# Assessment of Antifungal Activity of Bakuchiol on Oral-AssociatedCandida spp.

Authors: Mohd-Al-Faisal Nordin; Fathilah Abdul Razak; Wan Harun Himratul-Aznita
Journal: Evidence-Based Complementary and Alternative Medicine
 (2015)

---

## Abstract

Bakuchiol is an active component ofPsoralea glandulosa andPsoralea corylifolia, used in traditional Chinese medicine. The study aimed at investigating the antifungal activity of bakuchiol on planktonic and biofilm forms of orally associatedCandida species. The antifungal susceptibility testing was determined by the broth micro dilution technique. Growth kinetics and cell surface hydrophobicity (CSH) ofCandida were measured to assess the inhibitory effect of bakuchiol onCandida planktonic cells. Biofilm biomass and cellular metabolic activity were quantitatively estimated by the crystal violet (CV) and the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assays. AllCandida strains have been shown to be susceptible to bakuchiol with the MIC ranges from 12.5 to 100 μg/mL. Significant decrease in specific growth rates and viable counts demonstrates the inhibitory effect of bakuchiol onCandida planktonic cells. A brief exposure to bakuchiol also reduced CSH ofCandida (P
<
0.05), indicating altered surface properties of yeast cells towards hydrophobic interfaces. Biofilm biomass and cell metabolic activity were mostly decreased, except forC. glabrata (P
=
0.29). The antifungal properties of bakuchiol onCandida species in thisin vitro study may give insights into the application in therapeutic strategy againstCandida infections.


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## Body

## 1. Introduction

Given the fact that at least 50% of healthy individuals are the carriers ofCandida species, the commensal organisms are regarded as potentially pathogenic in susceptible hosts [1]. The infection is primarily depending on the immunological status of the host. Both local and systemic risk factors may result in weakened immune functions that mediateCandida colonisation on host surfaces [2].Candida species has a vibrant cell surface embedded with protein components that favour physical interaction to host surfaces. The adherence mechanisms are possibly mediated through both nonspecific and specific bindings. Previous studies reported that the cell surface hydrophobicity (CSH) ofCandida yeast cells is a putative virulence factor, and its expressed proteins may affect the CSH status ofCandida to interact with the cells and the surfaces [3, 4]. In addition, salivary pellicle components such as statherin and α-amylase and the complex bindings through lectin-like or protein-protein-type interactions have been known to be responsible for cell colonisation in disease progression [5, 6].The pathogenesis of oral candidiasis has been closely associated with the biofilm formation ofCandida on the oral surfaces [7]. Biofilm displays distinct biological properties compared to its planktonic counterparts which enable resistance to antifungal drugs.C. albicans biofilm consists of polysaccharide matrix-enclosed microcolonies of yeasts and hyphae with distinct biological properties from planktonic forms [8]. AlthoughC. albicans remains the principle etiological agent, reports on the prevalence of non-albicans Candida (NAC) species associated with invasive candidiasis reflect the significance of NAC in clinical samples.C. tropicalis,C. krusei, andC. glabrata are the predominant NAC isolates from neonatal candidemia [9–12]. Concern on the shift towards NAC species arising from antifungal resistance and side effects of conventional treatments has led the search for potential bioactive components from plants [10, 13].Natural products from plants have been subjected for treatment because they are rich in a numerous variety of secondary metabolites with antimicrobial properties [14]. This has prompted the research interest on novel mechanism of action of plant-derived bioactive compounds for better therapeutic strategies. Bakuchiol (Figure 1) is one of active components ofPsoralea glandulosa leaves, commonly used in folk medicine for the treatment of skin diseases caused by bacteria and fungi [15, 16]. It was also found in the seeds ofPsoralea corylifolia. The seed oil has been used for the treatment of leucoderma, psoriasis, and leprosy [17], and the crude extract has been suggested as a remedy for bone fractures, osteomalacia, and osteoporosis [18]. Bakuchiol has shown diverse therapeutic properties including antibacterial and anticancer activities [19–21]. It is also reported to exhibit effectiveness towards pathophysiologic features of acne [22], suggesting its potential use in cosmetic formulation. Therefore, the present study aimed at investigating the antifungal properties of bakuchiol on oral-associatedCandida species. The assessment of antifungal activity onCandida planktonic and biofilm was undertaken to characterise the mode of action of bakuchiol for the development of therapeutic agents specifically against candidal infections in the oral cavity.Figure 1
Chemical structure of bakuchiol.


## 2. Materials and Methods

### 2.1.Candida Strains and Bioactive Compound

Candida strains purchased from the American Type Culture Collection (ATCC),C. albicansATCC 14053,C. dubliniensis ATCC MYA-2975,C. glabrata ATCC 90030,C. krusei ATCC 14243,C. lusitaniae ATCC 64125, andC. tropicalis ATCC 13803, were used in the study. Cells were stored at −70°C as glycerol stocks and propagated by streaking a loopful of cells onto yeast peptone dextrose (YPD) agar (15% w/v yeast extract, 31% w/v peptone, 31% w/v dextrose, 23% w/v agar) and incubated overnight at 37°C. Bakuchiol is one of major components ofPsoralea corylifolia L. seed extract, identified by UV, IR, Mass, 1H, and 13C NMR spectra and melting point [23]. For thisin vitro study, bakuchiol (purity: ≥95% HPLC) purchased from ChromaDex Inc. was dissolved in 1% v/v dimethyl sulfoxide (DMSO) (a stock concentration of 1000 μg/mL) and stored at −20°C until use. Amphotericin B (Sigma-Aldrich; purity: ~80% HPLC) was used as a positive control in the experimental assays.


### 2.2. Culture Condition and Cell Inoculum

A single colony was inoculated into 10 mL YPD medium (20% w/v yeast extract, 40% w/v peptone, and 40% w/v dextrose) and grown overnight in an orbital shaker (150–180 rpm) at 30°C. Under this condition,Candida grows as budding yeast [24]. Cell cultures were harvested by centrifugation at 2000 ×g and washed with phosphate-buffered saline (PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride, and 137 mM sodium chloride, pH 7.2). A standard inoculum was then adjusted to 1 × 106 cells/mL (OD550 nm = 0.144).


### 2.3. Antifungal Susceptibility Testing

The minimum inhibitory concentration (MIC) assay was carried out by the standard broth microdilution method in YPD medium according to the Clinical and Laboratory Standards Institute (CLSI) reference method M27-A3 [25]. Inoculum of 1 × 103 yeast cells/mL was added to each well of microtiter plates containing different concentrations of bakuchiol which ranged from 1.5 to 100 μg/mL. The plates were incubated overnight at 37°C. The MIC endpoint was determined as the lowest concentration that caused a significant diminution (≥50% inhibition) of growth relative to the untreated groups [26]. Following this, the minimum fungicidal concentration (MFC) assay was carried out by spreading aliquots of 50 μL from the well showing no visible growth on YPD agar plates. Absence of viable growth following 24 to 48 h incubation indicated the MFC of compound on the respective strains.


### 2.4. Growth Kinetics

To analyse the effect of bakuchiol onCandida growth, the yeast cells (1 × 103 cells/mL) prepared in Section 2.2 were exposed to 0.5 × MIC of bakuchiol for 30 min. The treated cells were centrifuged, washed, and resuspended in PBS. These cells were then grown in YPD medium at 37°C for 18 h. At stipulated time intervals (3, 6, 9, and 12 h), the cell growth was measured at 550 nm. The growth of respectiveCandida species was distinguished by measuring the specific growth rate (μ) using the equation previously described [27]:(1)
μ
=
In
⁡
N
t
-
In
⁡
N
0
t
2
-
t
1
,where μ is the average specific growth rate, N
t represented the number of cells at log phase, N
0 represented the number of cells at zero time, t
2 was the time taken to reach plateau, and t
1 was the time when the cells entered the log phase. μ values were distinguished from the exponential phase between 6 h to 12 h, during which the cells appearing per unit time were proportional to the present population. The percent (%) inhibition in average specific growth rate following bakuchiol exposure was then calculated. The cell growth was further determined based on viable counts (CFU). After 12 h, 100 μL from each well was aspirated and serially diluted tenfold in sterile distilled water. 100 μL of each dilution was spread on YPD agar. Following 48 h incubation at 37°C, the CFU was enumerated.


### 2.5. Cell Surface Hydrophobicity

For this assay, the inoculum of 1 × 108 yeast cells/mL (OD550 nm = 0.5) was prepared. The hydrophobicity of untreated and bakuchiol-treated planktonic cells was determined by the biphasic hydrocarbon/aqueous method according to Anil et al. [28]. Aliquots 5 mL of inoculum were centrifuged at 8000 ×g, and the solution was discarded. The pellets were briefly exposed to 0.5 × MIC of bakuchiol for 15 min. The treated cells were centrifuged, washed, and resuspended in PBS. 4 mL from each sample was transferred into glass tubes, and the absorbance (A
0) was measured at 550 nm. Following this, 200 μL of hexadecane (Sigma-Aldrich) was added, vortexed vigorously, and left at room temperature for 30 min to allow for cells separated into biphasic state. The absorbance of the lower hydrophilic layer (A
1) was measured again. The CSH of eachCandida was expressed as the percentage decrease in optical density of the aqueous phase of the test using the following formula: (2)
Change  in
A
550
%
=
A
0
-
A
1
A
0
×
100
.


### 2.6. Biofilm Formation

Using the cell inoculum prepared in Section2.2, both single- and mixed-species biofilms ofCandida were allowed to form on commercially available polystyrene, flat-bottom 96-well microtiter plates (Thermo Scientific Nunc) [29]. For mixed-species biofilms, the cell suspension was instead replaced withC. albicans and each of NAC species at a ratio 1 : 1. The surface of wells was coated with 50 μL of clarified saliva and incubated for 90 min at 37°C. The saliva was then aspirated and 20 μL of cell suspension of the respective single and mixed species was added into each well to form the single and mixed biofilms. After 60 min of adhesion phase, 100 μL of YPD medium was added into each well, and the plates were incubated overnight in a rotary shaker at 37°C. After biofilm formation, the medium was aspirated, and the biofilms were gently washed with PBS to remove nonadherent cells. Biofilm biomass and the cellular metabolic activity were quantitatively measured using crystal violet staining and XTT reduction assay, respectively.


### 2.7. Biofilm Quantitation

#### 2.7.1. Crystal Violet Assay

Candida planktonic cells (Section 2.2) were exposed to 0.5 × MIC of bakuchiol for 30 min. Biofilms were then developed using these cells following the same procedure described in Section 2.6. After washing, the biofilms were fixed with ethanol and stained with 50 μL of 0.1% w/v crystal violet solution for 15 min without agitation. The biofilms were washed three times and destained with 95% v/v ethanol, following which 75 μL of the solution was transferred into new wells, and their absorbance was measured at 595 nm using a microtiter plate reader (SpectraMAX 340 Tunable Microplate Reader).


#### 2.7.2. XTT Reduction Assay

This assay was undertaken to examine the effect of bakuchiol on viability ofCandida cells within the biofilms, which relies on the reduction of yellow tetrazolium salt XTT by dehydrogenase enzymes of metabolically active cells yielding an orange-coloured, water-soluble formazan [30]. Biofilms established from inoculum prepared in Section 2.2 were exposed to 0.5 × MIC of bakuchiol for 2 h. Afterwards, a total of 100 μL XTT-menadione (10 : 1) solution, consisting of XTT sodium salt (Sigma-Aldrich) mixed with menadione (Sigma-Aldrich) solution, (1 mM in acetone; Sigma-Aldrich) was dispensed into each well. The plate was covered in aluminium foil and incubated in the dark for 2 h at 37°C. Following this, 75 μL of the solution was transferred into new wells, and the amount of colorimetric change (a reflection of the metabolic activity of biofilm cells) was measured at 490 nm.


### 2.8. Statistical Analysis

Statistical analyses were performed using SPSS software (version 18.0). An independentt-test was used to compare the significant differences between controls (untreated) and bakuchiol-treated samples. A P value of < 0.05 was considered statistically significant.


## 2.1.Candida Strains and Bioactive Compound

Candida strains purchased from the American Type Culture Collection (ATCC),C. albicansATCC 14053,C. dubliniensis ATCC MYA-2975,C. glabrata ATCC 90030,C. krusei ATCC 14243,C. lusitaniae ATCC 64125, andC. tropicalis ATCC 13803, were used in the study. Cells were stored at −70°C as glycerol stocks and propagated by streaking a loopful of cells onto yeast peptone dextrose (YPD) agar (15% w/v yeast extract, 31% w/v peptone, 31% w/v dextrose, 23% w/v agar) and incubated overnight at 37°C. Bakuchiol is one of major components ofPsoralea corylifolia L. seed extract, identified by UV, IR, Mass, 1H, and 13C NMR spectra and melting point [23]. For thisin vitro study, bakuchiol (purity: ≥95% HPLC) purchased from ChromaDex Inc. was dissolved in 1% v/v dimethyl sulfoxide (DMSO) (a stock concentration of 1000 μg/mL) and stored at −20°C until use. Amphotericin B (Sigma-Aldrich; purity: ~80% HPLC) was used as a positive control in the experimental assays.


## 2.2. Culture Condition and Cell Inoculum

A single colony was inoculated into 10 mL YPD medium (20% w/v yeast extract, 40% w/v peptone, and 40% w/v dextrose) and grown overnight in an orbital shaker (150–180 rpm) at 30°C. Under this condition,Candida grows as budding yeast [24]. Cell cultures were harvested by centrifugation at 2000 ×g and washed with phosphate-buffered saline (PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride, and 137 mM sodium chloride, pH 7.2). A standard inoculum was then adjusted to 1 × 106 cells/mL (OD550 nm = 0.144).


## 2.3. Antifungal Susceptibility Testing

The minimum inhibitory concentration (MIC) assay was carried out by the standard broth microdilution method in YPD medium according to the Clinical and Laboratory Standards Institute (CLSI) reference method M27-A3 [25]. Inoculum of 1 × 103 yeast cells/mL was added to each well of microtiter plates containing different concentrations of bakuchiol which ranged from 1.5 to 100 μg/mL. The plates were incubated overnight at 37°C. The MIC endpoint was determined as the lowest concentration that caused a significant diminution (≥50% inhibition) of growth relative to the untreated groups [26]. Following this, the minimum fungicidal concentration (MFC) assay was carried out by spreading aliquots of 50 μL from the well showing no visible growth on YPD agar plates. Absence of viable growth following 24 to 48 h incubation indicated the MFC of compound on the respective strains.


## 2.4. Growth Kinetics

To analyse the effect of bakuchiol onCandida growth, the yeast cells (1 × 103 cells/mL) prepared in Section 2.2 were exposed to 0.5 × MIC of bakuchiol for 30 min. The treated cells were centrifuged, washed, and resuspended in PBS. These cells were then grown in YPD medium at 37°C for 18 h. At stipulated time intervals (3, 6, 9, and 12 h), the cell growth was measured at 550 nm. The growth of respectiveCandida species was distinguished by measuring the specific growth rate (μ) using the equation previously described [27]:(1)
μ
=
In
⁡
N
t
-
In
⁡
N
0
t
2
-
t
1
,where μ is the average specific growth rate, N
t represented the number of cells at log phase, N
0 represented the number of cells at zero time, t
2 was the time taken to reach plateau, and t
1 was the time when the cells entered the log phase. μ values were distinguished from the exponential phase between 6 h to 12 h, during which the cells appearing per unit time were proportional to the present population. The percent (%) inhibition in average specific growth rate following bakuchiol exposure was then calculated. The cell growth was further determined based on viable counts (CFU). After 12 h, 100 μL from each well was aspirated and serially diluted tenfold in sterile distilled water. 100 μL of each dilution was spread on YPD agar. Following 48 h incubation at 37°C, the CFU was enumerated.


## 2.5. Cell Surface Hydrophobicity

For this assay, the inoculum of 1 × 108 yeast cells/mL (OD550 nm = 0.5) was prepared. The hydrophobicity of untreated and bakuchiol-treated planktonic cells was determined by the biphasic hydrocarbon/aqueous method according to Anil et al. [28]. Aliquots 5 mL of inoculum were centrifuged at 8000 ×g, and the solution was discarded. The pellets were briefly exposed to 0.5 × MIC of bakuchiol for 15 min. The treated cells were centrifuged, washed, and resuspended in PBS. 4 mL from each sample was transferred into glass tubes, and the absorbance (A
0) was measured at 550 nm. Following this, 200 μL of hexadecane (Sigma-Aldrich) was added, vortexed vigorously, and left at room temperature for 30 min to allow for cells separated into biphasic state. The absorbance of the lower hydrophilic layer (A
1) was measured again. The CSH of eachCandida was expressed as the percentage decrease in optical density of the aqueous phase of the test using the following formula: (2)
Change  in
A
550
%
=
A
0
-
A
1
A
0
×
100
.


## 2.6. Biofilm Formation

Using the cell inoculum prepared in Section2.2, both single- and mixed-species biofilms ofCandida were allowed to form on commercially available polystyrene, flat-bottom 96-well microtiter plates (Thermo Scientific Nunc) [29]. For mixed-species biofilms, the cell suspension was instead replaced withC. albicans and each of NAC species at a ratio 1 : 1. The surface of wells was coated with 50 μL of clarified saliva and incubated for 90 min at 37°C. The saliva was then aspirated and 20 μL of cell suspension of the respective single and mixed species was added into each well to form the single and mixed biofilms. After 60 min of adhesion phase, 100 μL of YPD medium was added into each well, and the plates were incubated overnight in a rotary shaker at 37°C. After biofilm formation, the medium was aspirated, and the biofilms were gently washed with PBS to remove nonadherent cells. Biofilm biomass and the cellular metabolic activity were quantitatively measured using crystal violet staining and XTT reduction assay, respectively.


## 2.7. Biofilm Quantitation

### 2.7.1. Crystal Violet Assay

Candida planktonic cells (Section 2.2) were exposed to 0.5 × MIC of bakuchiol for 30 min. Biofilms were then developed using these cells following the same procedure described in Section 2.6. After washing, the biofilms were fixed with ethanol and stained with 50 μL of 0.1% w/v crystal violet solution for 15 min without agitation. The biofilms were washed three times and destained with 95% v/v ethanol, following which 75 μL of the solution was transferred into new wells, and their absorbance was measured at 595 nm using a microtiter plate reader (SpectraMAX 340 Tunable Microplate Reader).


### 2.7.2. XTT Reduction Assay

This assay was undertaken to examine the effect of bakuchiol on viability ofCandida cells within the biofilms, which relies on the reduction of yellow tetrazolium salt XTT by dehydrogenase enzymes of metabolically active cells yielding an orange-coloured, water-soluble formazan [30]. Biofilms established from inoculum prepared in Section 2.2 were exposed to 0.5 × MIC of bakuchiol for 2 h. Afterwards, a total of 100 μL XTT-menadione (10 : 1) solution, consisting of XTT sodium salt (Sigma-Aldrich) mixed with menadione (Sigma-Aldrich) solution, (1 mM in acetone; Sigma-Aldrich) was dispensed into each well. The plate was covered in aluminium foil and incubated in the dark for 2 h at 37°C. Following this, 75 μL of the solution was transferred into new wells, and the amount of colorimetric change (a reflection of the metabolic activity of biofilm cells) was measured at 490 nm.


## 2.7.1. Crystal Violet Assay

Candida planktonic cells (Section 2.2) were exposed to 0.5 × MIC of bakuchiol for 30 min. Biofilms were then developed using these cells following the same procedure described in Section 2.6. After washing, the biofilms were fixed with ethanol and stained with 50 μL of 0.1% w/v crystal violet solution for 15 min without agitation. The biofilms were washed three times and destained with 95% v/v ethanol, following which 75 μL of the solution was transferred into new wells, and their absorbance was measured at 595 nm using a microtiter plate reader (SpectraMAX 340 Tunable Microplate Reader).


## 2.7.2. XTT Reduction Assay

This assay was undertaken to examine the effect of bakuchiol on viability ofCandida cells within the biofilms, which relies on the reduction of yellow tetrazolium salt XTT by dehydrogenase enzymes of metabolically active cells yielding an orange-coloured, water-soluble formazan [30]. Biofilms established from inoculum prepared in Section 2.2 were exposed to 0.5 × MIC of bakuchiol for 2 h. Afterwards, a total of 100 μL XTT-menadione (10 : 1) solution, consisting of XTT sodium salt (Sigma-Aldrich) mixed with menadione (Sigma-Aldrich) solution, (1 mM in acetone; Sigma-Aldrich) was dispensed into each well. The plate was covered in aluminium foil and incubated in the dark for 2 h at 37°C. Following this, 75 μL of the solution was transferred into new wells, and the amount of colorimetric change (a reflection of the metabolic activity of biofilm cells) was measured at 490 nm.


## 2.8. Statistical Analysis

Statistical analyses were performed using SPSS software (version 18.0). An independentt-test was used to compare the significant differences between controls (untreated) and bakuchiol-treated samples. A P value of < 0.05 was considered statistically significant.


## 3. Results

### 3.1. Antifungal Susceptibility of Planktonic Cells

The antifungal activity of bakuchiol againstCandida species was tabulated in Table 1. The MIC and MFC endpoints were ranged from 12.5 to 100 μg/mL.C. albicans andC. dubliniensis were found to be the most susceptible to bakuchiol, andC. glabratawas the least susceptible to bakuchiol.Table 1
Antifungal activity of bakuchiol againstCandidaspecies.
Antifungal susceptibility testa (µg/mL)
1
2
3
4
5
6
Bakuchiol
MIC
25
12.5
>100
50
50
50
MFC
50
25
>100
50
100
100
Amphotericin B
MIC
1.95
1.95
1.95
1.95
7.81
1.95
MFC
3.91
1.95
3.91
1.95
7.81
3.91
a(1) C. albicansATCC 14053, (2) C. dubliniensis ATCC MYA-2975, (3) C. glabrata ATCC 90030, (4) C. krusei ATCC 14243, (5) C. lusitaniae ATCC 64125, and (6) C. tropicalis ATCC 13803.


### 3.2. Effect of Bakuchiol on Cell Growth

A significant difference inCandida cell growth was observed after 6 h, which indicates the exponential stage ofCandida strains. Bakuchiol exhibited considerable growth inhibitory effect against most tested strains (Table 2). In detail, μ values ofC. albicans,C. dubliniensis, andC. lusitaniae were markedly reduced by >50% compared with the untreated yeast cells (P
<
0.05). On the other hand,C. glabrata andC. tropicalis were reduced to 38% and 35%, respectively. No significant differences were observed forC. krusei (11%). Based on CFU, the population ofC. albicans,C. dubliniensis,C. glabrata,C. krusei, andC. lusitaniae were decreased by 1.2- to 1.5-fold change (P
<
0.05) following exposure to bakuchiol. No significant differences were observed forC. tropicalis (P
=
0.19).Table 2
Changes in the specific growth rates (µ) and the viable counts (CFU) of Candidaspecies following bakuchiol exposure. The percentage denotes the inhibition of treated samples compared to the untreated ones.
Microorganism
Treatmenta
µ
(
%
)
Log10 CFU/mL (%)
C. albicans ATCC 14053
Untreated
1.73 ± 0.08
10.04 ± 0.03
Bakuchiol
0.70 ± 0.07 (59.5)∗
7.24 ± 0.08 (27.9)∗
Amphotericin B
0.49 ± 0.10 (71.7)∗
6.64 ± 0.05 (33.9)∗
C. dubliniensis ATCC MYA-2975
Untreated
0.98 ± 0.14
8.32 ± 0.28
Bakuchiol
0.45 ± 0.24 (54.1)∗
6.87 ± 0.42 (17.4)∗
Amphotericin B
0.74 ± 0.19 (24.5)∗
7.36 ± 0.09 (11.5)∗
C. glabrata ATCC 90030
Untreated
1.80 ± 0.06
10.13 ± 0.08
Bakuchiol
1.11 ± 0.03 (38.3)∗
8.34 ± 0.07 (17.7)∗
Amphotericin B
1.47 ± 0.13 (18.3)∗
9.20 ± 0.07 (9.2)∗
C. krusei ATCC 14243
Untreated
0.82 ± 0.20
8.63 ± 0.45
Bakuchiol
0.73 ± 0.15 (11)
5.72 ± 0.35 (33.7)∗
Amphotericin B
0.55 ± 0.12 (32.9)∗
6.32 ± 0.21 (26.8)∗
C. lusitaniae ATCC 64125
Untreated
1.09 ± 0.15
8.95 ± 0.36
Bakuchiol
0.42 ± 0.10 (61.5)∗
6.90 ± 0.16 (22.9)∗
Amphotericin B
0.98 ± 0.24 (10.1)
8.69 ± 0.50 (2.9)
C. tropicalis ATCC 13803
Untreated
1.57 ± 0.14
9.80 ± 0.43
Bakuchiol
1.02 ± 0.18 (35)∗
9.30 ± 0.23 (5.5)
Amphotericin B
0.91 ± 0.12 (42)∗
8.63 ± 0.08 (11.9)∗
aTest concentration was prepared at 0.5 × MIC. ∗
P
<
0.05 compared to the untreated samples.


### 3.3. Effect of Bakuchiol on Cell Surface Hydrophobicity

The effect of bakuchiol on cell surface hydrophobicity (CSH) ofCandida species was measured based on the percentage of cell adsorption to hexadecane (Figure 2). The percentages ofC. albicans,C. krusei, andC. tropicalis were significantly higher (P
<
0.05) than those of the otherCandida species. Findings show that the cell hydrophobicities were relatively compromised and decreased following brief exposure ofCandida planktonic cells to 0.5 × MIC of bakuchiol. The CSH ofC. albicans,C. dubliniensis,C. krusei, andC. tropicalis decreased within the range of 10% to 38% (P
<
0.05). Bakuchiol, however, exhibited least effect onC. glabrata andC. lusitaniae when compared with the untreated yeast cells.Figure 2
Cell surface hydrophobicity ofCandida species following bakuchiol exposure. Data are represented as mean ± SD of three independent experiments performed in triplicate. Amphotericin B used as a positive control. Asterisk (∗) denotes the significant difference between treated samples and the untreated ones (one-way ANOVA; P
<
0.05).


### 3.4. Effect of Bakuchiol onCandida Biofilm

Candida species were able to produce moderate-to-high degree of biomass after 24 h cultivated in a microtiter plate.C. albicans andC. tropicalis produced a dense biomass distinct fromC. dubliniensis,C. glabrata,C. krusei, andC. lusitaniae (P
<
0.05). Bakuchiol exposure showed significant reduction in mostCandida biomass production, except forC. glabrata (P
=
0.29) (Figure 3). Based on XTT assay, bothC. albicans andC. glabrata exhibited the highest XTT metabolic activity compared to the others. In mixed biofilms, XTT activity ofC. albicans andC. glabrata remains high (P
<
0.01) when compared to the other mixed culture biofilms (Table 3). Following bakuchiol exposure (2 h), XTT activity inC. albicans andC. tropicalis was reduced (P
<
0.05) relative to the untreated samples. No significant differences were observed on other single species biofilms. For mixed biofilms, onlyC. albicans andC. dubliniensis were markedly reduced by >50% (P
<
0.05), followed by mixedC. albicans andC. krusei biofilm in response to bakuchiol.Table 3
The metabolic activity of single and mixed biofilms when exposed to bakuchiol was measured. Values represent absorbance using XTT reduction assay.
Biofilms
Untreated
Bakuchiol
Amphotericin B
Single species
C. albicans
1.042 ± 0.049†
0.417 ± 0.085α
0.266 ± 0.046α,β
C. dubliniensis
0.279 ± 0.016
0.274 ± 0.029
0.174 ± 0.026α,β
C. glabrata
0.723 ± 0.040†
0.896 ± 0.096
0.353 ± 0.061α,β
C. krusei
0.467 ± 0.048
0.400 ± 0.085
0.228 ± 0.079α,β
C. lusitaniae
0.192 ± 0.016
0.182 ± 0.017
0.242 ± 0.030α,β
C. tropicalis
0.351 ± 0.037
0.243 ± 0.032α
0.376 ± 0.056β
Mixed species
C. albicans
+C. dubliniensis
0.435 ± 0.024
0.205 ± 0.015α
0.201 ± 0.023α
+ C. glabrata
1.023 ± 0.054†
0.962 ± 0.104
0.527 ± 0.036α,β
+ C. krusei
0.444 ± 0.014
0.315 ± 0.018α
0.330 ± 0.032β
+ C. lusitaniae
0.292 ± 0.013
0.246 ± 0.014
0.232 ± 0.025
+ C. tropicalis
0.289 ± 0.019
0.210 ± 0.025
0.198 ± 0.015β
†
P
<
0.01 compared to others in their respective groups; single and mixed species biofilms.
α
P
<
0.05 compared to the untreated samples.
β
P
<
0.05 compared to bakuchiol-treated samples.Figure 3
Absorbance values of crystal violet solutions obtained fromCandida biofilm formation following bakuchiol exposure. Data were represented as mean ± SD of three independent experiments performed in triplicate. Amphotericin B used as a positive control. Asterisk (∗) denotes the significant difference between treated samples and the untreated ones (one-way ANOVA, P
∗
<
0.05).


## 3.1. Antifungal Susceptibility of Planktonic Cells

The antifungal activity of bakuchiol againstCandida species was tabulated in Table 1. The MIC and MFC endpoints were ranged from 12.5 to 100 μg/mL.C. albicans andC. dubliniensis were found to be the most susceptible to bakuchiol, andC. glabratawas the least susceptible to bakuchiol.Table 1
Antifungal activity of bakuchiol againstCandidaspecies.
Antifungal susceptibility testa (µg/mL)
1
2
3
4
5
6
Bakuchiol
MIC
25
12.5
>100
50
50
50
MFC
50
25
>100
50
100
100
Amphotericin B
MIC
1.95
1.95
1.95
1.95
7.81
1.95
MFC
3.91
1.95
3.91
1.95
7.81
3.91
a(1) C. albicansATCC 14053, (2) C. dubliniensis ATCC MYA-2975, (3) C. glabrata ATCC 90030, (4) C. krusei ATCC 14243, (5) C. lusitaniae ATCC 64125, and (6) C. tropicalis ATCC 13803.


## 3.2. Effect of Bakuchiol on Cell Growth

A significant difference inCandida cell growth was observed after 6 h, which indicates the exponential stage ofCandida strains. Bakuchiol exhibited considerable growth inhibitory effect against most tested strains (Table 2). In detail, μ values ofC. albicans,C. dubliniensis, andC. lusitaniae were markedly reduced by >50% compared with the untreated yeast cells (P
<
0.05). On the other hand,C. glabrata andC. tropicalis were reduced to 38% and 35%, respectively. No significant differences were observed forC. krusei (11%). Based on CFU, the population ofC. albicans,C. dubliniensis,C. glabrata,C. krusei, andC. lusitaniae were decreased by 1.2- to 1.5-fold change (P
<
0.05) following exposure to bakuchiol. No significant differences were observed forC. tropicalis (P
=
0.19).Table 2
Changes in the specific growth rates (µ) and the viable counts (CFU) of Candidaspecies following bakuchiol exposure. The percentage denotes the inhibition of treated samples compared to the untreated ones.
Microorganism
Treatmenta
µ
(
%
)
Log10 CFU/mL (%)
C. albicans ATCC 14053
Untreated
1.73 ± 0.08
10.04 ± 0.03
Bakuchiol
0.70 ± 0.07 (59.5)∗
7.24 ± 0.08 (27.9)∗
Amphotericin B
0.49 ± 0.10 (71.7)∗
6.64 ± 0.05 (33.9)∗
C. dubliniensis ATCC MYA-2975
Untreated
0.98 ± 0.14
8.32 ± 0.28
Bakuchiol
0.45 ± 0.24 (54.1)∗
6.87 ± 0.42 (17.4)∗
Amphotericin B
0.74 ± 0.19 (24.5)∗
7.36 ± 0.09 (11.5)∗
C. glabrata ATCC 90030
Untreated
1.80 ± 0.06
10.13 ± 0.08
Bakuchiol
1.11 ± 0.03 (38.3)∗
8.34 ± 0.07 (17.7)∗
Amphotericin B
1.47 ± 0.13 (18.3)∗
9.20 ± 0.07 (9.2)∗
C. krusei ATCC 14243
Untreated
0.82 ± 0.20
8.63 ± 0.45
Bakuchiol
0.73 ± 0.15 (11)
5.72 ± 0.35 (33.7)∗
Amphotericin B
0.55 ± 0.12 (32.9)∗
6.32 ± 0.21 (26.8)∗
C. lusitaniae ATCC 64125
Untreated
1.09 ± 0.15
8.95 ± 0.36
Bakuchiol
0.42 ± 0.10 (61.5)∗
6.90 ± 0.16 (22.9)∗
Amphotericin B
0.98 ± 0.24 (10.1)
8.69 ± 0.50 (2.9)
C. tropicalis ATCC 13803
Untreated
1.57 ± 0.14
9.80 ± 0.43
Bakuchiol
1.02 ± 0.18 (35)∗
9.30 ± 0.23 (5.5)
Amphotericin B
0.91 ± 0.12 (42)∗
8.63 ± 0.08 (11.9)∗
aTest concentration was prepared at 0.5 × MIC. ∗
P
<
0.05 compared to the untreated samples.


## 3.3. Effect of Bakuchiol on Cell Surface Hydrophobicity

The effect of bakuchiol on cell surface hydrophobicity (CSH) ofCandida species was measured based on the percentage of cell adsorption to hexadecane (Figure 2). The percentages ofC. albicans,C. krusei, andC. tropicalis were significantly higher (P
<
0.05) than those of the otherCandida species. Findings show that the cell hydrophobicities were relatively compromised and decreased following brief exposure ofCandida planktonic cells to 0.5 × MIC of bakuchiol. The CSH ofC. albicans,C. dubliniensis,C. krusei, andC. tropicalis decreased within the range of 10% to 38% (P
<
0.05). Bakuchiol, however, exhibited least effect onC. glabrata andC. lusitaniae when compared with the untreated yeast cells.Figure 2
Cell surface hydrophobicity ofCandida species following bakuchiol exposure. Data are represented as mean ± SD of three independent experiments performed in triplicate. Amphotericin B used as a positive control. Asterisk (∗) denotes the significant difference between treated samples and the untreated ones (one-way ANOVA; P
<
0.05).


## 3.4. Effect of Bakuchiol onCandida Biofilm

Candida species were able to produce moderate-to-high degree of biomass after 24 h cultivated in a microtiter plate.C. albicans andC. tropicalis produced a dense biomass distinct fromC. dubliniensis,C. glabrata,C. krusei, andC. lusitaniae (P
<
0.05). Bakuchiol exposure showed significant reduction in mostCandida biomass production, except forC. glabrata (P
=
0.29) (Figure 3). Based on XTT assay, bothC. albicans andC. glabrata exhibited the highest XTT metabolic activity compared to the others. In mixed biofilms, XTT activity ofC. albicans andC. glabrata remains high (P
<
0.01) when compared to the other mixed culture biofilms (Table 3). Following bakuchiol exposure (2 h), XTT activity inC. albicans andC. tropicalis was reduced (P
<
0.05) relative to the untreated samples. No significant differences were observed on other single species biofilms. For mixed biofilms, onlyC. albicans andC. dubliniensis were markedly reduced by >50% (P
<
0.05), followed by mixedC. albicans andC. krusei biofilm in response to bakuchiol.Table 3
The metabolic activity of single and mixed biofilms when exposed to bakuchiol was measured. Values represent absorbance using XTT reduction assay.
Biofilms
Untreated
Bakuchiol
Amphotericin B
Single species
C. albicans
1.042 ± 0.049†
0.417 ± 0.085α
0.266 ± 0.046α,β
C. dubliniensis
0.279 ± 0.016
0.274 ± 0.029
0.174 ± 0.026α,β
C. glabrata
0.723 ± 0.040†
0.896 ± 0.096
0.353 ± 0.061α,β
C. krusei
0.467 ± 0.048
0.400 ± 0.085
0.228 ± 0.079α,β
C. lusitaniae
0.192 ± 0.016
0.182 ± 0.017
0.242 ± 0.030α,β
C. tropicalis
0.351 ± 0.037
0.243 ± 0.032α
0.376 ± 0.056β
Mixed species
C. albicans
+C. dubliniensis
0.435 ± 0.024
0.205 ± 0.015α
0.201 ± 0.023α
+ C. glabrata
1.023 ± 0.054†
0.962 ± 0.104
0.527 ± 0.036α,β
+ C. krusei
0.444 ± 0.014
0.315 ± 0.018α
0.330 ± 0.032β
+ C. lusitaniae
0.292 ± 0.013
0.246 ± 0.014
0.232 ± 0.025
+ C. tropicalis
0.289 ± 0.019
0.210 ± 0.025
0.198 ± 0.015β
†
P
<
0.01 compared to others in their respective groups; single and mixed species biofilms.
α
P
<
0.05 compared to the untreated samples.
β
P
<
0.05 compared to bakuchiol-treated samples.Figure 3
Absorbance values of crystal violet solutions obtained fromCandida biofilm formation following bakuchiol exposure. Data were represented as mean ± SD of three independent experiments performed in triplicate. Amphotericin B used as a positive control. Asterisk (∗) denotes the significant difference between treated samples and the untreated ones (one-way ANOVA, P
∗
<
0.05).


## 4. Discussion

The crude extract and secondary metabolites derived from plants serve as important fields of research for new antifungal agents [31]. The antimicrobial properties of bakuchiol have been reported in previous studies [19]. The present study was designed to assess the antifungal activity of bakuchiol onCandida species, commonly associated with oral infections.C. albicans andC. dubliniensis were shown to be susceptible to bakuchiol. Bakuchiol demonstrated the inhibitory effect onC. albicans,C. dubliniensis, andC. lusitaniae with the specific growth rates (μ values) reduced by more than 50% compared to the untreated samples. The efficacy of bakuchiol was further elucidated by the inhibition of CFU ofCandida cells and marked difference can be seen after 6 h inoculation.Candida yeast-like species may adopt adhesive hydrophobic interactions to mediate adherence to different host surfaces [32, 33], which is one of many types of adhesion mechanism demonstrated byCandida. The present study examined the affinity ofCandida planktonic cells for hydrophobic surfaces, that is, cell surface hydrophobicity based on the microbial adhesion to hydrocarbon (MATH) testing [34]. Previous studies claimed that CSH of planktonic cells positively correlates with biofilm formation [35]. Distinction in cell surface physicochemical properties and presence of carbohydrate moiety may influence the cell affinity for hydrophobic interface [33]. In this study, exposingCandida planktonic cells to bakuchiol (0.5 × MIC) has shown significant difference in the percentage of CSH, especially forC. albicans which were markedly reduced relative to the untreated samples. Bakuchiol could have made the cell surface area undergo transient changes and the impairment of cell hydrophobicity may lead to reduced adhesion to hydrophobic interfaces [32]. This assay demonstrated that bakuchiol exposure has a considerable effect towards the hydrophobic interactions ofCandida cells. The reduction in CSH following amphotericin B exposure was also reported in previous study [28].Cell adhesion and biofilm formation are the key areas for the antifungal treatment. The CSH assay has given insights to extend investigation onCandida biofilm formation. It is postulated that the impaired hydrophobic interactions may compromise the affinity of planktonic yeast cells to adhere to form biofilms. The crystal violet assay demonstrated that bakuchiol shows significant decreases in biofilm formation ofC. albicans,C. lusitaniae, andC. tropicalis. The planktonic cells ofC. dubliniensis,C. glabrata, andC. krusei were shown to be less susceptible to bakuchiol and slightly reduced the biomass production when compared to the untreated ones. The results show that bakuchiol may act as effective as amphotericin B in reducing biofilm formation.The efficacy of bakuchiol on the established biofilms was further evaluated through XTT reduction assay. The metabolic activities ofC. albicans,C. glabrata,C. krusei, andC. tropicalis were decreased following bakuchiol exposure. No significant differences were observed onC. dubliniensis andC. lusitaniae. This could possibly be due to their lower metabolic activity which may result in reduced response to the treatment. Meanwhile, for mixed-species biofilms, the metabolic rates are varying according to the sensitivity of differentCandida species within biofilms.C. albicans remains the principle species in biofilm formation due to germ tubes and hyphae formation. The hyphae-associated adhesins such as agglutinin-like sequence (ALS) and hyphal wall protein (HWP1) are crucial for adhesion [36, 37]. It is reported thatC. albicans had a positive impact on certain NAC species in biofilm formation compared to when growing alone [38]. The NAC species may benefit from the interaction withC. albicans in mixed biofilms which further increased the cell metabolic activities and were inherently resistant to antifungal treatment. In the present study, the coculture ofC. krusei andC. albicans was not highly proficient in forming dense biofilms. The cell number in mixed biofilm was also previously shown to be reduced when varying concentrations ofC. krusei were cocultured with a constant concentration ofC. albicans [36]. Johansson Wächtler et al. [37] reported that, unlikeC. albicans,C. krusei was unable to utilize salivary statherin and mimicking molecules as functional adhesion molecules on salivary pellicles and epithelial cells. This possibly explains the less dense biofilm formed betweenC. krusei andC. albicans. Bakuchiol treatment, however, markedly reduced the metabolic activities ofC. albicans cocultured withC. dubliniensis andC. tropicalis. The high susceptibility ofC. albicans to bakuchiol may have influenced the mixed species in biofilms. The coculture ofC. albicans andC. glabrata exhibited more resistance towards bakuchiol. This may indicate synergistic relationship between the two species towards antifungal resistance [39].


## 5. Conclusion

Bakuchiol exhibited antifungal activity against planktonic and biofilm forms ofCandida species. Findings show that bakuchiol inhibited the planktonic growth and reduced the adhesive capacity ofCandida.C. albicans and the NAC species, except forC. glabrata, have been shown to be susceptible to bakuchiol. Antifungal properties of bakuchiol in the present study could give insights for the development of a new therapeutic agent against the treatment ofCandida-associated infections.



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*Source: 918624-2015-11-08.xml*