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Introduction

The incidence of Candida infections has increased in patients who receive immunosuppressive therapy, cancer chemotherapy and transplantation. Among the Candida species, Candida albicans (C. albicans) is responsible for 50–70% of cases of invasive candidiasis in the majority of clinical settings (1,2). C. albicans, an opportunistic fungal pathogen that normally inhabits the mucous membranes of the gastrointestinal and female genital tracts (3), is able to attack the immunocompromised patients (4). When the normal microbial barrier is disrupted, C. albicans can invade the intestinal and female genital mucosal barriers and causes candidiasis and candidemia (5,6). A cross-sectional study has reported that Candida species isolation from the vaginal mucosa was more frequent in human immunodeficiency virus (HIV)-infected patients (29.7%) when compared with uninfected women (14.5%). C. albicans was the most prevalent pathogen isolated in HIV-infected (52.9%) and uninfected women (85.7%) (7). A previous study investigated 128 Candida isolates from South Africa and 126 Cameroonian Candida isolates. Of those, C. albicans was responsible for the highest percentage of them, with 82.8% of South African isolates and 73.0% of the Cameroonian isolates (8). In another study, of the 103 Candida species-infected oral mucosal isolates identified in HIV patients, C. albicans accounted for the majority of Candida species (77.7%) (9). A prospective observational study also examined the occurrence of oral Candida colonization among cancer patients in China between October 2012 and March 2013. C. albicans was the most common species isolated from patients, accounting for 30.8% in patients with pulmonary cancer (n=78), 33.7% in patients with digestive tract malignant tumor (n=101), and 12.7% in patients with hematopoietic system tumor (n=79). Cancer patients are a high-risk population for Candida colonization (10).

Traditional Chinese medicines (TCMs) remain a fundamental role in the treatment of various diseases due to their long history of clinical practice and reliable therapeutic efficacy. Berberine (Fig. 1A) is an isoquinoline quaternary alkaloid isolated from the traditional Chinese herb, Coptidis Rhizoma (also known as Huanglian), which includes several species: Coptis chinensis French, Coptis deltoidea and Coptis teetoides (11,12). Berberrubine (Fig. 1B) is isolated from the medicinal plant Berberis vulgaris (13), but is also readily derived from berberine by pyrolysis (14). Kim et al (15) studied the antimicrobial activity of berberine, berberrubine, 9-O-acylberberrubines and 9-O-alkylberberrubines. Berberrubine exhibited a relatively weaker anti-C. albicans activity [minimum inhibitory concentration (MIC) >128 µg/ml] than berberine (MIC = 128 µg/ml), while some of the acyl derivatives (decanoyl and lauroyl berberrubine chlorides) and the alkyl derivatives (heptyl, octyl, nonyl, decyl and undecyl) showed much stronger growth inhibition against C. albicans, with the MICs ranging from 1–4 µg/ml. Park et al (16) reported the antifungal activity of 13-(substituted benzyl) berberine and berberrubine derivatives. Among them, 13-(4-tertbutylbenzyl) and 13-(4-isopropyl benzyl) berberine derivatives exhibited the strongest antifungal activity against C. albicans (MICs = 4 µg/ml) when compared with berberine (MIC = 128 µg/ml). The 13-(4-tertbutylbenzyl) and 13-(4-isopropyl benzyl) berberrubines showed a better anti-C. albicans activity (MICs = 16 µg/ml) when compared with berberrubine (MIC >128 µg/ml).

Figure 1. Chemical structure of (A) berberine and (B) berberrubine.

TCMs can be used as complementary medicines to conventional drugs for a variety of disease treatments. Our previous study reported the complementary use of corilagin, a gallotannin identified in numerous plants, including Phyllanthus urinaria, with two chemotherapeutic drugs, cisplatin and doxorubicin, in order to lower the working concentration of these two agents and to obtain an increment in the anticancer effect. The IC50 values were ~3-fold reduced for cisplatin and ~4-fold decreased for doxorubicin with the Hep3B hepatoma cells in the presence of corilagin (17). The efficacy of standard antibiotics against bacterial strains was improved by the use of plant materials (18–21). TCMs as a complementary therapy with the standard antifungal drugs against fungal pathogens have emerged as a new choice for the treatment of infectious diseases. The present study utilizes the TCMs, berberine and berberrubine, as complementary agents with the standard antifungal drug, terbinafine, against the most common Candida species, C. albicans, responsible for the increased incidence of fungal infections.

Materials and methods

Chemical analysis

Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker/Varian 500 MHz Fourier transform spectrometer (Agilent Technologies, Santa Clara, CA, USA). 1H and 13C-NMR spectra were recorded relative to residual protiated solvent; a positive value of the chemical shift denoted a resonance downfield from tetramethylsilane (TMS, internal standard). J-values were in Hz. All the chemicals were purchased from commercial suppliers and used without further purification. All the reactions were monitored by analytical thin-layer chromatography (TLC) on Merck aluminum-precoated plates of silica gel 60 F254 with detection by spraying with 5% (w/v) dodecamolybdophosphoric acid in ethanol or 5% (w/v) ninhydrin in ethanol and subsequent heating.

Synthesis of natural berberrubine

Natural berberrubine was synthesized according to the reported procedure (22). Berberine chloride (2.01 g, 5.4 mmol) was heated at 190°C under reduced pressure for 2 h and the crude product was recrystallized from chloroform and hexane to obtain the title berberrubine in 83% yield as a dark red precipitate: Rf=0.29 (dichloromethane-methanol, 15:1); 1H-NMR (CDCl3) d9.20 (s, 1H), 7.58 (s, 1H), 7.30–7.24 (m, 2H), 6.75 (s, 1H), 6.51–6.49 (m, 1H), 6.06 (s, 2H), 4.42 (s, 2H), 3.89 (s, 3H), 3.09 (s, 2H); 13C-NMR (CDCl3) d167.7, 150.3, 149.1, 148.2, 145.9, 133.1, 131.5, 128.3, 122.2, 120.7, 120.2, 117.7, 108.4, 104.6, 103.3, 101.9, 56.2, 53.4, 28.6.

Determination of MIC and minimum fungicidal concentration (MFC)

C. albicans was obtained from American Type of Culture Collection (Manassas, VA, USA). The MIC values of berberine, berberrubine (kindly provided by Professor K.K.H. Lee) and terbinafine (both from Sigma-Aldrich, St. Louis, MO, USA) on C. albicans were determined by the broth dilution method. Briefly, different concentrations of berberine, berberrubine and terbinafine were loaded from a starting concentration of 100 µg/ml containing 1% dimethylsulfoxide (DMSO) as the vehicle and they were diluted serially. DMSO (1%) was used as a vehicle control. The fungal samples were subsequently incubated at 35°C for 48 h. The minimum concentrations of berberine, berberrubine and terbinafine, which induced a complete growth inhibition would be recorded as their MIC values (16,23). For the determination of MFC, 10 µl of the 48 h incubated medium was removed and plated. MFC was recorded at a concentration where no colony of fungal growth was observed. When MFC/MIC <4, the compound or combination would be considered as fungicidal, while when MFC/MIC ≥4 the compound or combination would be considered as fungistatic. In each case, three independent experiments were conducted and each experiment was carried out in triplicates.

Sensitization test

For the sensitization investigation experiment, the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay was employed (24). Briefly, C. albican cells were seeded at day 0 in the 96-well microplate. Terbinafine was added at 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 and 0.2 µg/ml, respectively, while berberine and berberrubine were added at 100 µg/ml together with terbinafine. After 48 h of incubation, MTS (Promega, Madison, WI, USA)/phenazine methosulfate (PMS) as electron coupling agent mixed solution was added. Lastly, optical absorbance was determined at 490 nm using a microplate reader (Perkin Elmer Victor V) according to the manufacturer's protocol. To determine MIC and MFC values from the sensitization test, no MTS/PMS was added and the experimental procedures were conducted as mentioned before. In each case, three independent experiments were conducted and each experiment was carried out in triplicates.

Zone of inhibition study

C. albicans was used to study the effectiveness of berberine alone (100 µg), berberine (100 µg) plus terbinafine (6 µg), berberrubine alone (100 µg), and berberrubine (100 µg) plus terbinafine (6 µg) against its growth in culture. C. albicans was diluted with yeast mold broth and plated on the yeast mold agar plate, and the holes were created on the agar using a sterile transfer pipette. For the tested samples, 1% DMSO (as negative control) and 6 µg terbinafine (as positive control) were placed in the holes of agar. The plates were subsequently incubated at 35°C for 48 h and the inhibition zones (mm, in terms of diameter) of fungi on the agar plates were recorded (23,25,26).

Results and Discussion

Determination of MIC, MFC and sensitization assay

Terbinafine is an allylamine agent with a broad spectrum of antifungal activity. It interferes with the biosynthesis of ergosterol, an essential component of fungal cell membranes, via inhibition of the fungal enzyme squalene epoxidase. In the in vitro susceptibility tests, terbinafine have been shown to possess primarily fungicidal activity against dermatophytes, moulds and certain dimorphic fungi, but only fungistatic activity against C. albicans (27). The cell death mechanisms of berberine against C. albicans have been fully addressed in the previous studies. They involved the ability to impair mitochondrial function, generation of reactive oxygen species, targeting cell wall integrity pathway and also affecting heat shock transcription factor, HSF1 (28). The MIC and MFC values of terbinafine against C. albicans were determined as 6 and 24 µg/ml, respectively. As MFC/MIC was equal to 4, it was considered to be fungistatic. For berberine and berberrubine, their MIC values were >100 µg/ml. No MFC value was determined. These findings were consistent with the results reported previously (16). However, berberine and berberrubine at 100 µg/ml could not improve the MFC, and the MFC of terbinafine could significantly potentiate the antifungal activity of terbinafine on C. albicans as determined by the MTS/PMS assay. Berberine and berberrubine at 100 µg/ml could effectively assist the antifungal potential of terbinafine (Fig. 2). Notably, the complementary activity of berebrine with terbinafine was much stronger than that of berberrubine when terbinafine was loaded from 3.13, 1.56, 0.78, 0.39 and 0.2 µg/ml. Therefore, berberine was more effective in amplifying the antifungal action of terbinafine when compared with its analogue, berberrubine.

Figure 2. Sensitization of Candida albicans to terbinafine by berberine and berberrubine. Berberine and berberrubine could assist the antifungal action of the standard antifungal drug, terbinafine. The complementary activity of berebrine with terbinafine was much stronger than that of berberrubine. In each case, three independent experiments were conducted and a mean value was obtained. Each experiment was carried out in triplicates. Results are shown as mean ± standard deviation from three independent experiments.

Zone of inhibition study

Previous studies have reported that berberine in combination with fluconazole or miconazole showed a synergistic antifungal activity against C. albicans with larger inhibition zones in the agar diffusion tests (29–31). In order to evaluate the sensitivity of C. albicans to the combination of terbinafine and berberine or berberrubine, agar diffusion assays were conducted to determine their inhibition zones on the agar plates. Terbinafine alone at 6 µg possessed certain fungistatic effect against C. albicans (inhibition zone, 14.67±3.06 mm), whereas berberine or berberrubine alone showed small or no level of growth inhibition of the fungi (Fig. 3). The combination of 100 µg berberine and 6 µg terbinafine showed an increased inhibition zones in size (inhibition zone, 25.67±2.52 mm) when compared with terbinafine alone. The result may suggest that a synergistic fungistatic activity against C. albicans may occur between the combined drugs even if the MIC and MFC of this combination were not improved. However, the combination of 100 µg berberrubine and 6 µg terbinafine did not enhance the fungal sensitivity, with no significant enlargement in size of inhibition zone. No growth inhibition of C. albicans could be found in 1% DMSO (vehicle control).

Figure 3. Inhibition zone (mm, in terms of mean diameter ± standard deviation).

In conclusion, the present study reports the complementary application of berberine and berberrubine with terbinafine against the opportunistic fungal pathogen, C. albicans. A recent study has demonstrated that berebrine can assist fluconazole to kill fluconazole-resistant C. albicans (32). The present results further indicate the possible sensitization of various pathogenic fungi to other standard drugs by berberine and berberrubine, with the purpose of obtaining an increment in the antifungal potency.

Acknowledgements

The present study was supported by the Innovation Technology Commission to ABCT and HKPU [with the grant codes FRG/14-15/021, 30-14-121 and 38-40-116 (Dr C.H.C.)], as well as grant no. 03-16-176 (Professor Z.X.B.). Professor R.G. received a grant from AIRC (contract no. IG 13575).

References