Synergistic Antifungal Activity of Magnoliae Cortex and Syzyii Flos against Candida albicans

Plants and their extracts library were prepared in the previous study (Ham and Kim, 2018). Candidaalbicans KCTC 7965 was purchased from Korean Collection for Type Cultures in Korea Research Institute of Bioscience and Biotechnology (Jeongeup, Korea) and stored at −80℃ in a mixture with 15% glycerol. YPD medium was made with 1% yeast extract, 2% peptone, and 2% dextrose. YPD plate was made with YPD medium and 1.5% agar. SDA plate was made with 3% Sabouraud Dextrose Broth (catalog number: BD 238230, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and 1.5% agar. RPMI-1640 medium was made with 1.04% RPMI-1640 medium (catalog number: R6504, Merck KGaA, Darmstadt, Germany) and 0.165 M 3-(N-morpholino)propanesulfonic acid as a buffer. The analytical standards, eugenol (catalog number: E51791) and magnolol (catalog number: D3971), were purchased from Sigma-Aldrich Co., Inc. (St. Louis, MO, United States) and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), respectively.

Each extract of 50 mg was dissolved in 1 mL of methanol and incubated at 50℃ for 5 min. After vortexing, the dissolved extract sample was centrifuged at 13,000 rpm for 10 min. The transparent supernatant was used for further experiments. The supernatant with 20 μL was loaded 5 times onto paper disc (Catalog number: 49005040, Advantec MFS, Inc., Dublin, CA, USA) and dried. As a control, 20 μL of methanol, the solvent in which the extract was dissolved, was loaded 5 times and dried.

C. albicans stored at -80℃ were inoculated on YPD plates and incubated at 26℃ for 2 days. A single colony was incubated in 5 mL of YPD medium and incubated at 26℃ and 250 rpm for 1 day. Absorbance of the pre-cultured cells was measured and diluted with YPD medium to make the concentration of the initial cells (Abs₆₀₀ = 1). After the diluted cells with 0.1 mL was spread on YPD plate, a paper disc with an extract was placed in the center of the plates and incubated at 26℃ for 1 days. After incubation, it was judged that an extract having a growth inhibition zone more than 1 mm around the paper had an antifungal activity. The experiments were repeated 3 times per extract and the average value showing the diameter of the growth-inhibiting zone with the standard deviation was calculated.

After the transparent sample solution was prepared by the method described in the previous paragraph, it was diluted according to the test concentration and 50 μL of the diluted sample was mixed in 5 mL of YPD medium. The control YPD medium was treated with 50 μL of methanol. The pre-cultured C. albicans was inoculated to have an Abs₆₀₀ value of 0.05 and then cultured at 26℃ and 250 rpm for 18 hours. The cell concentration was measured with a spectrophotometer (Optizen 2120UV, Mecasys Co., Ltd., Daejeon, Korea) at an absorbance of 600 nm. The relative growth was calculated by comparing the cell growth to the control.

In order to evaluate the synergistic effect of the selected extracts, sample concentrations that inhibited the growth of C. albicans by 20% were determined. The determined concentrations for extracts are presented in the result section. Extracts at the indicated concentrations were prepared by mixing 50 μL of one extract and 50 μL of the other extract in 5 mL of YPD medium. The control group was treated with 100 μL of 99.5% methanol, the solvent in which the extract was dissolved. Three different control cultures were used: culture with 100 μL of methanol; culture with 50 μL of the first extract; and culture with 50 μL of the other extract. The pre-cultured C. albicans was inoculated to have an Abs₆₀₀ value of 0.05 and then cultured at 26℃ and 250 rpm for 18 hours. The cell concentration of the culture medium was measured with a spectrophotometer (Optizen 2120UV). Relative growth was calculated and compared to the cell growth of the control.

To measure the antifungal activity on C. albicans, CLSI M27-A2 broth microdilution methods (NCCLS, 2002) using checkerboard combinations was employed. C. albicans stored at -80℃ were streaked on SDA plates and incubated at 35℃ for 1 day. A single colony was suspended in 0.145 mol/L saline and vortexed for 15 seconds. The suspended colony was measured by absorbance at a wavelength of 530 nm with a spectrophotometer (Optizen 2120UV) and adjusted with saline to 0.5 McFarland turbidity in order to prepare a C. albicans suspension. The inoculated concentration of C. albicans in the culture for measuring the antifungal activity was 1 × 10³ ~ 5 × 10³ CFU/mL. Both tested extracts were dissolved separately in RPMI 1640 medium. Two dissolved extracts were mixed in 50 μL each. As a control, 50 μL of each sample and 50 μL of RPMI 1640 medium were used. After inoculation, cells were incubated at 35℃ for 2 days. The sunk cells were suspended by pipetting and cell turbidity of wells was measured at 530 nm with a microplate reader (Synergy LX, BioTek Instruments, Inc., Winooski, VT, USA).

Growth inhibition (GI %) was calculated by the equation stated below. The cell density was measured by absorbance at a wavelength of 530 nm with a spectrophotometer.

GI (%) = (Cell density of control – Cell density of sample) / Cell density of control × 100

Compared with the antifungal activity as GI % of each treatment, it was judged that there was a synergistic antifungal activity when the antifungal activity of the mixture were 2 times or more than individual two treatments (Chusri et al., 2014).

High-performance liquid chromatography used for the analysis of eugenol and magnolol was composed with vacuum degasser & mixer (catalog number: SDV40A), gradient column oven (catalog number: CTS30), auto-sampler (catalog number: YL9150 Alias), solvent delivery pump (catalog number: SP930D), and UV / Vis detector with dual wavelength (catalog number: YL9120) from YL Instruments Co., Ltd. (Anyang, Korea).

YMC-Triart C18 (catalog number: TA12S05-2546WT, YMC Korea Co., Ltd, Seongnam, Korea) was used for the analytic column. All samples were filtered with SEPARA syringeless filter vial (catalog number: MV32ANPPV002FC01, GVS North America, Inc., Sanford, ME, USA) prior to analysis. Results were analyzed with Autochro-3000 software (version 2.0.0) program from YL Instruments Co., Ltd.

The operating conditions for the analysis of eugenol were the same as those of a previous study (Seal, 2016). The mobile phase was acetonitrile (solvent A) and 1% (v/v) acetic acid (solvent B). The solvent ratio (solvent A : solvent B) was 10:90 at 0 minute, 40:60 at 28 minute, 60:40 at 39 minute, 90:10 at 50 minute, 10:90 at 55 minute, and 10:90 at 65 minute. The wavelength for eugenol analysis was 272 nm, speed of the mobile phase was 0.7 mL/min, column temperature was 28℃, and analysis time was 65 minutes.

The conditions for the magnolol analysis were the same as those of a previous study (Kim and Kim, 2004). The mobile phase for magnolol was acetonitrile and 2% (v/v) acetic acid, at the ration of 50:50. The analysis conditions for magnolol were 289 nm of wavelength for detection, 1 mL/min for speed of the mobile phase, 26℃ for column temperature, and 60 minutes for analysis time.

Antifungal activity against C. albicans was measured by disk diffusion test using a methanol extract library of 280 edible plants listed in the Korean Pharmacopoeia or Herbal/Korean Traditional Products from National Institute of Food and Drug Safety Evaluation. The growth inhibition activity of 17 selected plant extracts is shown in Table 1. The size of the clear zone was 34.8 mm by Cinnamomi Cortex, 19.8 mm by Cinnamomi Ramulus, 19.8 mm by Magnoliae Cortex, and 16.8 mm by Syzygii Flos. The antifungal effect according to the concentration was observed in four plant extracts, with a diameter of ≥ 15 mm in the inhibition area (Fig. 1). Three plant extracts, Cinnamomi Cortex (Fig. 1A), Magnoliae Cortex (Fig. 1C), and Syzygii Flos (Fig. 1D), showed ≥ 78% growth inhibitory activity against C. albicans at a concentration of 0.5 g/L. Cinnamomi Cortex and Cinnamomi Ramulus is a bark and young branch, respectively, of Cinnamomum cassia Presl. However, the growth inhibitory effect of Cinnamomi Ramulus extract on C. albicans was much less than that of Cinnamomi Cortex extract. Cinnamomi Cortex extract showed no cell growth at 0.5 g/L. trans-Cinnamaldehyde as an antifungal compound from Cinnamomi Cortex inhibited chitin synthases, the cell wall synthesizing enzymes of Saccharomyces cerevisiae, in vitro (Bang et al., 2000a). Cinnamaldehyde was reported to be an antifungal chemical in Cinnamomi Ramulus (Wan et al., 2017). Magnolol and honokiol from Magnoliae Cortex inhibited the growth of seven pathogenic fungi (Chen et al., 2019) and magnolol had a synergistic antifungal activity with azoles against C. albicans (Sun et al., 2015). Eugenol from Syzygii Flos synergistically inhibited the growth of C. albicans with nystatin (Silva et al., 2017). A chemical-genetic profile analysis suggested that eugenol interfered with two permeases for the transport of aromatic and branched chain amino acids in the cytoplasmic membrane of S. cerevisiae (Darvishi et al., 2013).

Fig. 1. Antifungal activity against C. albicans according to the concentration of the selected four plant extracts, (A) Cinnamomi Cortex extract, (B) Cinnamomi Ramulus extract, (C) Magnoliae Cortex extract, and (D) Syzygii Flos extract. The values are average of three independent experiments.

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Synergistic growth inhibitory effect on C. albicans was tested using a mixture of two extracts among the four tested extracts in Fig. 1 (Fig. 2). The test concentration of the four selected extracts in Fig. 2 were determined to have GI 20% activity from Fig. 1, with 0.06 g/L for Cinnamomi Cortex, 0.3 g/L for Cinnamomi Ramulus, 0.06 g/L for Magnoliae Cortex, and 0.12 g/L for Syzygii Flos. Inhibitory activity against the growth of C. albicans was measured for six combinations of four selected plant extracts. Although the five combinations showed just the sum of their respective inhibitory effects, the combination of Magnoliae Cortex extract and Syzygii Flos extract showed ≥ GI 60%. The inhibitory activity above the sum of their respective inhibitory effects suggest a synergistic effect by mixing the two tested extracts.

Fig. 2. Antifungal activity of combination of two antifungal plant extracts against C. albicans. C.C.: Cinnamomi Cortex extract (0.06 g/L); C.R.: Cinnamomi Ramulus extract (0.3 g/L); M.C.: Magnoliae Cortex extract (0.06 g/L); S.F.: Syzygii Flos extract (0.12 g/L). The values are average of three independent experiments.

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Magnolol is known as an antifungal compound in Magnoliae Cortex against C. albicans (Chen et al., 2019; Sun et al., 2015) and eugenol is an antifungal compound in Syzygii Flos extract (Darvishi et al., 2013). Using a high-performance liquid chromatography analysis, the Magnoliae Cortex extract contained 6.44% magnolol, with a retention time of about 41.1 min, while Syzygii Flos extract contained 56.68% eugenol, with a retention time of about 49.8 min.

Both magnolol and eugenol, the assay standard compounds, inhibited the growth of C. albicans (Fig. 3). The growth inhibitory effect was shown in proportion to the concentration of magnolol and C. albicans growth was completely suppressed by magnolol in concentrations ≥ 0.09 mM (24 mg/L) (Fig. 3A). This result is consistent with the inhibitory concentration of magnolol on the biofilm formation of C. albicans in a previous study (Zhou et al., 2017).

Fig. 3. Antifungal activity of magnolol and eugenol against C. albicans. The values are average of six independent experiments.

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Eugenol did not inhibit the growth of C. albicans until 0.76 mM (0.125 g/L), but showed complete inhibition at concentrations above 3.05 mM (0.5 g/L) (Fig. 3B). This result is twice the value of 0.25 g/L in planktonic growth of C. albicans (Tampieri et al., 2005) and is consistent with the minimal inhibitory concentration of 0.5 g/L for sessile C. albicans in a previous study (He et al., 2007). The main component of Syzygii Flos is eugenol (Pinto et al., 2009) and the antifungal activity of eugenol on fungi other than C. albicans have been reported in previous studies (Chami et al., 2004; Cheng et al., 2008; de Oliveira Pereira et al., 2013). The antifungal activity of magnolol against C. albicans was about 34 times stronger than that of eugenol.

To test the synergistic growth inhibition of magnolol and eugenol, a checker board experiment was performed at various concentrations of magnolol (0–24 mg/L) and eugenol (0–0.3 g/L). The synergistic concentration range was determined to have more than twice the inhibitory activity of all the results of the individual compounds. The synergistic growth inhibitory concentration range was 8–24 mg/L for magnolol and 0.25–0.3 g/L for eugenol. The concentration ratio of magnolol and eugenol for the synergistic inhibitory activity was 1:25–1:61.

Considering the content of magnolol in Magnoliae Cortex and eugenol in Syzygii Flos extract, the antifungal activity on C. albicans by combination of magnolol and eugenol was estimated to contribute 28–48% of the antifungal activity by combination of Magnoliae Cortex extract and Syzygii Flos extract. This estimated value suggests that there are additional compounds with antifungal activity in addition to magnolol and eugenol in the Magnoliae Cortex and Syzygii Flos extract, respectively. In a previous study, it was reported that honokiol and obovatol, in addition to magnolol, were chemical components present in Magnoliae Cortex (Shen et al., 2009). Honokiol had an antifungal activity on C. albicans (Bang et al., 2000b). Obovatols inhibited the cell wall synthesis of S. cerevisiae (Hwang et al., 2002). In addition to eugenol, Syzygii Flos had phenolic compounds (Shan et al., 2005) and these phenolic compounds, including caffeic acid (Ma and Ma, 2015), ferulic acid (Shi et al., 2016), elagic acid (Li et al., 2015), and salicylic acids (Amborabé et al., 2002), showed antifungal activities.

With the recent increase in reports of azole resistance of C. albicans (Whaley et al., 2017), it has become very important to discover and develop new antifungal compounds, as well as reduce the use of antifungal agents by mixing antifungal agents. Moreover, synergistic antifungal activities had been reported in previous studies, such as oil from Ocimum basilicum var. Maria Bonita with fluconazole (Cardoso et al., 2016), ethanolic extract of the leaves of Ocimium gratisimum L. with ketoconazole and nystatin (Nweze and Eze, 2009), Melaleuca alternifolia, Origanum vulgare, and Pelargonium graveolens essential oils with amphotericin B (Rosato et al., 2008), and thionin-like peptide from Capsicum annuum fruits with fluconazole (Taveira et al., 2016). In this study, the synergistic action between the extracts of Magnoliae Cortex and extract of Syzygii Flos was suggested. In this study, we proposed an antifungal treatment that utilizes increased antifungal activity while minimizing the amount of use by combining only plant extracts without using antifungal compounds.