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Synergistic effects of tea polyphenol epigallocatechin 3-O-gallate and azole drugs against oral Candida isolates
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MYCMED-865; No. of Pages 10 Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Research Paper
Synergistic effects of tea polyphenol epigallocatechin 3-O-gallate and azole drugs against oral Candida isolates J.M. Behbehani a,*, M. Irshad b,*, S. Shreaz b, M. Karched b a b
Department of Restorative Sciences, Faculty of Dentistry, Health Sciences Center, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait Department of Bioclinical Sciences, Faculty of Dentistry, Health Sciences Center, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 October 2018 Received in revised form 27 January 2019 Accepted 31 January 2019 Available online xxx
Background. – The antifungal drug resistance has become an emerging problem in the management of candida infections worldwide. The objective of this study was to examine the efficacy of epigallocatechin 3-O-gallate (EGCG) alone and in combination with fluconazole/ketoconazole drugs against oral Candida isolates. Methods. – Minimum inhibitory concentration (MIC) and minimum fungicidal concentrations (MFC) of EGCG against 60 oral Candida isolates and 4 ATCC strains were determined. Synergism of EGCG with azole drugs was evaluated by checkerboard micro-dilution method and calculated fractional inhibitory concentration index (FICI). Candida cells’ ultrastructure was studied by electron microscopy. Results. – MIC and MFC values of EGCG were in the range of 3.91–15.63 and 15.63–31.25 mg/mL, respectively. Minimum biofilm inhibitory concentration (MBIC) range of EGCG (62.5–125 mg/mL), was less than the ketoconazole (64–256 mg/mL) and fluconazole (128–512 mg/mL). The combination of EGCG with fluconazole/ketoconazole exhibited synergistic effects (SFICI 0.50). EGCG with azole drugs showed high sensitivity against the tested isolates in growth curve assays. Against the biofilm, the susceptibility of fluconazole/ketoconazole significantly increased (3 to 5 fold), after combination with EGCG (MBIC/4) (P 0.001). Electron microscopy of EGCG treated cells showed deformation of cell structure, ruptured cell wall and release of intracellular content. In molecular docking experiments, a strong interaction was observed between EGCG and fungal cell membrane molecule ergosterol. Conclusion. – We conclude that EGCG synergistically enhanced the antifungal potential of azole drugs. The synergistic potential of EGCG might be helpful in preventing the development of drug resistance, in lowering the drug dosage, and thus minimizing adverse effects. C 2019 Published by Elsevier Masson SAS.
Keywords: Oral candidiasis Epigallocatechin 3-O-gallate Antifungal Synergism Microscopic analysis
1. Introduction Candida species are commensal yeast of the mucocutaneous surface of the human oral cavity. In healthy adult population, oral carriage of Candida spp. is reported to be 30–45% [1]. Over the past three decades, incidence of systemic candidiasis has considerably increased and has become a leading cause of mycosis-associated mortality worldwide [2]. The antifungal drugs used currently are reported to have moderate to sever toxicity effect on the patients’ health [3]. Also, resistance to the most common antifungal drugs has
* Corresponding authors at: Faculty of Dentistry, Kuwait University, P.O. Box. 24923, Safat 13110, Kuwait. E-mail addresses: [email protected] (J.M. Behbehani), [email protected] (M. Irshad).
increased in recent years [2,4]. Therefore, there is a need of novel, less cytotoxic molecules that can be used as antifungal therapeutics. Green tea or tea is a widely consumed beverage of Camellia sinensis var. sinensis and Camellia sinensis var. assamica plant leaves [5]. It has long been believed that drinking of tea beverage has health benefits and increases longevity [5]. Green tea or tea beverage contains rich amount of polyphenols, of which epigallocatechin 3-O-gallate (EGCG) (Fig. 1), is the most abundant [6]. The green tea beverage of 237 mL contained about 30–130 mg EGCG [6]. A study reported that ingestion of EGCG was safe and well-tolerated by the healthy human volunteers at the oral doses of 800 mg/day, which is equivalent to about 8 to 16 cups of green tea a day [7]. EGCG has many pharmacological properties including antibacterial and antifungal properties [8,9]. In the present study, we evaluated the anticandidal potential of EGCG alone and in combination with azole antifungal drugs against
https://doi.org/10.1016/j.mycmed.2019.01.011 C 2019 Published by Elsevier Masson SAS. 1156-5233/
Please cite this article in press as: Behbehani JM, et al. Synergistic effects of tea polyphenol epigallocatechin 3-O-gallate and azole drugs against oral Candida isolates. Journal De Mycologie Me´dicale (2019), https://doi.org/10.1016/j.mycmed.2019.01.011
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7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS). Overnight culture of Candida was diluted in medium to obtain a final inoculum of 2.5 103 cells/mL. An aliquot of 100 mL was added to each well of a 96-well U-bottom tissue culture plate. The treatment concentration range of EGCG was 0.98– 250 mg/mL, and for fluconazole and ketoconazole, the range was 0.50–128 mg/mL. For positive growth control, medium without drugs was used. The cell growth in the wells was observed visually after incubation at 37 8C for 24 and 48 hrs. MIC is the lowest concentration of a test compounds at which no visible growth was found [11,12]. The minimum fungicidal concentration (MFC) was evaluated by inoculation of 20 mL content from the wells with clear broth/no visible growth (MIC, 2MIC and 4MIC) on the sabouraud dextrose agar (SDA) plate. The inoculated plates were incubated at 37 8C for 24–48 hrs. MFC was defined as a concentration of drug at which no visible growth was observed on the SDA plates [12]. All of the assays were performed independently three times. 2.4. Inhibition of mature Candida biofilm Fig. 1. Chemical structure of epigallocatechin 3-O-gallate (EGCG).
the planktonic and mature sessile biofilms of oral Candida isolates. We used techniques like confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy techniques to understand mechanisms of action of EGCG. 2. Materials and methods 2.1. Chemicals EGCG (epigallocatechin gallate or epigallocatechin 3-gallate or epigallocatechin 3-O-gallate) (purity 98%, Catalogue No. 1236700), fluconazole and ketoconazole were purchased from Sigma-Aldrich, St. Louis, MO, USA. The other chemicals and culture medium constituents were also purchased from Sigma-Aldrich, USA. 2.2. Sample collection Oral Candida samples were collected from dental patients at the Kuwait University Dental Clinic (KUDC), by using a standard oral rinse technique [10]. The collected samples were cultured in YPD medium, containing yeast extract (1% w/v), peptone (2% w/ v) and dextrose (2% w/v), and incubated at 37 8C. The Candida spp. were identified by using CHROMagar medium (CHROMagar Candida, France) and VITEK-2 system assays (bioMerieux, Craponne, France). As reported in our previous study, we confirmed the identity of eight Candida spp. from the collected oral samples, i.e., C. albicans (n = 33), C. dubliniensis (n = 13), C. tropicalis (n = 3), C. glabrata (n = 7), C. krusei (n = 1), C. lusitaniae (n = 1), C. kefyr (n = 1), and C. parapsilosis (n = 1). Four ATCC strains (C. albicans ATCC 24433, C. glabrata ATCC 15126; C. parapsilosis ATCC 90018 and C. krusei ATCC 6258), were obtained from the Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait. 2.3. Minimum inhibitory and fungicidal concentration Clinical Laboratory Standards Institute (CLSI) method for the yeasts (M27-A3) was used for evaluation of minimum inhibitory concentration (MIC) [11]. Test was carried out in RPMI-164 medium containing L-glutamine, without sodium bicarbonate (Cat. No. R6504, Sigma-Aldrich, USA), and buffered to pH
A standardized method was used in this study. The mature biofilms of Candida were formed in 96-well polystyrene microtiter plates (Corning Inc., Corning, NY, USA). Different concentrations of EGCG and standard drug were prepared separately in RPMI 1640 medium using serial dilution method and added to each well (200 mL/well). The medium without test agents was added to the controll wells. After treatment, microtitre plate was re-incubated at 37 C for 24 hrs. After incubation period, each well was washed carefully with sterile PBS (pH 7). Biofilm metabolic activity was quantified by MTT assay following manufacturer’s instructions, but with a slight modification. Briefly, MTT (1 mg/mL) was freshly prepared in PBS and menadione (10:1 v/v) was added before use. Menadione (0.4 mM) solution was prepared in acetone and stored at 4 8C. MTTmenadione solution was added to each well (60 mL/well) and incubated at 37 C for 3 hrs. The MTT-formazan product was formed by the live cells, which was dissolved in DMSO (200 mL) and optical density was measured at 570 nm (Microplate Reader, BioTek Instruments, Winooski, USA). An inhibition of metabolic activity of biofilm was calculated by the equation:
Inhibitionð%Þ ¼
ðControlTestÞ 100 Control
The minimum biofilm inhibitory concentration (MBIC) of a test agent was defined as the lowest concentration at which 80% metabolic activity of biofilms was inhibited as compared to the control. The assays were performed in triplicate and three independent experiments were performed. 2.5. Synergistic effect of EGCG and azole drugs on Candida planktonic cells To study the synergistic effect of EGCG and drugs, we used checkerboard microtiter plate method [13]. Different combination of EGCG and fluconazole or ketoconazole drugs was prepared in RPMI medium. The concentration of EGCG was in the range of 0.98 to 62.5 mg/mL and for fluconazole or ketoconazole drugs, the range was 1 to 128 mg/mL. Candida cells ( 2.5 103 cells/mL) were added to each well and incubated at 37 8C for 48 hrs. The growth of Candida cells was read visually. The synergistic effects of EGCG and drugs were defined as the lowest concentration that prevented visible growth. All assays were performed independently three times.
Please cite this article in press as: Behbehani JM, et al. Synergistic effects of tea polyphenol epigallocatechin 3-O-gallate and azole drugs against oral Candida isolates. Journal De Mycologie Me´dicale (2019), https://doi.org/10.1016/j.mycmed.2019.01.011
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To evaluate interaction between EGCG and drugs, the fractional inhibitory concentration index (FICI) was calculated. FICI of EGCG = MIC (EGCG with drug)/MIC (EGCG alone) and FICI of drug = MIC (Drug with EGCG)/MIC (Drug alone). The sum of both; SFICI 0.5 exhibited synergetic interaction, FICI > 0.5–4 exhibited no interaction and FICI > 4 exhibited antagonistic interaction between EGCG and drug.
refractivity, partition coefficient (log P), polar surface area and atom count were calculated and matched with Lipinski’s rule of five [16]. Docking studies of EGCG with ergosterol were carried out by using AutoDock 4.2 Tools (Scripps Research Institute, USA) [17]. Ergosterol (PDB ID: 2AIB) X-ray crystal structure was obtained from the Protein Data Bank [RCSB Protein Data Bank., http://www.rcsb.org/pdb].
2.6. Combination effect of EGCG and azole drugs on the growth of Candida cells
2.11. Statistical analysis
The combination effect of drugs on the growth of Candida was examined. Fresh overnight growth of Candida cells ( 1 10 6 ; optical density A 595 = 0.1) was inoculated into YPD medium. The MIC and sub-MIC values of EGCG alone or EGCG with drug was added to the medium. The cells were incubated at 37 C and 120 rpm, using IKA KS 3000 Shaking Incubator (Staufen, Germany). The optical density of cells was recorded per hour by using Eppendorf Spectrophotometer (Hamburg, Germany).
The experiments were performed in triplicate. Statistical analysis was performed by SPSS software (SPSS Inc., USA). The significant differences between the groups were analyzed by ANOVA and Tuky HSD test. The value P 0.05 was considered statistically significant differences.
3. Results 3.1. Minimum inhibitory concentration (MIC) and fungicidal concentration (MFC)
2.7. Synergistic effects of EGCG and azole drugs on Candida biofilms Candida biofilms were formed in polystyrene 96-well plates, as previously described. The checkerboard micro-dilution method was used to prepare different combination of EGCG and drug in RPMI-164 medium [13]. The drug combinations were prepared in fresh a sterile plate and then added into the corresponding biofilm wells (200 mL/well). The plate was incubated at 37 8C for 48 hrs, after which, the plate was washed twice with PBS. The metabolic activity of biofilm was evaluated by the MTT-menadione reduction assay as described in the previous section. The assays were performed independently three times. 2.8. Confocal scanning laser microscopy The effect of EGCG on Candida cell membranes was assessed by using florescence dye SYTO-9 and Propidium iodide (PI) (LIVE/ DEAD1 BacLightTM, USA). Candida cells ( 1 106) were inoculated in YPD medium and treated with EGCG. The treated and untreated cells were incubated at 37 8C with constant shaking (120 rpm) for 12 hrs. After incubation period, cells were washed twice in PBS (pH-7) and then florescence dye was added at a concentration of one mg/mL. To study cell membrane permeabilization, 10 mL cell suspension was added on a glass slide and dye uptake was visualized by confocal microscopy (ZEISS LSM 500).
EGCG showed antifungal activity against 60 oral isolates and 4 ATCC strains of Candida spp. The MIC ranges of EGCG were 3.91 to 15.63 mg/mL against the planktonic cells of all Candida isolates. Among the tested isolates, the MIC of EGCG was as follows: 3.91 mg/mL for 4 isolates, 7.81 mg/mL for 23 isolates, and 15.63 mg/mL for the rest 33 isolates. The MIC ranges of fluconazole and ketoconazole were 4 to 128 mg/mL and 4 to 32 mg/mL, respectively. The MFC values of EGCG were 15.63 to 31.25 mg/mL against all isolates. The MIC and MFC values of EGCG against the specific strains are given in Table 1. 3.2. Effects of EGCG in combination with fluconazole or ketoconazole against planktonic Candida cells To test the combined activity, we assessed various combination concentrations of EGCG and fluconazole or ketoconazole using checkerboard titer assay. The combination of EGCG with fluconazole or ketoconazole showed a remarkable decrease in the MIC values compared to each drug alone (Table 2). The result showed MIC of fluconazole decreased from 4–64 to 1–8 mg/mL, whereas the MIC of ketoconazole decreased from 4–32 to 1–8 mg/mL (P < 0.01). The results showed that the combinations of EGCG/ ketoconazole exerted synergistic effects against the planktonic cells of Candida spp. (S FICI 0.50). However, combination of EGCG/fluconazole exerted either synergism or no interaction (S FICI, 0.375 to 0.625).
2.9. Scanning and transmission electron microscopy EGCG treated cells ( 1 10 6) were observed under scanning electron microscopy (SEM) (JEOL, CarryScope JCM 5700) and transmission electron microscopy (TEM [JEOL, JEM 1200 EXZ]). The MIC value of EGCG was used to observed effect on the ultrastructure of the Candida cells. The treated samples were washed twice in PBS and fixed in glutaraldehyde (3%) and processed for SEM/TEM analysis as described previously [14]. Other details can be obtained from the method by Mares [15]. 2.10. EGCG and ergosterol docking study The likeliness of EGCG molecule was assessed theoretically by using computer-added MarvinSketch 5.8.2 program. The parameters like molecular mass, hydrogen bond donors, acceptors, molar
Table 1 MIC and MFC values of EGCG against Candida spp. Strain/isolates (CDC NO./ATCC)
C. C. C. C. C. C. C. C.
dubliniensis (CDC 27963) albicans (CDC 27974) glabrata (CDC 27845) albicans (CDC 28304) glabrata (CDC 28398) dubliniensis (CDC 28551) albicans (ATCC 24433) glabrata (ATCC 15126)
EGCG (mg/mL) MIC
MFC
15.63 15.63 15.63 7.81 15.63 7.81 15.63 15.63
31.25 31.25 31.25 31.25 15.63 15.63 31.25 31.25
EGCG: epigallocatechin 3-O-gallate; CDC: Comprehensive Dental Care Number; ATCC: American Type Culture Collection; MIC: Minimum Inhibitory Concentration (no visible growth); MFC: Minimum Fungicidal Concentration (no visible growth).
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Table 2 Antifungal effects of EGCG alone and in combination with fluconazole (FLC) and ketoconazole (KTC) against Candida planktonic cells. Strain/isolates (CDC NO./ATCC)
C. dubliniensis (CDC 27963) C. albicans (CDC 27974) C. glabrata (CDC 27845) C. albicans (CDC 28304) C. glabrata (CDC 28398) C. dubliniensis (CDC 28551) C. albicans (ATCC 24433) C. glabrata (ATCC 15126)
MIC (mg/mL) Antifungal
Antifungal (Alone/combo)b
EGCG (Alone/combo)b
SFICI
Fluconazole
32/4
15.63/3.91
0.375
Ketoconazole Fluconazole
8/2 64/8
15.63/3.91 15.63/3.91
0.500 0.375
Ketoconazole Fluconazole
8/2 16/4
15.63/1.95 15.63/3.91
0.375 0.500
Ketoconazole Fluconazole
8/2 64/8
15.63/1.95 7.81/3.91
0.375 0.625a
Ketoconazole Fluconazole
32/8 16/4
7.81/1.95 15.63/3.91
0.500 0.500
Ketoconazole Fluconazole
8/1 4/1
15.63/3.91 7.81/1.95
0.375 0.500
Ketoconazole Fluconazole
4/1 64/4
7.81/1.95 15.63/3.91
0.500 0.313
Ketoconazole Fluconazole
16/2 16/4
15.63/3.91 15.63/3.91
0.375 0.500
Ketoconazole
16/4
15.63/3.91
0.500
EGCG: epigallocatechin 3-O-gallate; CDC: Comprehensive Dental Care Number; ATCC: American Type Culture Collection; FIC: Fractional Inhibitory Concentration Index. Tukey HSD multiple comparisons test exhibited significance differences between the MIC value of FLC alone vs. FLC with EGCG (P = 0.0001) and KTC alone vs. KTC with EGCG (P = 0.003). a No interaction. b Significant differences in ANOVA test (P < 0.01).
3.3. Combination effects of EGCG and fluconazole or ketoconazole on the growth of Candida cells The growth of Candida spp. was evaluated in the presence of EGCG alone or in combination with azole drugs. The growth pattern was plotted against time as shown in Fig. 2(A–D). EGCG alone at MIC and sub-MIC value showed a significant inhibitory effect on the growth of C. albicans CDC 27974, C. dubliniensis CDC 27963, C. glabrata CDC 27845, and C. albicans ATCC 24433, respectively. The synergistic combination of EGCG (MIC/4) with fluconazole (MIC/4 or MIC/8 or MIC/16) or EGCG (MIC/4 or MIC/8) with ketoconazole (MIC/4 or MIC/8) showed a significant inhibitory effect against the tested Candida spp. (Fig. 2A–D). The Candida cells without any treatment showed a normal pattern of growth such as lag phase, active exponential phase and stationary phase. In conclusion, EGCG showed synergistic effect with fluconazole or ketoconazole drugs leading to a fungicidal activity at lowered concentration. Treatment of all 4 species with the EGCG (MIC) alone resulted in 95.13% reduction in optical density, whereas in synergistic combinations [(EGCG-Fluconazole) and (EGCG-ketoconazole)] resulted in 92.27% and 97.51% reduction in optical density, as compared to the control. 3.4. Inhibition of mature Candida biofilm The activity of EGCG was tested on mature biofilm of selected oral isolates and two ATCC Candida strains. The biofilm metabolic activity was quantified by the MTT assay. The metabolic activity of mature biofilm decreased in a concentration dependent manner (Fig. 3). The minimum biofilm inhibitory concentration (MBIC) ranges of EGCG against the all tested isolates/strain were 62.5 to
125 mg/mL (Table 3). In comparison, antifungal activity of EGCG against the Candida planktonic cells was two to three fold higher than the mature biofilm at the same concentration (P < 0.05). 3.5. Combination effect of EGCG in combination with fluconazole or ketoconazole on the Candida biofilms To determine metabolic activity of the mature Candida biofilms, 48 hrs old biofilms were treated with EGCG/fluconazole or EGCG/ ketoconazole combinations. The MBIC values of fluconazole and ketoconazole against the biofilms of all tested Candida spp. were in the range of 128 to 512 mg/mL and 64 to 512 mg/mL, respectively (Table 3). The results indicate that the mature biofilms of Candida spp. were resistant to these drugs. When EGCG was combined with fluconazole or ketoconazole drugs, the MBIC values of drugs and EGCG decreased many fold indicating a synergistic effect in all most all the cases (P = 0.0001). 3.6. Cell membrane permeability Confocal laser scanning microscopy (CLSM) was used to explore individual cells stained with SYTO9 alone or SYTO9/propidium iodide. CLSM results showed the membrane-impermeant propidium iodide uptake by the EGCG treated Candida cells. Propidium iodide is a membrane impermeable red dye and is therefore excluded by healthy cells. However, SYTO9 is a membrane permeant green dye and therefore stained all live and dead cells (Fig. 4). In Fig. 4, CLSM images in left panel show the untreated C. albicans CDC 27974, C. dubliniensis CDC 27963 and C. glabrata CDC 27845 cells which is stained green only. In the treated samples, the images clearly show that Syto9 was taken up
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Fig. 2. Effect of epigallocatechin 3-O-gallate (EGCG) alone and in synergistic combination with fluconazole (FLC) and ketoconazole (KTC) on the growth of Candida spp. The graph represents the growth curves of (A) C. dubliniensis CDC 27963, (B) C. albicans CDC 27974, (C) C. glabrata CDC 27845, and (D) C. albicans ATCC 24433, in presence of EGCG (MIC and sub-MIC) alone and in synergistic combination with FLC or KTC drugs (as given in Table 2). Growth curve plotted against optical density at 595 nm versus time (hrs).
by both live and dead cells, whereas propidium iodide was taken up only the dead cells. The results confirm that the Candida cells membrane was disrupted by EGCG treatment. 3.7. Scanning electron microscopy (SEM) SEM micrographs of EGCG treated and untreated Candida cells are shown in Fig. 5. C. albicans CDC 27974, and C. dubliniensis CDC 27963 were treated with MIC values of EGCG for 12 hrs (Fig. 5). The images showed drastic morphological changes in treated cells compared to the untreated cells. The micrograph of untreated cells showed oval in shape and with smooth cell surfaces and polar bud scars (Fig. 5A–B). While treated cells showed deformed cells shape with convoluted and irregular cells surfaces. Most of the treated cells showed deep furrows and wrinkles of the cell surface (Fig. 5C–D). 3.8. Transmission electron microscopy (TEM) The ultrathin sections of EGCG treated Candida cells showed irregular breakage in the cell wall and cell membrane in
contrast to the untreated cells. In Fig. 6, TEM micrograph of untreated and treated C. albicans CDC 27974 and C. dubliniensis CDC 27963 cells are shown. The untreated cells showed typical cell morphology and had uniform central density (Fig. 6A–B). However, EGCG treated cells (12 hrs) showed severe damage in cell wall and plasma membrane, like rupturing and swelling at many places (Fig. 6C–F). The disintegration of the cell wall structure indicates release of cytoplasmic content and cell death. 3.9. Ergosterol and EGCG likeness and docking studies The molecular property of EGCG molecule has all of the drug likeness criteria as defined by Lipinski (Table 4). The docking experiments showed strong interaction between EGCG and ergosterol molecules (Fig. 7). In this study, docking energy of interaction between both molecules was 27.17 kJ/mole. A total of six hydrogen bonds formed between the hydroxyl groups of EGCG and the polar surface of ergosterol (Fig. 7b). The interaction between EGCG and ergosterol were also by Van der Waals bonding (Fig. 7c).
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Fig. 3. Effect of epigallocatechin 3-O-gallate (EGCG) on preformed biofilm of Candida spp. The mature Candida biofilm was grown in the polystyrene surface microliter plate. Different concentrations of EGCG were treated in triplicate, and then further incubated for 24 hrs. After incubation periods, the extent of biofilm was estimated by the MTT reduction assay. The data represents (mean SD) of three experiments. All eight strains have significant correlation along with concentration (P < 0.05).
Table 3 Antifungal effects of EGCG alone and in combination with fluconazole (FLC) and ketoconazole (KTC) against Candida biofilms. Strain/isolates (CDC NO./ATCC)
C. dubliniensis (CDC 27963) C. albicans (CDC 27974) C. glabrata (CDC 27845) C. albicans (CDC 28304) C. glabrata (CDC 28398) C. dubliniensis (CDC 28551) C. albicans (ATCC 24433) C. glabrata (ATCC 15126)
MIC80 (mg/mL) Antifungal
Antifungal (Alone/combo)b
EGCG (Alone/combo)b
S FICI
Fluconazole
256/16
62.5/15.63
0.313
Ketoconazole Fluconazole
256/16 512/32
62.5/15.63 125/62.5
0.313 0.563a
Ketoconazole Fluconazole
512/32 128/16
125/31.25 62.5/15.63
0.313 0.375
Ketoconazole Fluconazole
64/8 256/32
62.5/15.63 125/31.25
0.375 0.375
Ketoconazole Fluconazole
256/16 256/16
125/31.25 62.5/15.63
0.313 0.313
Ketoconazole Fluconazole
256/16 128/16
62.5/15.63 62.5/15.63
0.313 0.375
Ketoconazole Fluconazole
64/8 512/16
62.5/15.63 125/62.5
0.375 0.531a
Ketoconazole Fluconazole
256/8 256/8
125/31.25 62.5/15.63
0.281 0.281
Ketoconazole
256/8
62.5/15.63
0.281
EGCG: epigallocatechin 3-O-gallate; CDC: Comprehensive Dental Care Number; ATCC: American Type Culture Collection; FICI: Fractional Inhibitory Concentration Index. Tukey HSD test exhibited significance differences between the MIC value of FLC alone vs. FLC with EGCG (P = 0.0001) and KTC alone vs. KTC with EGCG (P = 0.0001). a No interaction. b Significant differences in ANOVA test (P < 0.001).
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Fig. 4. Confocal scanning laser microscopy (CSLM) images of Candida spp. treated with epigallocatechin 3-O-gallate (EGCG) (MIC) for 12 hrs (right panel) and compared with untreated control cells (left panel). To membrane damage cells were stained with PI (red signals).
4. Discussion Natural antifungal compounds in combination with antimycotics have recently been paid much attention by researchers. It is expected that the combination therapy with natural products provides promising rapid and synergistic potency of drug activity, lowering the risk of drug resistance and reduces dosing toxicity of
drug. In the present study, EGCG exhibited strong antifungal potential; tested against 60 oral Candida isolates and 4 ATCC Candida strains. EGCG inhibited the growth of both C. albicans and non-albicans species. In addition, we found that the minimum inhibitory concentration (MIC) of EGCG varied with isolates. The MIC value of EGCG obtained in the present study is low and consistent with the previous reports tested against the Candida
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Fig. 5. Scanning electron micrograph of Candida cells treated with epigallocatechin 3-O-gallate (EGCG) (MIC). A. Untreated C. albicans CDC 27974. B. Untreated C. dubliniensis CDC 27963. C. Treated C. albicans CDC 27974. D. Treated C. dubliniensis CDC 27963. (Panel 3) enlarged view of a selected portion of C and D. (1, 2) Untreated cells are indicating polar bud scar, an oval shape and smooth cell surfaces (3, 4, 5, 6) deformed cells with convoluted and irregular surfaces and formation of deep furrows.
Fig. 6. Transmission electron micrograph of Candida cells treated with epigallocatechin 3-O-gallate (EGCG) (MIC). (A) untreated C. albicans CDC 27974 (B) untreated C. dubliniensis CDC 27963 (C, D) treated C. albicans CDC 27974. (E, F) treated C. dubliniensis CDC 27963. (1) Rupturing of both cell wall and plasma membrane. (2) Disintegration of the cell wall. (3) Oozing out of intracellular content. (4) Undulating cell wall (5) deposition of lipid globules (6) deterioration of cytoplasm. (7) Disintegrated cell wall (8) swelling of the cell wall (9) disrupted cells wall. The values in parentheses represent the number of cells observed for each sample.
planktonic cells [18,19]. However, in one particular study, MIC range of EGCG was much higher than the present study [20]. The higher MIC values in this study might be due to differences in the purity of the compound, origin of the strains and growth conditions. This study demonstrated synergistic inhibitory effects of EGCG and azole antifungal drugs against planktonic and mature sessile biofilms of Candida cells. The treatment of azole drugs along with
EGCG showed decrease of MIC value of fluconazole and ketoconazole in comparison to the effect of these drugs alone (Table 2). Our results are consistent with previous studies in which EGCG enriched the fungicidal potential of amphotericin-B, fluconazole and miconazole tested against the Candida planktonic cells [20,21]. The eradication of Candida biofilm is one of the most challenging tasks in fungal therapeutics, beacause of its reduced
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Table 4 Theoretical molecular properties of EGCG. The values in green are in agreement with Lipinski’s values and those in red are outside the range of the values set by Lipinski.
EGCG Lipinski’s values
Molecular mass/u
Log P
Hydrogen bond acceptor
Hydrogen bond donor
Molar refractivity (m3M1)
Polar surface area/A˚2
Atom count
458.372 180 to 500
3.08 0.4 to +5.6
10 Up to 10
8 Up to 5
111.75 40–130
197.37 Up to 140
51 20–70
EGCG: epigallocatechin 3-O-gallate.
Fig. 7. a–c: docking images of epigallocatechin gallate with ergosterol showing various kinds of interactions possible between ergosterol and epigallocatechin gallate: a: represents the docked image of epigallocatechin gallate with ergosterol visualized by PyMol; b: represents the hydrogen bonding interactions between epigallocatechin gallate and ergosterol; c: shows the hydrophobic residues involved in the interaction between epigallocatechin gallate and ergosterol.
susceptibility to antimycotic drugs [22]. Similar to an earlier study, our results showed that Candida biofilms were more resistant to fluconazole and ketoconazole drugs in comparison to the planktonic counterparts [20,23]. In the present study, EGCG alone destroyed the biofilm morphology and inhibited the maintenance of mature biofilm at the MIC value. The mean MBIC value of EGCG against the randomly selected six oral Candida spp. was 83.33 mg/ mL and against the two ATCC strains it was 93.75 mg/mL. The fungicidal potential of EGCG against Candida biofilms increased when treated in combination with fluconazole or ketoconazole. Similarly, fluconazole or ketoconazole fungicidal potential increased when combined with EGCG. The MBIC value of ketoconazole/EGCG or ketoconazole/EGCG decreased 3 to 4-fold in comparison to the inhibitory effect of those drugs alone. The results indicate that the EGCG can significantly improve the efficacy of fluconazole and ketoconazole drugs against preformed sessile biofilms. Ning et al. [20] also reported that the MBIC values of fluconazole decreased two to four times in combination with EGCG. In summary, our results indicate that EGCG could significantly inhibit the mature biofilm alone or in combination with azole antifungal drugs. The effect of EGCG alone on the membrane integrity of Candida cells was examined by the CLMS, SEM and TEM. This is the first study to report an effect of EGCG on ultrastructure of Candida cells. Confocal laser microscopy showed the intensity of red propidium iodide inside the treated dead cells or cells with deformed membrane, but not the untreated cells (Fig. 4). This is the first line of evidences that the antifungal activity of EGCG against Candida might be by making a lesion on the cell membrane or by causing loss of cell membrane integrity [24,25]. The second level of evidences is established by the SEM and TEM micrographs. The untreated cells clearly have normal oval shape having smooth cell surface with polar bud scars (Fig. 5A–B). However, treated cells showed deformed cells and receding of cytoplasm leading to lysis of cells (Fig. 5C–D). Similar type of cell membrane alterations has been reported in C. albicans cells treated by antifungal agents [26]. TEM observation showed that cell organelles were damaged in treated Candida cells (Fig. 6C and E). These observations were consistent with the damages observed in C. albicans cells treated with magnolol and garlic oil [27]. Also TEM micrograph confirmed
the damage of cells, such as breaking of both cell wall and plasma membrane (Fig. 6D and F). The collapse of cell wall leads to change of membrane permeability, osmotic imbalance and finally resulted to cell death [28]. Computational tools have been used for target identification and drug discovery [29]. Since several years, fungal cell wall ergosterol macromolecule has been used as target for antifungal drug discovery [30]. In the present study, results of docking experiments exhibited strong binding between EGCG and ergosterol. The interaction between both molecules may create pores in fungal cell membranes and result in loss of essential ions such as potassium and protons and other molecules, eventually leading to death of fungal cells. 5. Conclusion The results from the present study strongly support the antifungal potential of EGCG against different Candida isolates. The synergistic effect of EGCG with fluconazole and ketoconazole against the planktonic and mature Candida biofilms might be useful in reducing the antimycotics drug dose, prevent drug resistance development and side effects. From the obtained results, we suggest to examine the fungicidal potentiality of EGCG in an in vitro model. Ethical approval Ethical approved from the Health Sciences Center Ethical Committee, Kuwait University (Ref: VDR/EC/2347). Disclosure of interest The authors declare that they have no competing interest. Acknowledgements The authors are thankful to the Office of the Vice President for Research, Kuwait University, for financial support (Grant No. DR04/14). We thank the Oral Microbiology Research Labora-
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tory at the Faculty of Dentistry for utilizing the instruments, laboratory space and other resources (SRUL 01/14). We sincerely acknowledge the Nisha Sina Philip (Technician) Nanoscopy Science Centre (Kuwait University, Khaldiya Campus) for SEM and TEM analysis and Mohammad Arshad (Research Assistant), Research Core Facility (Kuwait University, Jabriya Campus) for the assistance in CSLM analysis. References [1] Akpan A, Morgan R. Oral candidiasis. Postgrad M J 2002;78:455–9. [2] Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Candida glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiol Rev 2012;36:288– 305. [3] Laniado-Laborin R, Cabrales-Vargas MN. Amphotericin: side effects and toxicity. Rev Iberoam Micol 2009;26:223–7. [4] Pfaller MA, Messer SA, Hollis RJ, Boyken L, Tendolkar S, Kroeger J, et al. Variation in susceptibility of bloodstream isolates of Candida glabrata to fluconazole according to patient age and geographic location in the United States in 2001 to 2007. J Clin Microbiol 2009;47:3185–90. [5] Al-salafe R, Irshad M, Abdulghani HM. Does green tea help to fight against obesity? An overview of the epidemiological reports. Austin J Clin Med 2014;1:1–11. [6] Higdon JV, Frei B. Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions. Crit Rev Food Sci Nutr 2003;43:89–143. [7] Chow HH, Cai Y, Hakim IA, Crowell JA, Shahi F, Brooks CA, et al. Pharmacokinetics and safety of green tea polyphenols after multiple-dose administration of epigallocatechin gallate and polyphenon E in healthy individuals. Clin Cancer Res 2003;9:312–9. [8] Cui Y, Oh YJ, Lim J, Youn M, Lee I, Pak HK, et al. AFM study of the differential inhibitory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negative bacteria. Food Microbiol 2012;1:80–7. [9] Chen M, Zhai L, Arendrup MC. In vitro activity of 23 tea extractions and epigallocatechin gallate against Candida species. Med Mycol 2015;53:194–8. [10] Samaranayke LP, MacFarlane TW, Lamey PJ, Ferguson MM. A comparison of oral rinse and imprint sampling techniques for the detection of yeast, coliform and Staphylococcus aureus carriage in the oral cavity. J Oral Path 1986;15:386– 8. [11] Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts, approved standard – third edition, CLSI document M27-A3. Wayne, Penn, USA: Clinical and Laboratory Standards Institute; 2008. [12] Yang LF, Liu X, Lv LL, Ma ZM, Feng XC, Ma TH. Dracorhodin perchlorate inhibits biofilm formation and virulence factors of Candida albicans. J Mycol Med 2018;28:36–44. [13] Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 2003;52:1.
[14] Shreaz S, Bhatia R, Khan N, Muralidhar S, Manzoor N, Khan LA. Influences of cinnamic aldehydes on H+ extrusion activity and ultrastructure of Candida. J Med Microbiol 2013;62:232–40. [15] Mares D. Electron microscopy of Microsporum cookie after in vitro treatment with protoanemonin: a combined SEM and TEM study. Mycopathologia 1998;108:37–46. [16] Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 2001;46:3–26. [17] Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, et al. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem 1998;19:1639–62. [18] Park BJ, Park JC, Taguchi H, Fukushima K, Hyon SH, Takatori K. Antifungal susceptibility of epigallocatechin 3-O-gallate (EGCg) on clinical isolates of pathogenic yeasts. Biochem Biophys Res Commun 2006;347:401–5. [19] Navarro-Martı´nez MD, Garcı´a-Ca´novas F, Rodrı´guez-Lo´pez JN. Tea polyphenol epigallocatechin-3-gallateepigallocatechin 3-O-gallate inhibits ergosterol synthesis by disturbing folic acid metabolism in Candida albicans. J Antimicrob Chemother 2006;57:1083–92. [20] Ning Y, Ling J, Wu CD. Synergistic effects of tea catechin epigallocatechin gallate and antimycotics against oral Candida species. Arch Oral Biol 2015;60:1565–70. [21] Anand J, Rai N. Anticandidal synergistic activity of green tea catechins, antimycotics and copper sulphate as a mean of combinational drug therapy against candidiasis. J Mycol Med 2017;27:33–45. [22] Mathe´ L, Van Dijck P. Recent insights into Candida albicans biofilm resistance mechanisms. Curr Genet 2013;59:251–64. [23] Melo AS, Bizerra FC, Freymu¨ller E, Arthington-Skaggs BA, Colombo AL. Biofilm production and evaluation of antifungal susceptibility amongst clinical Candida spp. isolates, including strains of the Candida parapsilosis complex. Med Mycol 2011;49:253–62. [24] Liao RS, Rennie RP, Talbot JA. Sublethal injury and resuscitation of Candida albicans after amphotericin-B treatment. Antimicrob Agents Chemother 2003;47:1200–6. [25] Wei MK, Wu QP, Huang Q, Wu JL, Zhang JM. Plasma membrane damage to Candida albicans caused by chlorine dioxide (ClO2). Lett Appl Microbiol 2008;47:67–73. [26] Kim KS, Kim YS, Han I, Kim MH, Jung MH. Quantitative and qualitative analyses of the cell death process in Candida albicans treated by antifungal agents. Plos One 2011;6:e28176. [27] Li WR, Shi QS, Dai HQ, Liang Q, Xie XB, Huang XM, et al. Antifungal activity, kinetics and molecular mechanism of action of garlic oil against Candida albicans. Sci Rep 2016;6:22805. [28] Lee HS, Kim Y. Antifungal activity of Salvia miltiorrhiza against Candida albicans is associated with the alteration of membrane permeability and (1,3)-b-D-Glucan synthase activity. J Microbiol Biotechnol 2016;26:610–7. [29] Katsila T, Spyroulias GA, Patrinos GP, Matsoukas MT. Computational approaches in target identification and drug discovery. Comput Struct Biotechnol J 2016;14:177–84. [30] Ghannoum MA, Rice LB. Antifungal agents, mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev 1999;12:501–17.
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Available online at
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Research Paper
Synergistic effects of tea polyphenol epigallocatechin 3-O-gallate and azole drugs against oral Candida isolates J.M. Behbehani a,*, M. Irshad b,*, S. Shreaz b, M. Karched b a b
Department of Restorative Sciences, Faculty of Dentistry, Health Sciences Center, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait Department of Bioclinical Sciences, Faculty of Dentistry, Health Sciences Center, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 October 2018 Received in revised form 27 January 2019 Accepted 31 January 2019 Available online xxx
Background. – The antifungal drug resistance has become an emerging problem in the management of candida infections worldwide. The objective of this study was to examine the efficacy of epigallocatechin 3-O-gallate (EGCG) alone and in combination with fluconazole/ketoconazole drugs against oral Candida isolates. Methods. – Minimum inhibitory concentration (MIC) and minimum fungicidal concentrations (MFC) of EGCG against 60 oral Candida isolates and 4 ATCC strains were determined. Synergism of EGCG with azole drugs was evaluated by checkerboard micro-dilution method and calculated fractional inhibitory concentration index (FICI). Candida cells’ ultrastructure was studied by electron microscopy. Results. – MIC and MFC values of EGCG were in the range of 3.91–15.63 and 15.63–31.25 mg/mL, respectively. Minimum biofilm inhibitory concentration (MBIC) range of EGCG (62.5–125 mg/mL), was less than the ketoconazole (64–256 mg/mL) and fluconazole (128–512 mg/mL). The combination of EGCG with fluconazole/ketoconazole exhibited synergistic effects (SFICI 0.50). EGCG with azole drugs showed high sensitivity against the tested isolates in growth curve assays. Against the biofilm, the susceptibility of fluconazole/ketoconazole significantly increased (3 to 5 fold), after combination with EGCG (MBIC/4) (P 0.001). Electron microscopy of EGCG treated cells showed deformation of cell structure, ruptured cell wall and release of intracellular content. In molecular docking experiments, a strong interaction was observed between EGCG and fungal cell membrane molecule ergosterol. Conclusion. – We conclude that EGCG synergistically enhanced the antifungal potential of azole drugs. The synergistic potential of EGCG might be helpful in preventing the development of drug resistance, in lowering the drug dosage, and thus minimizing adverse effects. C 2019 Published by Elsevier Masson SAS.
Keywords: Oral candidiasis Epigallocatechin 3-O-gallate Antifungal Synergism Microscopic analysis
1. Introduction Candida species are commensal yeast of the mucocutaneous surface of the human oral cavity. In healthy adult population, oral carriage of Candida spp. is reported to be 30–45% [1]. Over the past three decades, incidence of systemic candidiasis has considerably increased and has become a leading cause of mycosis-associated mortality worldwide [2]. The antifungal drugs used currently are reported to have moderate to sever toxicity effect on the patients’ health [3]. Also, resistance to the most common antifungal drugs has
* Corresponding authors at: Faculty of Dentistry, Kuwait University, P.O. Box. 24923, Safat 13110, Kuwait. E-mail addresses: [email protected] (J.M. Behbehani), [email protected] (M. Irshad).
increased in recent years [2,4]. Therefore, there is a need of novel, less cytotoxic molecules that can be used as antifungal therapeutics. Green tea or tea is a widely consumed beverage of Camellia sinensis var. sinensis and Camellia sinensis var. assamica plant leaves [5]. It has long been believed that drinking of tea beverage has health benefits and increases longevity [5]. Green tea or tea beverage contains rich amount of polyphenols, of which epigallocatechin 3-O-gallate (EGCG) (Fig. 1), is the most abundant [6]. The green tea beverage of 237 mL contained about 30–130 mg EGCG [6]. A study reported that ingestion of EGCG was safe and well-tolerated by the healthy human volunteers at the oral doses of 800 mg/day, which is equivalent to about 8 to 16 cups of green tea a day [7]. EGCG has many pharmacological properties including antibacterial and antifungal properties [8,9]. In the present study, we evaluated the anticandidal potential of EGCG alone and in combination with azole antifungal drugs against
https://doi.org/10.1016/j.mycmed.2019.01.011 C 2019 Published by Elsevier Masson SAS. 1156-5233/
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7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS). Overnight culture of Candida was diluted in medium to obtain a final inoculum of 2.5 103 cells/mL. An aliquot of 100 mL was added to each well of a 96-well U-bottom tissue culture plate. The treatment concentration range of EGCG was 0.98– 250 mg/mL, and for fluconazole and ketoconazole, the range was 0.50–128 mg/mL. For positive growth control, medium without drugs was used. The cell growth in the wells was observed visually after incubation at 37 8C for 24 and 48 hrs. MIC is the lowest concentration of a test compounds at which no visible growth was found [11,12]. The minimum fungicidal concentration (MFC) was evaluated by inoculation of 20 mL content from the wells with clear broth/no visible growth (MIC, 2MIC and 4MIC) on the sabouraud dextrose agar (SDA) plate. The inoculated plates were incubated at 37 8C for 24–48 hrs. MFC was defined as a concentration of drug at which no visible growth was observed on the SDA plates [12]. All of the assays were performed independently three times. 2.4. Inhibition of mature Candida biofilm Fig. 1. Chemical structure of epigallocatechin 3-O-gallate (EGCG).
the planktonic and mature sessile biofilms of oral Candida isolates. We used techniques like confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy techniques to understand mechanisms of action of EGCG. 2. Materials and methods 2.1. Chemicals EGCG (epigallocatechin gallate or epigallocatechin 3-gallate or epigallocatechin 3-O-gallate) (purity 98%, Catalogue No. 1236700), fluconazole and ketoconazole were purchased from Sigma-Aldrich, St. Louis, MO, USA. The other chemicals and culture medium constituents were also purchased from Sigma-Aldrich, USA. 2.2. Sample collection Oral Candida samples were collected from dental patients at the Kuwait University Dental Clinic (KUDC), by using a standard oral rinse technique [10]. The collected samples were cultured in YPD medium, containing yeast extract (1% w/v), peptone (2% w/ v) and dextrose (2% w/v), and incubated at 37 8C. The Candida spp. were identified by using CHROMagar medium (CHROMagar Candida, France) and VITEK-2 system assays (bioMerieux, Craponne, France). As reported in our previous study, we confirmed the identity of eight Candida spp. from the collected oral samples, i.e., C. albicans (n = 33), C. dubliniensis (n = 13), C. tropicalis (n = 3), C. glabrata (n = 7), C. krusei (n = 1), C. lusitaniae (n = 1), C. kefyr (n = 1), and C. parapsilosis (n = 1). Four ATCC strains (C. albicans ATCC 24433, C. glabrata ATCC 15126; C. parapsilosis ATCC 90018 and C. krusei ATCC 6258), were obtained from the Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait. 2.3. Minimum inhibitory and fungicidal concentration Clinical Laboratory Standards Institute (CLSI) method for the yeasts (M27-A3) was used for evaluation of minimum inhibitory concentration (MIC) [11]. Test was carried out in RPMI-164 medium containing L-glutamine, without sodium bicarbonate (Cat. No. R6504, Sigma-Aldrich, USA), and buffered to pH
A standardized method was used in this study. The mature biofilms of Candida were formed in 96-well polystyrene microtiter plates (Corning Inc., Corning, NY, USA). Different concentrations of EGCG and standard drug were prepared separately in RPMI 1640 medium using serial dilution method and added to each well (200 mL/well). The medium without test agents was added to the controll wells. After treatment, microtitre plate was re-incubated at 37 C for 24 hrs. After incubation period, each well was washed carefully with sterile PBS (pH 7). Biofilm metabolic activity was quantified by MTT assay following manufacturer’s instructions, but with a slight modification. Briefly, MTT (1 mg/mL) was freshly prepared in PBS and menadione (10:1 v/v) was added before use. Menadione (0.4 mM) solution was prepared in acetone and stored at 4 8C. MTTmenadione solution was added to each well (60 mL/well) and incubated at 37 C for 3 hrs. The MTT-formazan product was formed by the live cells, which was dissolved in DMSO (200 mL) and optical density was measured at 570 nm (Microplate Reader, BioTek Instruments, Winooski, USA). An inhibition of metabolic activity of biofilm was calculated by the equation:
Inhibitionð%Þ ¼
ðControlTestÞ 100 Control
The minimum biofilm inhibitory concentration (MBIC) of a test agent was defined as the lowest concentration at which 80% metabolic activity of biofilms was inhibited as compared to the control. The assays were performed in triplicate and three independent experiments were performed. 2.5. Synergistic effect of EGCG and azole drugs on Candida planktonic cells To study the synergistic effect of EGCG and drugs, we used checkerboard microtiter plate method [13]. Different combination of EGCG and fluconazole or ketoconazole drugs was prepared in RPMI medium. The concentration of EGCG was in the range of 0.98 to 62.5 mg/mL and for fluconazole or ketoconazole drugs, the range was 1 to 128 mg/mL. Candida cells ( 2.5 103 cells/mL) were added to each well and incubated at 37 8C for 48 hrs. The growth of Candida cells was read visually. The synergistic effects of EGCG and drugs were defined as the lowest concentration that prevented visible growth. All assays were performed independently three times.
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To evaluate interaction between EGCG and drugs, the fractional inhibitory concentration index (FICI) was calculated. FICI of EGCG = MIC (EGCG with drug)/MIC (EGCG alone) and FICI of drug = MIC (Drug with EGCG)/MIC (Drug alone). The sum of both; SFICI 0.5 exhibited synergetic interaction, FICI > 0.5–4 exhibited no interaction and FICI > 4 exhibited antagonistic interaction between EGCG and drug.
refractivity, partition coefficient (log P), polar surface area and atom count were calculated and matched with Lipinski’s rule of five [16]. Docking studies of EGCG with ergosterol were carried out by using AutoDock 4.2 Tools (Scripps Research Institute, USA) [17]. Ergosterol (PDB ID: 2AIB) X-ray crystal structure was obtained from the Protein Data Bank [RCSB Protein Data Bank., http://www.rcsb.org/pdb].
2.6. Combination effect of EGCG and azole drugs on the growth of Candida cells
2.11. Statistical analysis
The combination effect of drugs on the growth of Candida was examined. Fresh overnight growth of Candida cells ( 1 10 6 ; optical density A 595 = 0.1) was inoculated into YPD medium. The MIC and sub-MIC values of EGCG alone or EGCG with drug was added to the medium. The cells were incubated at 37 C and 120 rpm, using IKA KS 3000 Shaking Incubator (Staufen, Germany). The optical density of cells was recorded per hour by using Eppendorf Spectrophotometer (Hamburg, Germany).
The experiments were performed in triplicate. Statistical analysis was performed by SPSS software (SPSS Inc., USA). The significant differences between the groups were analyzed by ANOVA and Tuky HSD test. The value P 0.05 was considered statistically significant differences.
3. Results 3.1. Minimum inhibitory concentration (MIC) and fungicidal concentration (MFC)
2.7. Synergistic effects of EGCG and azole drugs on Candida biofilms Candida biofilms were formed in polystyrene 96-well plates, as previously described. The checkerboard micro-dilution method was used to prepare different combination of EGCG and drug in RPMI-164 medium [13]. The drug combinations were prepared in fresh a sterile plate and then added into the corresponding biofilm wells (200 mL/well). The plate was incubated at 37 8C for 48 hrs, after which, the plate was washed twice with PBS. The metabolic activity of biofilm was evaluated by the MTT-menadione reduction assay as described in the previous section. The assays were performed independently three times. 2.8. Confocal scanning laser microscopy The effect of EGCG on Candida cell membranes was assessed by using florescence dye SYTO-9 and Propidium iodide (PI) (LIVE/ DEAD1 BacLightTM, USA). Candida cells ( 1 106) were inoculated in YPD medium and treated with EGCG. The treated and untreated cells were incubated at 37 8C with constant shaking (120 rpm) for 12 hrs. After incubation period, cells were washed twice in PBS (pH-7) and then florescence dye was added at a concentration of one mg/mL. To study cell membrane permeabilization, 10 mL cell suspension was added on a glass slide and dye uptake was visualized by confocal microscopy (ZEISS LSM 500).
EGCG showed antifungal activity against 60 oral isolates and 4 ATCC strains of Candida spp. The MIC ranges of EGCG were 3.91 to 15.63 mg/mL against the planktonic cells of all Candida isolates. Among the tested isolates, the MIC of EGCG was as follows: 3.91 mg/mL for 4 isolates, 7.81 mg/mL for 23 isolates, and 15.63 mg/mL for the rest 33 isolates. The MIC ranges of fluconazole and ketoconazole were 4 to 128 mg/mL and 4 to 32 mg/mL, respectively. The MFC values of EGCG were 15.63 to 31.25 mg/mL against all isolates. The MIC and MFC values of EGCG against the specific strains are given in Table 1. 3.2. Effects of EGCG in combination with fluconazole or ketoconazole against planktonic Candida cells To test the combined activity, we assessed various combination concentrations of EGCG and fluconazole or ketoconazole using checkerboard titer assay. The combination of EGCG with fluconazole or ketoconazole showed a remarkable decrease in the MIC values compared to each drug alone (Table 2). The result showed MIC of fluconazole decreased from 4–64 to 1–8 mg/mL, whereas the MIC of ketoconazole decreased from 4–32 to 1–8 mg/mL (P < 0.01). The results showed that the combinations of EGCG/ ketoconazole exerted synergistic effects against the planktonic cells of Candida spp. (S FICI 0.50). However, combination of EGCG/fluconazole exerted either synergism or no interaction (S FICI, 0.375 to 0.625).
2.9. Scanning and transmission electron microscopy EGCG treated cells ( 1 10 6) were observed under scanning electron microscopy (SEM) (JEOL, CarryScope JCM 5700) and transmission electron microscopy (TEM [JEOL, JEM 1200 EXZ]). The MIC value of EGCG was used to observed effect on the ultrastructure of the Candida cells. The treated samples were washed twice in PBS and fixed in glutaraldehyde (3%) and processed for SEM/TEM analysis as described previously [14]. Other details can be obtained from the method by Mares [15]. 2.10. EGCG and ergosterol docking study The likeliness of EGCG molecule was assessed theoretically by using computer-added MarvinSketch 5.8.2 program. The parameters like molecular mass, hydrogen bond donors, acceptors, molar
Table 1 MIC and MFC values of EGCG against Candida spp. Strain/isolates (CDC NO./ATCC)
C. C. C. C. C. C. C. C.
dubliniensis (CDC 27963) albicans (CDC 27974) glabrata (CDC 27845) albicans (CDC 28304) glabrata (CDC 28398) dubliniensis (CDC 28551) albicans (ATCC 24433) glabrata (ATCC 15126)
EGCG (mg/mL) MIC
MFC
15.63 15.63 15.63 7.81 15.63 7.81 15.63 15.63
31.25 31.25 31.25 31.25 15.63 15.63 31.25 31.25
EGCG: epigallocatechin 3-O-gallate; CDC: Comprehensive Dental Care Number; ATCC: American Type Culture Collection; MIC: Minimum Inhibitory Concentration (no visible growth); MFC: Minimum Fungicidal Concentration (no visible growth).
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Table 2 Antifungal effects of EGCG alone and in combination with fluconazole (FLC) and ketoconazole (KTC) against Candida planktonic cells. Strain/isolates (CDC NO./ATCC)
C. dubliniensis (CDC 27963) C. albicans (CDC 27974) C. glabrata (CDC 27845) C. albicans (CDC 28304) C. glabrata (CDC 28398) C. dubliniensis (CDC 28551) C. albicans (ATCC 24433) C. glabrata (ATCC 15126)
MIC (mg/mL) Antifungal
Antifungal (Alone/combo)b
EGCG (Alone/combo)b
SFICI
Fluconazole
32/4
15.63/3.91
0.375
Ketoconazole Fluconazole
8/2 64/8
15.63/3.91 15.63/3.91
0.500 0.375
Ketoconazole Fluconazole
8/2 16/4
15.63/1.95 15.63/3.91
0.375 0.500
Ketoconazole Fluconazole
8/2 64/8
15.63/1.95 7.81/3.91
0.375 0.625a
Ketoconazole Fluconazole
32/8 16/4
7.81/1.95 15.63/3.91
0.500 0.500
Ketoconazole Fluconazole
8/1 4/1
15.63/3.91 7.81/1.95
0.375 0.500
Ketoconazole Fluconazole
4/1 64/4
7.81/1.95 15.63/3.91
0.500 0.313
Ketoconazole Fluconazole
16/2 16/4
15.63/3.91 15.63/3.91
0.375 0.500
Ketoconazole
16/4
15.63/3.91
0.500
EGCG: epigallocatechin 3-O-gallate; CDC: Comprehensive Dental Care Number; ATCC: American Type Culture Collection; FIC: Fractional Inhibitory Concentration Index. Tukey HSD multiple comparisons test exhibited significance differences between the MIC value of FLC alone vs. FLC with EGCG (P = 0.0001) and KTC alone vs. KTC with EGCG (P = 0.003). a No interaction. b Significant differences in ANOVA test (P < 0.01).
3.3. Combination effects of EGCG and fluconazole or ketoconazole on the growth of Candida cells The growth of Candida spp. was evaluated in the presence of EGCG alone or in combination with azole drugs. The growth pattern was plotted against time as shown in Fig. 2(A–D). EGCG alone at MIC and sub-MIC value showed a significant inhibitory effect on the growth of C. albicans CDC 27974, C. dubliniensis CDC 27963, C. glabrata CDC 27845, and C. albicans ATCC 24433, respectively. The synergistic combination of EGCG (MIC/4) with fluconazole (MIC/4 or MIC/8 or MIC/16) or EGCG (MIC/4 or MIC/8) with ketoconazole (MIC/4 or MIC/8) showed a significant inhibitory effect against the tested Candida spp. (Fig. 2A–D). The Candida cells without any treatment showed a normal pattern of growth such as lag phase, active exponential phase and stationary phase. In conclusion, EGCG showed synergistic effect with fluconazole or ketoconazole drugs leading to a fungicidal activity at lowered concentration. Treatment of all 4 species with the EGCG (MIC) alone resulted in 95.13% reduction in optical density, whereas in synergistic combinations [(EGCG-Fluconazole) and (EGCG-ketoconazole)] resulted in 92.27% and 97.51% reduction in optical density, as compared to the control. 3.4. Inhibition of mature Candida biofilm The activity of EGCG was tested on mature biofilm of selected oral isolates and two ATCC Candida strains. The biofilm metabolic activity was quantified by the MTT assay. The metabolic activity of mature biofilm decreased in a concentration dependent manner (Fig. 3). The minimum biofilm inhibitory concentration (MBIC) ranges of EGCG against the all tested isolates/strain were 62.5 to
125 mg/mL (Table 3). In comparison, antifungal activity of EGCG against the Candida planktonic cells was two to three fold higher than the mature biofilm at the same concentration (P < 0.05). 3.5. Combination effect of EGCG in combination with fluconazole or ketoconazole on the Candida biofilms To determine metabolic activity of the mature Candida biofilms, 48 hrs old biofilms were treated with EGCG/fluconazole or EGCG/ ketoconazole combinations. The MBIC values of fluconazole and ketoconazole against the biofilms of all tested Candida spp. were in the range of 128 to 512 mg/mL and 64 to 512 mg/mL, respectively (Table 3). The results indicate that the mature biofilms of Candida spp. were resistant to these drugs. When EGCG was combined with fluconazole or ketoconazole drugs, the MBIC values of drugs and EGCG decreased many fold indicating a synergistic effect in all most all the cases (P = 0.0001). 3.6. Cell membrane permeability Confocal laser scanning microscopy (CLSM) was used to explore individual cells stained with SYTO9 alone or SYTO9/propidium iodide. CLSM results showed the membrane-impermeant propidium iodide uptake by the EGCG treated Candida cells. Propidium iodide is a membrane impermeable red dye and is therefore excluded by healthy cells. However, SYTO9 is a membrane permeant green dye and therefore stained all live and dead cells (Fig. 4). In Fig. 4, CLSM images in left panel show the untreated C. albicans CDC 27974, C. dubliniensis CDC 27963 and C. glabrata CDC 27845 cells which is stained green only. In the treated samples, the images clearly show that Syto9 was taken up
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Fig. 2. Effect of epigallocatechin 3-O-gallate (EGCG) alone and in synergistic combination with fluconazole (FLC) and ketoconazole (KTC) on the growth of Candida spp. The graph represents the growth curves of (A) C. dubliniensis CDC 27963, (B) C. albicans CDC 27974, (C) C. glabrata CDC 27845, and (D) C. albicans ATCC 24433, in presence of EGCG (MIC and sub-MIC) alone and in synergistic combination with FLC or KTC drugs (as given in Table 2). Growth curve plotted against optical density at 595 nm versus time (hrs).
by both live and dead cells, whereas propidium iodide was taken up only the dead cells. The results confirm that the Candida cells membrane was disrupted by EGCG treatment. 3.7. Scanning electron microscopy (SEM) SEM micrographs of EGCG treated and untreated Candida cells are shown in Fig. 5. C. albicans CDC 27974, and C. dubliniensis CDC 27963 were treated with MIC values of EGCG for 12 hrs (Fig. 5). The images showed drastic morphological changes in treated cells compared to the untreated cells. The micrograph of untreated cells showed oval in shape and with smooth cell surfaces and polar bud scars (Fig. 5A–B). While treated cells showed deformed cells shape with convoluted and irregular cells surfaces. Most of the treated cells showed deep furrows and wrinkles of the cell surface (Fig. 5C–D). 3.8. Transmission electron microscopy (TEM) The ultrathin sections of EGCG treated Candida cells showed irregular breakage in the cell wall and cell membrane in
contrast to the untreated cells. In Fig. 6, TEM micrograph of untreated and treated C. albicans CDC 27974 and C. dubliniensis CDC 27963 cells are shown. The untreated cells showed typical cell morphology and had uniform central density (Fig. 6A–B). However, EGCG treated cells (12 hrs) showed severe damage in cell wall and plasma membrane, like rupturing and swelling at many places (Fig. 6C–F). The disintegration of the cell wall structure indicates release of cytoplasmic content and cell death. 3.9. Ergosterol and EGCG likeness and docking studies The molecular property of EGCG molecule has all of the drug likeness criteria as defined by Lipinski (Table 4). The docking experiments showed strong interaction between EGCG and ergosterol molecules (Fig. 7). In this study, docking energy of interaction between both molecules was 27.17 kJ/mole. A total of six hydrogen bonds formed between the hydroxyl groups of EGCG and the polar surface of ergosterol (Fig. 7b). The interaction between EGCG and ergosterol were also by Van der Waals bonding (Fig. 7c).
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Fig. 3. Effect of epigallocatechin 3-O-gallate (EGCG) on preformed biofilm of Candida spp. The mature Candida biofilm was grown in the polystyrene surface microliter plate. Different concentrations of EGCG were treated in triplicate, and then further incubated for 24 hrs. After incubation periods, the extent of biofilm was estimated by the MTT reduction assay. The data represents (mean SD) of three experiments. All eight strains have significant correlation along with concentration (P < 0.05).
Table 3 Antifungal effects of EGCG alone and in combination with fluconazole (FLC) and ketoconazole (KTC) against Candida biofilms. Strain/isolates (CDC NO./ATCC)
C. dubliniensis (CDC 27963) C. albicans (CDC 27974) C. glabrata (CDC 27845) C. albicans (CDC 28304) C. glabrata (CDC 28398) C. dubliniensis (CDC 28551) C. albicans (ATCC 24433) C. glabrata (ATCC 15126)
MIC80 (mg/mL) Antifungal
Antifungal (Alone/combo)b
EGCG (Alone/combo)b
S FICI
Fluconazole
256/16
62.5/15.63
0.313
Ketoconazole Fluconazole
256/16 512/32
62.5/15.63 125/62.5
0.313 0.563a
Ketoconazole Fluconazole
512/32 128/16
125/31.25 62.5/15.63
0.313 0.375
Ketoconazole Fluconazole
64/8 256/32
62.5/15.63 125/31.25
0.375 0.375
Ketoconazole Fluconazole
256/16 256/16
125/31.25 62.5/15.63
0.313 0.313
Ketoconazole Fluconazole
256/16 128/16
62.5/15.63 62.5/15.63
0.313 0.375
Ketoconazole Fluconazole
64/8 512/16
62.5/15.63 125/62.5
0.375 0.531a
Ketoconazole Fluconazole
256/8 256/8
125/31.25 62.5/15.63
0.281 0.281
Ketoconazole
256/8
62.5/15.63
0.281
EGCG: epigallocatechin 3-O-gallate; CDC: Comprehensive Dental Care Number; ATCC: American Type Culture Collection; FICI: Fractional Inhibitory Concentration Index. Tukey HSD test exhibited significance differences between the MIC value of FLC alone vs. FLC with EGCG (P = 0.0001) and KTC alone vs. KTC with EGCG (P = 0.0001). a No interaction. b Significant differences in ANOVA test (P < 0.001).
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Fig. 4. Confocal scanning laser microscopy (CSLM) images of Candida spp. treated with epigallocatechin 3-O-gallate (EGCG) (MIC) for 12 hrs (right panel) and compared with untreated control cells (left panel). To membrane damage cells were stained with PI (red signals).
4. Discussion Natural antifungal compounds in combination with antimycotics have recently been paid much attention by researchers. It is expected that the combination therapy with natural products provides promising rapid and synergistic potency of drug activity, lowering the risk of drug resistance and reduces dosing toxicity of
drug. In the present study, EGCG exhibited strong antifungal potential; tested against 60 oral Candida isolates and 4 ATCC Candida strains. EGCG inhibited the growth of both C. albicans and non-albicans species. In addition, we found that the minimum inhibitory concentration (MIC) of EGCG varied with isolates. The MIC value of EGCG obtained in the present study is low and consistent with the previous reports tested against the Candida
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Fig. 5. Scanning electron micrograph of Candida cells treated with epigallocatechin 3-O-gallate (EGCG) (MIC). A. Untreated C. albicans CDC 27974. B. Untreated C. dubliniensis CDC 27963. C. Treated C. albicans CDC 27974. D. Treated C. dubliniensis CDC 27963. (Panel 3) enlarged view of a selected portion of C and D. (1, 2) Untreated cells are indicating polar bud scar, an oval shape and smooth cell surfaces (3, 4, 5, 6) deformed cells with convoluted and irregular surfaces and formation of deep furrows.
Fig. 6. Transmission electron micrograph of Candida cells treated with epigallocatechin 3-O-gallate (EGCG) (MIC). (A) untreated C. albicans CDC 27974 (B) untreated C. dubliniensis CDC 27963 (C, D) treated C. albicans CDC 27974. (E, F) treated C. dubliniensis CDC 27963. (1) Rupturing of both cell wall and plasma membrane. (2) Disintegration of the cell wall. (3) Oozing out of intracellular content. (4) Undulating cell wall (5) deposition of lipid globules (6) deterioration of cytoplasm. (7) Disintegrated cell wall (8) swelling of the cell wall (9) disrupted cells wall. The values in parentheses represent the number of cells observed for each sample.
planktonic cells [18,19]. However, in one particular study, MIC range of EGCG was much higher than the present study [20]. The higher MIC values in this study might be due to differences in the purity of the compound, origin of the strains and growth conditions. This study demonstrated synergistic inhibitory effects of EGCG and azole antifungal drugs against planktonic and mature sessile biofilms of Candida cells. The treatment of azole drugs along with
EGCG showed decrease of MIC value of fluconazole and ketoconazole in comparison to the effect of these drugs alone (Table 2). Our results are consistent with previous studies in which EGCG enriched the fungicidal potential of amphotericin-B, fluconazole and miconazole tested against the Candida planktonic cells [20,21]. The eradication of Candida biofilm is one of the most challenging tasks in fungal therapeutics, beacause of its reduced
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Table 4 Theoretical molecular properties of EGCG. The values in green are in agreement with Lipinski’s values and those in red are outside the range of the values set by Lipinski.
EGCG Lipinski’s values
Molecular mass/u
Log P
Hydrogen bond acceptor
Hydrogen bond donor
Molar refractivity (m3M1)
Polar surface area/A˚2
Atom count
458.372 180 to 500
3.08 0.4 to +5.6
10 Up to 10
8 Up to 5
111.75 40–130
197.37 Up to 140
51 20–70
EGCG: epigallocatechin 3-O-gallate.
Fig. 7. a–c: docking images of epigallocatechin gallate with ergosterol showing various kinds of interactions possible between ergosterol and epigallocatechin gallate: a: represents the docked image of epigallocatechin gallate with ergosterol visualized by PyMol; b: represents the hydrogen bonding interactions between epigallocatechin gallate and ergosterol; c: shows the hydrophobic residues involved in the interaction between epigallocatechin gallate and ergosterol.
susceptibility to antimycotic drugs [22]. Similar to an earlier study, our results showed that Candida biofilms were more resistant to fluconazole and ketoconazole drugs in comparison to the planktonic counterparts [20,23]. In the present study, EGCG alone destroyed the biofilm morphology and inhibited the maintenance of mature biofilm at the MIC value. The mean MBIC value of EGCG against the randomly selected six oral Candida spp. was 83.33 mg/ mL and against the two ATCC strains it was 93.75 mg/mL. The fungicidal potential of EGCG against Candida biofilms increased when treated in combination with fluconazole or ketoconazole. Similarly, fluconazole or ketoconazole fungicidal potential increased when combined with EGCG. The MBIC value of ketoconazole/EGCG or ketoconazole/EGCG decreased 3 to 4-fold in comparison to the inhibitory effect of those drugs alone. The results indicate that the EGCG can significantly improve the efficacy of fluconazole and ketoconazole drugs against preformed sessile biofilms. Ning et al. [20] also reported that the MBIC values of fluconazole decreased two to four times in combination with EGCG. In summary, our results indicate that EGCG could significantly inhibit the mature biofilm alone or in combination with azole antifungal drugs. The effect of EGCG alone on the membrane integrity of Candida cells was examined by the CLMS, SEM and TEM. This is the first study to report an effect of EGCG on ultrastructure of Candida cells. Confocal laser microscopy showed the intensity of red propidium iodide inside the treated dead cells or cells with deformed membrane, but not the untreated cells (Fig. 4). This is the first line of evidences that the antifungal activity of EGCG against Candida might be by making a lesion on the cell membrane or by causing loss of cell membrane integrity [24,25]. The second level of evidences is established by the SEM and TEM micrographs. The untreated cells clearly have normal oval shape having smooth cell surface with polar bud scars (Fig. 5A–B). However, treated cells showed deformed cells and receding of cytoplasm leading to lysis of cells (Fig. 5C–D). Similar type of cell membrane alterations has been reported in C. albicans cells treated by antifungal agents [26]. TEM observation showed that cell organelles were damaged in treated Candida cells (Fig. 6C and E). These observations were consistent with the damages observed in C. albicans cells treated with magnolol and garlic oil [27]. Also TEM micrograph confirmed
the damage of cells, such as breaking of both cell wall and plasma membrane (Fig. 6D and F). The collapse of cell wall leads to change of membrane permeability, osmotic imbalance and finally resulted to cell death [28]. Computational tools have been used for target identification and drug discovery [29]. Since several years, fungal cell wall ergosterol macromolecule has been used as target for antifungal drug discovery [30]. In the present study, results of docking experiments exhibited strong binding between EGCG and ergosterol. The interaction between both molecules may create pores in fungal cell membranes and result in loss of essential ions such as potassium and protons and other molecules, eventually leading to death of fungal cells. 5. Conclusion The results from the present study strongly support the antifungal potential of EGCG against different Candida isolates. The synergistic effect of EGCG with fluconazole and ketoconazole against the planktonic and mature Candida biofilms might be useful in reducing the antimycotics drug dose, prevent drug resistance development and side effects. From the obtained results, we suggest to examine the fungicidal potentiality of EGCG in an in vitro model. Ethical approval Ethical approved from the Health Sciences Center Ethical Committee, Kuwait University (Ref: VDR/EC/2347). Disclosure of interest The authors declare that they have no competing interest. Acknowledgements The authors are thankful to the Office of the Vice President for Research, Kuwait University, for financial support (Grant No. DR04/14). We thank the Oral Microbiology Research Labora-
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