecules in the vapor phase does not require direct contact suggests that such EOs could be used for the prevention and treatment of respiratory diseases. ²³ In addition, vaporized EOs could be used to protect food against the spread of microorganisms and extend their shelf life.^24–26 Studies have shown that the antibacterial effects of vaporized EOs include the degradation of the cell wall and cell membranes, structural changes to membrane proteins, leakage of cytolymph, condensation of the cytoplasm, and alterations in nuclear activity.^25,27,28 The formation of mycelia is critical for the virulence and biofilm development of C albicans: the mycelium form is more invasive than the yeast form.^29,30 The mycelial form can more easily invade the mucosa, causing mucosal infection or break through the mucosa, leading to candidiasis and disseminated candidiasis. However, fluconazole, the most common antifungal drug in clinical use, has no significant inhibitory effect on germ tube formation. ⁷ Therefore, the development of antifungal drugs to inhibit the biological process of mycelia transformation and prevent the transition of C albicans from yeast to biofilm is imperative to overcome drug resistance. ³¹ In this study, we found that concentrations of 0.125 mg/mL to 1 mg/mL (below the MIC values) of liquid-phase borneol (60%-99%) reduced the germ tube formation of C albicans ATCC 10231. Similar results have been reported using the EOs of Thymus camphoratus, ⁷ Bupleurum rigidum subsp. paniculatum (Brot.) H.Wolff, ³² Pelargonium capitatum and Cymbopogon martini, ³³ Bursera morelensis Ramirez, ³⁴ desert wormwood, ³⁵ and Lavandula angustifolia. ³⁶

Our study also found that borneol inhibited between 33.7% and 58.2% of the preformed biofilm activity at concentrations between 0.25 and 4 mg/mL. Other studies have shown that linalool and terpinen-4-ol, found in T camphoratus and Thymus carnosus EOs, were also able to disrupt preformed C albicans biofilms. ⁷ Moreover, thymol and terpinen-4-ol could inhibit the viability and membrane integrity of C albicans biofilms.^37,38

Our study demonstrated the antifungal activity of natural borneol against planktonic C albicans cells and its inhibition of preformed biofilms. However, further efforts are required to fully elucidate the mechanism of action of borneol at the molecular and biochemical levels, and its in vivo effects and toxicity, to determine whether it can be safely used as an antifungal drug. We believe that the translation of this research into the clinical field will lead to a reduction in Candida infections.

Materials and Methods

Chemicals and Strains

Natural borneol was purchased from Xinhuang Natural Borneol, Hunan, China. The C albicans reference strain ATCC 10231 was purchased from Anto Corporation, Henan, China. Ten clinical isolates were obtained from the clinical laboratory of Changzhou Cancer Hospital, Changzhou, China. Sabouraud dextrose broth (SDB) and Sabouraud dextrose agar (SDA) were purchased from Hangzhou Tianhe, Hangzhou, China. The strains were kept at −80°C in 20% glycerol broth.

Determination of Anti-C albicans Activity of Vapor-Phase Borneol

The effect of vapor-phase natural borneol on C albicans was studied with the inverted Petri dish method, as previously reported, ²² with some modifications. The 0.5 McFarland standard cell suspensions were taken and 100 μL was spread evenly on a 60-mm SDA Petri dish. A total of 500 μL of borneol solution in anhydrous ethanol, at concentrations of 5.4, 10.8, and 21.6 mg/mL, were placed on the surface of 60-mm-diameter sterile filter paper discs. After solvent evaporation, each disc was placed on the lid of an SDA Petri dish. The dish was sealed with Parafilm tape, and MIC values were calculated after 24 h of incubation at 37°C. The MIC, determined by comparison with the control (discs treated with absolute ethanol without borneol), was defined as the lowest concentration of borneol that inhibited the visible growth of C albicans, expressed by mass/plate space volume, that is, mg/cm³. The space inside the sealed Petri dish was calculated to be 27 cm³ of air. All tests were repeated 3 times.

Determination of the MIC of Borneol Using the Broth Dilution Method

The MIC of natural borneol was determined using the microbroth dilution method, as previously reported, ³⁹ with some modifications. A 0.5 McFarland standard cell suspension of C albicans was diluted 1:1000 in SDB and 100 μL was placed in a 96-well plate. Thereafter, 100 μL each of 8, 4, and 2 mg/mL borneol solution in 5% v/v Tween 80 was added to the 96-well plate and incubated at 37°C for 24 h. SDB medium containing 5% Tween 80 alone was used as a control. All tests were repeated 3 times. The MIC was determined as the lowest concentration of borneol that resulted in no visible growth of C albicans.

Determination of the MIC of Borneol Using the agar Dilution Method

The MIC of borneol against C albicans was also determined using an agar dilution assay. ³⁹ SDA plates with borneol concentrations of 1, 2, and 4 mg/mL in 5% v/v Tween 80 were prepared and inoculated with 10³ CFU of each C albicans strain. An SDA medium containing 5% Tween 80 was used as a control. All the plates were incubated at 37°C. The MIC was determined as the lowest concentration of borneol that prevented visible growth of the C albicans strains.

The IC₅₀ of natural borneol was determined using the microbroth dilution method, as previously reported, ⁴⁰ with some modifications. A 0.5 McFarland cell suspension was diluted 1:1000 in SDB and 100 μL was placed in a 96-well plate. Then, 100μL of, either 4, 3, 2, or 1 mg/mL of borneol solution (in 5% v/v Tween 80) was added to the 96-well plate and incubated at 37°C for 24 h. SDB medium containing 5% Tween 80 alone was used as a control. The absorbance was measured at 600 nm. The IC₅₀ value was expressed as the mean ± standard deviation (SD) of 3 independent experiments. Inhibition (%) of borneol in percent was calculated as [(A control−A sample)/A control] × 100, where A control is the absorbance of the control, and A sample is the absorbance of the borneol. The IC₅₀ was calculated using the linear relation between the borneol concentration and probability of the percentage of borneol inhibition.

Effects of sub-MIC of Natural Borneol on C albicans Germ Tube Formation

The assay was carried out as described by Alves et al. ³⁹ The density of the C albicans suspension was adjusted to 10⁶ CFU/mL, and 990 μL was added to each test tube. Ten μL borneol was added to each test tube to give final borneol concentrations of 1, 0.5, 0.25, 0.125, 0.063, and 0.031 mg/mL. The test tubes were then incubated for 3 h at 37°C. Germ tube formation was recorded under an optical microscope (40 × ). A solution of 1% v/v Tween 80 was used as the positive control. The results were expressed as the mean ± SD of 3 independent experiments.

Effects of Borneol on the Preformed Biofilm of C albicans

The effect of borneol on the preformed biofilm of C albicans was measured using triphenyltetrazolium chloride (TTC) as a metabolic indicator. ⁴¹ One hundred μL of C albicans cell suspension of 10⁶/mL in SDB was added to a 96-well plate and incubated at 37°C for 24 h. The supernatant was then removed, and the plates was washed 3 times with PBS to remove planktonic bacteria and unbound cells. One hundred μL of culture medium containing different concentrations (4, 2, 1, 0.5, 0.25, 0.13, 0.06, and 0.03 mg/ mL) of borneol, and 0.05% TTC, was added, before the plates were incubated for 24 h at 37°C and absorbance was measured at 500 nm. Culture medium without borneol was used as a control. The results were expressed as the mean ± SD of 3 independent experiments.

Statistical Analysis

Statistical significance was determined using one-way analysis of variance (ANOVA), followed by Dunnett's post hoc test. Statistical analyses were performed using SPSS19. P values of P < .05 were considered as significant.