Analysis of chemical composition and in vitro antidermatophyte activity of ethanol extracts of Dryopteris fragrans (L.) Schott

Author’s Accepted Manuscript Analysis of chemical composition and in vitro antidermatophyte activity of ethanol extracts of Dryopteris fragrans (L.) Schott Xueping Liu, Jiayuan Liu, Tao Jiang, Lili Zhang, Yixi Huang, Jiangfan Wan, Guoqiang Song, Haoqi Lin, Zhibin Shen, Chunping Tang www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(18)31316-3 https://doi.org/10.1016/j.jep.2018.07.030 JEP11457

To appear in: Journal of Ethnopharmacology Received date: 14 April 2018 Revised date: 23 July 2018 Accepted date: 27 July 2018 Cite this article as: Xueping Liu, Jiayuan Liu, Tao Jiang, Lili Zhang, Yixi Huang, Jiangfan Wan, Guoqiang Song, Haoqi Lin, Zhibin Shen and Chunping Tang, Analysis of chemical composition and in vitro antidermatophyte activity of ethanol extracts of Dryopteris fragrans (L.) Schott, Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2018.07.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Analysis of chemical composition and in vitro antidermatophyte activity of ethanol extracts of Dryopteris fragrans (L.) Schott

a

a

c

a

a

a

a

a

Xueping Liu , Jiayuan Liu , Tao Jiang , Lili Zhang , Yixi Huang , Jiangfan Wan , Guoqiang Song , Haoqi Lin , Zhibin a*

a,b*

Shen , Chunping Tang

a

School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, Guangdong,

China. b

Guangdong Provincial Engineering Center of Topical Precise Drug Delivery System, Guangdong Pharmaceutical

University, Guangzhou 510006, Guangdong, China. c

Laboratory Animal Center, Guangdong Pharmaceutical University, Guangzhou 510006, Guangdong, China.

[email protected]

[email protected]

*

Corresponding authors.

Abstract Ethnopharmacological relevance Dryopteris fragrans (L.) Schott is a deciduous perennial herb, which has been used traditionally for treatment of ringworm infections and others skin diseases in the north of China. Aim of the study To characterize the chemical composition, evaluate the antifungal activity and explore possible mechanisms

about action of ethanol extracts of D. fragrans. Materials and methods The chemical components in the ethanol extracts of D. fragrans were detected by high-performance liquid chromatography coupled with electrospray ionization and quadruple time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS/MS). The minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of the ethanol extracts of D. fragrans were determined by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 method against 62 isolates of dermatophytes. The kinetics of fungal kill, synergy testing by checkerboard dilution and quantitation of sterol by ultra-performance liquid chromatography (UPLC) on Trichophyton rubrum and Trichophyton mentagrophytes were also investigated. Results Fourteen derivatives of phloroglucinol were identified in the ethanol extracts of D. fragrans. The MIC of the ethanol extracts of D. fragrans ranged from 0.059 to 3.780 mg/mL while MFC ranged from 0.118 to 3.780 mg/mL. The ethanol extracts of D. fragrans exerted fungicidal activity after 12 h of incubation against Trichophyton rubrum while it required 36 h of incubation against Trichophyton mentagrophytes at concentrations of 8×MIC. In synergy testing, the interaction between miconazole (MCZ) and terbinafine (TBF) with the ethanol extracts of D. fragrans proved to be indifferent by testing fractional inhibitory concentration (FIC) values. Sterol in samples of fungal cells treated with the ethanol extracts of D. fragrans was significantly reduced. Conclusions The ethanol extracts of D. fragrans had antifungal and fungicidal activity against dermatophytes and was likely a strain-dependent fungicidal agent. Interaction between drugs was indifferent on tested isolates. The inhibition of ergosterol biosynthesis was one of the antifungal mechanisms of the ethanol extracts of D. fragrans. These results showed that the ethanol extracts of D. fragrans could be explored for promising antifungal drugs. Dozens of

phloroglucinol derivatives may contribute to high antifungal activity of the ethanol extracts of D. fragrans.

Keywords: Dryopteris fragrans (L.) Schott; HPLC-ESI-QTOF-MS/MS; phloroglucinol derivative; antifungal activity; antifungal mechanism

1 Introduction Dermatophytosis affects about 25% of the world population with increasing incidence, which reflects a significant public health problem still unsolved (Peres et al., 2010). Among the fungal species isolated from skin infections, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis and Microsporum gypseum are the most common dermatophytes in the medical cases of tinea in unguium, pedis, cruris and corporis (Farokhipor et al., 2018). The availability of marketed antidermatophytic drugs, including the azole and allylamine, becomes subject to numerous clinically significant restrictions due to the development of side effects and resistance of these drugs (Ghannoum, 2016; Peres et al., 2010). Moreover, the aged and transplant recipients as well as chemotherapy patients are at a high-risk for having fungal infections (Baddley et al., 2011). Therefore, new strategies of effective therapies, preventions of dermatophytosis and novel clinically useful antifungal agents are in urgent demanded (Farokhipor et al., 2018). Dryoptens fragrans (L.) Schott, a deciduous perennial herb, has been used to treat tinea manuum, tinea pedis, psoriasis, dermatitis, skin rashes and acne by the local people lived in Heilongjiang province in the north of China for many decades (Wenxin and Xin, 2014). Besides, this plant has been applied in the treatment of senile skin pruritus and various fungal skin diseases in clinical trials (Xianjun, 2013). Previous studies had found that the most effective fraction of D. fragrans was the 95%-ethanol extract mainly contained derivatives of

phloroglucinol (Huaqian et al., 2012). Aspidin BB, a typical phloroglucinol derivative from D. fragrans, showed antifungal (Yang et al., 2017) and antibacterial activities (Gao et al., 2016). Previous research had evaluated antifungal activities against several dermatophytes with the ethanol extracts of D. fragrans (Yixi et al., 2016). However, its chemical composition and more activities of the ethanol extracts of D. fragrans still uncovered. In the present study, the chemical composition of the ethanol extracts of D. fragrans was analyzed and the antifungal spectrum and possible mechanisms of action of the ethanol extracts of D. fragrans were investigated. 2 Materials and methods 2.1 Plant material and collection The sample of D. fragrans was collected in Aug. 2016 in Wu-Da-Lian-Chi, Heilongjiang province, China, and identified by Prof. De-Lian Zhang (D.-L.Z., Harbin University of Commerce, China). The voucher specimen (registration number: XLMJ-201608) of this plant was deposited in the School of Traditional Chinese Medicine, Guangdong Pharmaceutical University. 2.2 Preparation of the ethanol extracts of D. fragrans 2.2.1 Preparation of sample for antifungal evaluation The air-dried sample (20 g) was powdered and extracted twice by 95% (v/v) ethanol (200 mL) at room temperature (24 h for each time). In addition, extraction solvent filtered and concentrated by evaporation under reduced pressure to 1.890 g. Phloroglucinol content in the ethanol extracts of D. fragrans was determined by ultraviolet spectrophotometry and confirmed at 58%. Then sample was dissolved in 95% (v/v) ethanol to 387 mg/mL (5 mL) and stored at 4°C. 2.2.2 Preparation of sample for chemical analysis The ethanol extracts of D. fragrans was eluted by the 95% ethanol eluent of macroporous resin to 10 mL approximately, evaporated to dryness by an evaporating dish on a hot water bath, dissolved by acetonitrile.

Supersonic instrument and 0.22 µm membrane were used for assisted dissolution and purification of the ethanol extracts of D. fragrans. 2.3 Antifungal agents Miconazole (MCZ, Sigma-Aldrich Co.) and terbinafine (TBF, Sigma-Aldrich Co.) were dissolved in dimethyl sulfoxide with the concentration of 6.4 mg/mL for positive controls. Fluconazole (FCZ, Sigma-Aldrich Co.) was prepared in sterile water with the concentration of 6.4 mg/mL and used as a quality control drug. 2.4 Fungal isolates A total of 62 dermatophyte isolates, including 23 standard isolates and 39 clinical isolates, were tested by the Institute of Dermatology, Chinese Academy of Medical Sciences, Nanjing, China. All isolates were managed by clinical examination and specimen collection. The isolates were identified by direct microscopic examination, observed macroscopic features after culturing on the Sabouraud dextrose agar medium (OXOID corporation, the United Kingdom) and rice grains medium (3 g rice, 10 mL water), and detected by extracellular enzymatic activities(Gendy et al., 2016). Prior to the antifungal susceptibility tests, each isolate was sub-cultured on Sabouraud Dextrose Agar (SDA) or Yeast Extract Peptone Dextrose Agar (10 g of yeast extract, 20 g of peptone, 20 g of glucose, and 20 g of agar per liter AOBOX corporation, China) (Candida parapsilosis only). T. rubrum CMCC (F) T1d and T. mentagrophytes CMCC (F) T5c were used in time-killing kinetics, synergy testing and sterol assay. 2.5 Chemical components analysis 2.5.1 HPLC Conditions Chromatographic separation of the ethanol extracts of D. fragrans was operated by XBridgeTM BEH C18 column (4.6×100 mm, 2.5 μm) equipped with acetonitrile (A)-0.5% trifluoroacetic acid (B) as mobile phase with gradient elution (0-2 min: 10%-20% A, 2-10 min: 20%-40% A, 10-16 min: 40%-40.5% A, 16-20 min: 40.5%-40.9% A,

20-22 min: 40.9%-41% A, 22-24 min: 41%-41.1% A, 24-28 min: 41.1%-45% A, 28-33 min: 45%-60% A, 33-41 min: 60%-75% A, 41-53 min: 75%-77% A, 53-56 min: 77%-80% A, 56-58 min: 80%-90% A, 58-60 min: 90%-100% A) at 35℃ in the velocity of 0.8 mL/min.

2.5.2 MS conditions The mass spectrometer was operated in positive ion mode with ESI source. The following parameters settings were used: capillary voltage of 1.4 kV and 1.3 kV, cone voltage of 40 V and 23 V, ion source temperature at 120℃, gas temperature of desolvent at 350℃, cone-hole gas flow rate of 50.0 L/h, desolvent gas flow rate of 6.0 L/h, collision energy of 35 eV, ion energy of 1 V, mass scan range of 100-2000 m/z. The spectrometer was gathered every 0.2 s.

2.6 Susceptibility tests As M38-A2 mentioned, MIC, the lowest concentration of an antimicrobial agent that causes a specified reduction in visible growth of a microorganism in broth dilution susceptibility test. The MFC was determined as the lowest concentration of the extract that did not allow the growth of any fungal colony on the solid medium after the incubation period. MIC and MFC were determined by the broth microdilution method (M38-A2), 3

following the instructions established by CLSI. Fungal suspensions were diluted to twofold final inoculums of 1×10 3

~ 3×10 CFU/mL by RPMI-1640 medium with L-glutamine and without sodium bicarbonate (Gibco corporation, America). These dilutions were used in the test at a pH of 7.0 ± 0.1 with 3- (N- morpholino) propanesulfonic buffer (Sigma-Aldrich Corporation, China) along with 1 mol/L NaOH. The dilutions of the ethanol extracts of D. fragrans were prepared in RPMI-1640 medium ranging from 0.007 to 3.780 mg/mL. The plates were cultured at 35°C for 96 h. The MIC of the ethanol extracts of D. fragrans was defined as the lowest concentration that prevented any discernable growth (100% inhibition) compared with the growth control (drug-free) well by visual inspection. Using cumulative MIC percentage curves allowed visual analysis of MIC distribution. Cumulative percentage curves

of the ethanol extracts of D. fragrans for T. rubrum, T. mentagrophytes and 62 isolates of dermatophytes were calculated. After MIC were read, 20 µL of sample was removed from all wells of no visible growth to SDA plates and incubated at 28°C for 7 d. The MFC were defined as the lowest concentration of drugs inhibiting 100% growth as compared to the growth control. Each isolate was tested in triplicate. 2.7 Time-killing kinetics Time-killing kinetic of the ethanol extracts of D. fragrans was evaluated by the method based on the 4

previous studies with minor modifications (Simonetti et al., 2014). The inoculums of 1×10 CFU/mL was incubated with the ethanol extracts of D. fragrans at 1×MIC, 2×MIC, 4×MIC and 8×MIC along with positive controls (with TBF and MCZ). Growth controls contained RPMI-1640 medium with the relevant isolates without drugs. The cultures were incubated at 28°C with shaking. Further samples were removed from the cultures at 0, 12, 24, 36, 48, 60 and 72 h of incubation for viability counting. A 100 µL sample was taken out from each culture flask and diluted tenfold with sterile water, and a 100 µL aliquot was plated on a SDA plate. Then colonies were counted after incubation at 28°C for 5 to 7 d. When colony counts were suspicious that it would be < 1000 CFU/mL, 30 µL samples were taken out from the culture flask and plated without dilution. The limit of quantification is 100 CFU/mL based on these methods. Colony counts were evaluated by a reduction of ≤ 99.9% of CFU/mL with respect to starting inoculates. Every test was repeated three times. 2.8 Synergy testing Interactions of the ethanol extracts of D. fragrans with TBF and MCZ were assessed by a checker-board method. Briefly, testing was performed in the same medium used for susceptibility tests. After determination of the MIC of the ethanol extracts of D. fragrans, MCZ and TBF against dermatophytes, 10 dilutions were prepared to obtain the fourfold final concentration. In the 96-well microtiter plates, 50 µL aliquots of each the ethanol extracts

of D. fragrans dilution were combined with other 50 µL of either MCZ or TBF dilutions. Then, 100 µL of fungal 3

3

suspensions (2×10 ~ 6×10 CFU/mL) was transferred to each well of sterile 96-well U-bottomed plates and incubated at 35°C for 96 h. The MIC endpoint was 100% of growth inhibition with respect to the control well. The interactions between the ethanol extracts of D. fragrans and antifungals were quantitatively evaluated by determining the fractional inhibitory concentration (FIC). The calculation formula of FIC was: FIC= [MICA in combination/MICA]/ [MICB in combination/MICB]. The interactions were classified as synergism if the FIC was ≤ 0.5, indifference if the FIC was > 0.5 and ≤ 4.0, and antagonism if the FIC was > 4.0 (Simonetti et al., 2014). Every test was repeated three times. 2.9 Sterol assay The total intracellular sterols of dermatophytes were extracted based on early reports with slight 3

3

modifications (Zhi-Jian et al., 2009). Briefly, fungal suspensions of the 2 isolates of 1×10 ~ 3×10 CFU/mL with 100 mL of RPMI-1640 medium were prepared. The cultures were incubated for 3 days with shaking (150 rpm) at 35°C and mycelia pellets ware obtained. Every 5 mL of inoculate suspensions containing 0.5 × MIC, 1 × MIC and 2 × MIC of the ethanol extracts of D. fragrans along with a negative control (without drug) and a positive control (with TBF) were prepared. Concentration of each sample was tested in triplicate and the cultures were incubated at 35°C for 24 h. The stationary-phase cells were collected by centrifugation at 1000 g for 5 min and the supernatant was discarded. Ten milliliters of water were added to each centrifuge tube containing. The mycelia pellets were washed by sterile distilled twice. Then the net weight of the cell pellet was determined. Three milliliters of fresh 25% potassium hydroxide were added to each tube and the tubes were placed in 85°C water bath for 1 h. Then the pellets were allowed to cool to room temperature. Sterols were then extracted by 1 mL sterile distilled water and 3 mL N-heptane with vigorous vortex mixing for 3 min. The N-heptane layer was evaporated to dryness on water

bath. Appropriate methanol was added to the dried sample and then ultrasonic treatment (25°C, 40 KHz) for 5 min. Next, the samples were dissolved with methanol to 5 mL and filtered (0.22 μm). The sterols of dermatophytes were determined by UPLC- photo-diode array (detection wavelength at 281 nm) as earlier report with slight modifications (Mingyue et al., 2012). 2.10 Statistical analysis Data were analyzed with GraphPad Prism Version 5.01 and IBM SPSS Statistics 20.0 software and expressed as mean ± standard. One-way analysis of variance (ANOVA) was carried out on data of susceptibility test, synergy test and sterol assay. Data of three independent experiments, synergy test and sterol assay were furthermore analyzed *

by Bonferroni’s multiple comparisons test. Levels of statistical significance at P＜0.05,

**

P＜0.01,

***

P＜0.001

were used. A value of P＜0.05 was considered statistically significant. 3 Results and discussion The qualitative analysis of chemical constituents in elution solution of the ethanol extracts of D. fragrans was operated by HPLC-ESI-MS. the spectrum of the total ion current of (+) ESI/MS was shown in Fig. 1. The chemical constituents of the ethanol extracts of D. fragrans was tentatively identified and characterized by comparison with references (Ito et al., 1997; Li et al., 2012; Peng et al., 2016; Yang et al., 2017). Fourteen compounds belonging to phloroglucinol were identified and shown in Tab. 1. Based on the M38-A2 method, the results of the MIC and MFC of the ethanol extracts of D. fragrans showed a boarder antifungal effect against the different species, such as Trichophyton spp., Microsporum spp. and Epidermophyton spp. as listed in Tab. 2. The MIC of the ethanol extracts of D. fragrans ranged from 0.059 to 3.780 mg/mL while MFC ranged from 0.118 to 3.780 mg/mL. By comparing the antifungal activity among specimens of each genus, the activity of the ethanol extracts of D. fragrans was significant different against T. rubrum and T. mentagrophytes, while the values of MIC and MFC against M. canis and M. gypseum did not show significantly

different. The cumulative percentage curves of the ethanol extracts of D. fragrans for dermatophytes were shown in Fig. 2. The MIC of the ethanol extracts of D. fragrans had broader distribution in two major causes, T. rubrum and T. mentagrophytes, than other specimens. The MIC of total dermatophytes showed a same tendency. Tab. 3 clear showed MIC and MFC values of each isolate. It had been known for many years that the ethanol extracts of D. fragrans aqueous-extracts inhibited the growth of fungi in vitro (Hongzhi et al., 2005). Thus, primarily, we had focused on exploring the optimal extraction method of D. fragrans to achieve a better antidermatophyte effect in an agar dilution assay (Huaqian et al., 2012). The results showed that its ethanol extract had the better antifungal effect. However, these non-standardized either qualitative or quantitative tests prevented the comparison of results. Therefore, we had compared the agar dilution assay with the standardized method (M38-A2) in measuring the susceptibility of filamentous fungi (Yixi et al., 2016). In this study, we used the M38-A2 method for determination of the MIC and MFC against 62 isolates of dermatophytes, confirming the results obtained in our previous studies (Huaqian et al., 2012). Our data demonstrated that the ethanol extracts of D. fragrans exerted both fungistatic and fungicidal activities against 62 isolates of dermatophytes. The MIC of the ethanol extracts of D. fragrans encompassed a wide range, which suggested that MIC was strain-dependent. The variation of susceptibility seen in dermatophytes also indicated the importance of detecting the causative fungi to select effective antifungal agents in each case and to prevent development of resistance (Tamura et al., 2014).

MIC and MFC not only could not provide the duration of fungistatic action and fungicidal speed, but also could not predict whether the increased concentration could enhance the fungicidal speed (Leite et al., 2014). In order to clarify the dynamic relationship between concentration and activity over time, we delved into whether the ethanol extracts of D. fragrans had fungistatic or fungicidal action by time-killing kinetics assay. The representative killing experiments were reported in Fig. 3. The ethanol extracts of D. fragrans at 8×MIC exerted a

fungicidal activity upon 12 h of incubation against T. rubrum CMCC (F) T1d, while it required 36 h of incubation against T. mentagrophytes CMCC (F) T5c. The ethanol extracts of D. fragrans at 4×MIC exerted a fungicidal activity at 36 h of incubation against either T. rubrum CMCC (F) T1d or T. mentagrophytes CMCC (F) T5c. With regard to T. rubrum CMCC (F) T1d and T. mentagrophytes CMCC (F) T5c, the ethanol extracts of D. fragrans at 1× MIC had the lowest effect on the delay of its growth. These results showed that the ethanol extracts of D. fragrans exerted a concentration-dependent fungicidal effect. FIC index usually be used to evaluate synergy action among antifungal or antibacterial drugs. In this study, synergy test of antifungal drugs was performed against T. rubrum CMCC (F) T1d and T. mentagrophytes CMCC (F) T5c and the results were presented in Tab. 4. Fig. 4 proved the dynamic of interaction between the ethanol extracts of D. fragrans with MCZ and TBF. The result showed that interaction between drugs was indifferent on T. rubrum CMCC (F) T1d and T. mentagrophytes CMCC (F) T5c. However, synergy was not achieved. Simultaneously, no antagonistic interaction was observed in combination of the ethanol extracts of D. fragrans with MCZ and TBF against T. rubrum CMCC(F)T1d and T. mentagrophytes CMCC(F)T5c (Simonetti et al., 2014). However, the statistical difference between MIC values of drug alone and combination might indicated cooperative effect (Geary, 2013; Julie and Mickael, 2015). The MIC of the ethanol extracts of D. fragrans alone against T. rubrum CMCC (F) T1d was 0.473 ± 0.000 mg/mL, the MIC combined with TBF was 0.236 ± 0.000 mg/mL (P = 0 < 0.05). The statistical difference between MIC values of DF alone and the MIC combined with TBF against T. rubrum CMCC (F) T1d might indicated cooperative effect. The MIC of the ethanol extracts of D. fragrans combined with MCZ did not show statistical difference when against T. rubrum CMCC (F) T1d (0.315 ± 0.137 mg/mL, P = 0.079 > 0.05). The combined MIC of the ethanol extracts of D. fragrans and TBF or MCZ was 0.315 ± 0.137 mg/mL (P = 0.079 > 0.05), both did not show statistical difference compared the MIC of the ethanol extracts of D. fragrans alone (0.236 ± 0.000 mg/mL) when against T. mentagrophytes CMCC (F) T5c.

Our study previously indicated that the structure of cell wall and cell membranes of T. rubrum had both significantly changed after treated with the ethanol extracts of D. fragrans (Jieying et al., 2013). Considering the possible interference of the ethanol extracts of D. fragrans on fungal membrane composition, the sterol quantitation was tested to investigate its action on ergosterol biosynthesis as shown in Fig. 5. The ergosterol content of samples treated for 24 h was evaluated by UPLC. Compared with the control sample, the ergosterol quantitation of all samples treated with the ethanol extracts of D. fragrans (0.5 to 2 times of the MIC) against T. mentagrophytes were significantly reduced (P＜0.01 or P＜0.001), while the samples treated with the ethanol extracts of D. fragrans at 1×MIC and 2×MIC against T. rubrum CMCC (F) T1d were also reduced (P＜ 0.01). Besides, TBF was a more effective inhibitor against T. rubrum CMCC (F) T1d and T. mentagrophytes CMCC (F) T5c (P＜0.001). Ergosterol, an important component of the fungal cell membrane, was responsible for the maintenance of cell membrane structure and function. Our results showed that ergosterol in samples of fungal cells treated with the ethanol extracts of D. fragrans were significantly decreased and suggested that the ethanol extracts of D. fragrans was likely to exert its efficacy by inhibiting ergosterol biosynthesis. However, due to the complex composition of the ethanol extracts of D. fragrans, the targets on cell membranes of the compounds isolated from the ethanol extracts of D. fragrans against dermatophyes should be further investigated to elucidate the underlying mechanism of antifungal action.

Conclusion Our study demonstrated that phloroglucinol derivatives were the major compounds in the ethanol extracts of D. fragrans. The ethanol extracts of D. fragrans exerted both fungistasis and fungicidal activities against dermatophytes in susceptibility tests and time-killing kinetics. Besides, sterol quantitation assay indicated that the

inhibition of ergosterol biosynthesis was one of the antifungal mechanisms of the ethanol extracts of D. fragrans. However, in order to develop the ethanol extracts of D. fragrans into an alternative antifungal agent in the future, a further research is still needed to elucidate the antifungal effect of the ethanol extracts of D. fragrans, specifically its bioactive constituents and even its efficacy in vivo. Moreover, potential antifungal activity of phloroglucinol derivatives in the ethanol extracts of D. fragrans should be surveyed.

Acknowledgements This work was supported by grants from Science and Technology Projects of Guangdong Provincial Department of Science and Technology of China (Grant No. 2015B020234009), the funding of the Innovative and Strong School Project of Guangdong pharmaceutical university, Guangdong province, China (Grant No. 2016KZDXM039), special funds for cultivating scientific and technological innovation of college students in Guangdong province (Grant No. pdjh2017a0260), and from National Scientific and Technological Project of Traditional Chinese Medicine Industry (Grant No. 201507004). I also thank the reviewers for their helpful comments.

Conflict of interest The authors declare no conflict of interest.

References Baddley, J.W., Winthrop, K.L., Patkar, N.M., Delzell, E., Beukelman, T., Xie, F., Chen, L., Curtis, J.R., 2011. Geographic distribution of endemic fungal infections among older persons, United States. Emerging infectious diseases 17(9), 1664-1669. Chen, W.H., Song, G.Q., Jia, X.Z., Tang, C.P., Feng, D.K., Shen, Z.B., 2017. Isolation and antifungal activities of a pair of isomers of phloroglucinol derivatives from Dryopteris fragrans. Chinese Traditional & Herbal Drugs 48(3), 433-436.

Farokhipor, S., Ghiasian, S.A., Nazeri, H., Kord, M., Didehdar, M., 2018. Characterizing the clinical isolates of dermatophytes in Hamadan city, Central west of Iran, using PCR-RLFP method. Journal de Mycologie Médicale. Feng, Q.I., Wang, E.L., 2007. Studies on active constituents in rhizome of Dryopteris crassirhizoma. Journal of Tianjin Medical University. Gao, C., Guo, N., Li, N., Peng, X., Wang, P., Wang, W., Luo, M., Fu, Y.J., 2016. Investigation of antibacterial activity of aspidin BB against Propionibacterium acnes. Archives of Dermatological Research 308(2), 79-86. Geary, N., 2013. Understanding synergy. Am J Physiol Endocrinol Metab 304(3), E237-E253. Gendy, S.G.E., Seddek, N.H., Mohammed, S.M., Gendy, S.G.E., Seddek, N.H., Mohammed, S.M., Gendy, S.G.E., Seddek, N.H., Mohammed, S.M., 2016. Activity of Some Natural Oils on Dermatophytes Isolated from Assuit University Hospitals. Ghannoum, M., 2016. Azole Resistance in Dermatophytes. Journal of the American Podiatric Medical Association 106(1), 79-86. Hisada, S., Inoue, O., Inagaki, I., 1973. Phloroglucinol derivatives of Dryopteris sieboldii. Phytochemistry 12(8), 2055-2055. Hongzhi, L., Zhibin, S., Ying, Z., Miao, J., Delian, Z., 2005. The effect of Dryopteris fragrans ointment on the ringworm of rat model Jorunal of Chinese Medicinal Materials 28(10), 941-942. Huaqian, F., Zhibin, S., Yanfen, C., Jieying, W., Chaoyan, Y., LiangWannian, Chunping, T., 2012. Study on Antifungal Susceptibility of Different Extract of Dryopteris fragrans. Journal of Chinese Medicinal Materials 35(12), 1981-1985. Ishiguro, K., Yamaki, M., Kashihara, M., Takagi, S., 1987. Saroaspidin A, B, and C: additional antibiotic compounds from Hypericum japonicum. Planta Medica 53(05), 415-417. Ito, H., Muranaka, T., Mori, K., Jin, Z.X., Yoshida, T., 1997. DRYOFRAGIN AND ASPIDIN PB, PISCICIDAL COMPONENTS FROM DRYOPTERIS FRAGRANS. Chem Pharm Bull 45, 1720-1722. Jieying, W., Qinyuan, Z., Yuyuan, H., Zhibin, S., 2013. Inhibitory effect of Dryopteris Fragrans L． against Trichophyton Rubrum and its mechanism. Anhui Medical and Pharmaceutical Journal 17(6), 919-920. Julie, F., Mickael, G., 2015. Analysis of drug combinations: current methodological landscape. Pharmacology Research & Perspectives 3(3). Lee, H.B., Kim JCLee, S.M., 2009. Antibacterial activity of two phloroglucinols, flavaspidic acids AB and PB, from Dryopteris crassirhizoma. Archives of Pharmacal Research 32(5), 655. Leite, M.C., Bezerra, A.P., de Sousa, J.P., Guerra, F.Q., Lima, E.O., 2014. Evaluation of Antifungal Activity and Mechanism of Action of Citral against Candida albicans. Evidence-Based Complementray and Alternative Medicine,2014,(2014-8-27) 2014(1), 378280. Li, X.J., Fu, Y.J., Luo, M., Wang, W., Zhang, L., Zhao, C.J., Zu, Y.G., 2012. Preparative separation of dryofragin and aspidin BB from Dryopteris fragrans extracts by macroporous resin column chromatography. Journal of Pharmaceutical & Biomedical Analysis 61(5), 199-206. Mingyue, Z., Zhijian, L., Gulnar·Dawuti, Silafu·Aibai, 2012. Determination of Lanosterol in Mycelium of Trichophyton rubrum by High Performance Liquid Chromatography. Lishizhen Medicine and Materia Medica Research 23(12), 2980-2981. Peng, B., Bai, R.F., Li, P., Han, X.Y., Wang, H., Zhu, C.C., Zeng, Z.P., Chai, X.Y., 2016. Two new glycosides from Dryopteris fragrans with anti-inflammatory activities. Journal of Asian Natural Products Research 18(1), 59-64. Peres, N.T.d.A., Maranhão, F.C.A., Rossi, A., Martinez-Rossi, N.M., 2010. Dermatophytes: host-pathogen interaction and antifungal resistance. An Bras Dermatol 85(5), 657-667. Shen, Z.B., Luo, W.Y., Yan, Y.S., Zhu, J.F., 2006. [Studies on phloroglucinol derivatives of Dryopteris fragrans L]. Journal of Chinese Medicinal Materials 29(6), 560.

Simonetti, O., Silvestri, C., Arzeni, D., Cirioni, O., Kamysz, W., Conte, I., Staffolani, S., Orsetti, E., Morciano, A., Castelli, P., Scalise, A., Kamysz, E., Offidani, A.M., Giacometti, A., Barchiesi, F., 2014. In vitro activity of the protegrin IB-367 alone and in combination compared with conventional antifungal agents against dermatophytes. Mycoses 57(4), 233-239. Tamura, T., Asahara, M., Yamamoto, M., Yamaura, M., Matsumura, M., Goto, K., Rezaei-Matehkolaei, A., Mirhendi, H., Makimura, M., Makimura, K., 2014. In vitro susceptibility of dermatomycoses agents to six antifungal drugs and evaluation by fractional inhibitory concentration index of combined effects of amorolfine and itraconazole in dermatophytes. Microbiology and immunology 58(1), 1-8. Wenxin, L., Xin, L., 2014. Research Progress on Dryopteris fragrans ( L.) Schott. Journal of Anhui Agri 42(4), 985-988，993. Wenzhao, D.U., Song, G., Liu, H., Feng, D., Shen, Z., 2016. Study on phloroglucinol derivatives of Dryopteris fragrans( L.) Schott. Journal of Guangdong Pharmaceutical University. Widén, C.-J., Faden, R.B., Lounasmaa, M., Vida, G., Euw, J.V., Reichstein, T., 1973. Die phloroglucide von neun dryopteris‐arten aus kenya sowie der d. oligodonta (desv.) pic.‐serm. und d. “dilatata” von den canarischen inseln. Helvetica Chimica Acta 56(7), 2125-2151. Widén, C.J., Lounasmaa, M., Sarvela, J., 1975. Phloroglucinol derivatives of eleven Dryopteris species from Japan. Planta Medica 28(2), 144-164. Xianjun, Y., 2013. Curative observation of Dryopteris fragrans on 48 cases with stubborn tinea pedis. Drugs & Clinic 28(1), 57-58. Yang, S.-h., Chen, W.-h., Shan, F., Jia, X.-z., Deng, R.-r., Tang, C.-p., Shen, Z.-b., 2017. Antifungal Activity of Aspidin BB from Dryopteris fragrans against Trichophyton rubrum Involved Inhibition of Ergosterol Biosynthesis. Chinese Herbal Medicines 9(1), 63-68. Yixi, H., Zhibin, S., Tao, J., Yanfen, C., Jiayuan, L., Lili, Z., Chunping, T., 2016. Antifungal effect of the active fraction of Dryopteris fragrans on Trichophyton rubrum in vitro. Journal of Guangdong Pharmaceutical University 32(1), 18. Zhi-Jian, L.I., GulinaDawuti, SilafuAibai, Hui-Xia, A.N., Xiao, W., 2009. Determination of Ergosterol in Mycelium of Trichophyton Rubrum by High Performance Liquid Chromatography. Journal of Shihezi University. Zuo, L., Wang, H.Q., Chen, R.Y., 2005. Chemical constituents in roots of Dryopteris championii. Chinese Traditional & Herbal Drugs.

Fig. 5

Graphical Abstract

Table. 1 Components analysis of the ethanol extracts of D. fragrans Peak

RT

[M+H]

+

(min) 1

22.6

447.2064

2

molecular

MS Fragments

Compound

formula

(Relative Abundance)

Name

C24H30O8

447 (100), 237 (20), 224, 219 (100),

albaspidin PB

209 (10), 193, 181, 179, 165 2

38.8

433.2010

C23H28O8

reference

(Widén et al., 1973)

447 (100), 237 (75), 225 (60), 224,

disflavaspidic

(Chen et al.,

219 (100), 209 (50), 208, 197 (12),

acid PB

2017)

flavaspidic

(Hisada et al.,

acid AB

1973)

196, 165 3

39.3

419.1854

C22H25O8

419 (100), 223 (38), 211 (100), 209 (50), 197 (13), 193 (13), 181, 165

4

41.0

419.1848

C22H26O8

419 (100) 223 (70), 211 (60), 210,

albaspidin AP

209 (50), 208, 197 (100), 193 5

43.4

433.2023

C23H28O8

433 (100) 223 (100), 211 (98), 205

Wang, 2007) flavaspidic

(Lee and Kim

acid PB

JCLee, 2009)

methylene-bis-

(Widén et al.,

aspidinol BB

1975)

447 (100) 237 (38), 225 (20), 224,

flavaspidic

(Lee and Kim

223 (70), 211 (100), 193 (30), 179,

acid BB

JCLee, 2009)

saroaspidin A

(Ishiguro et al.,

(52), 1 9 3 (50), 179, 167 6

43.6

461.2432

C25H32O8

461 (50) 237 (100), 225 (35), 219 (32), 207 (35)

7

44.6

447.2181

C24H30O8

(Feng and

177 8

45.4

447.2184

C24H30O8

447 (100) 237 (50), 225 (20), 224, 223 (32), 211 (100), 1 93 (10), 181

9

49.7

433.2036

C23H28O8

433 (100) 237 (98), 225 (100), 224,

1987) aspidin AB

(Zuo et al.,

209 (54), 208, 197 (12), 193, 181 10

50.9

433.2024

C23H28O8

433 (100) 237 (100), 225 (98), 209

2005) albaspidin PP

(55), 197 (10), 196, 167 (10) 11

52.6

447.2190

C24H30O8

447 (100) ,223 (32), 211 (100), 209

(Wenzhao et al., 2016)

aspidin PB

(Shen et al.,

(20),197 (50), 193, 181 12

53.2

459.3287

C28H42O5

2006)

459 (100) 251 (5), 237 (100), 235,

dryofragin

(Shen et al.,

225 (5), 221, 217, 209, 207, 203,

2006)

193, 191, 181 13

53.8

461.2347

C25H32O8

461 (100) 237 (100), 225 (70), 193

albaspidin BB

(Wenzhao et al., 2016)

14

55.1

461.2358

C25H32O8

461 (100) , 223 (50), 211 (100), 209

aspidin BB

(Yang et al.,

(24), 197 (39), 193, 181

2017)

RT: retention time

Table. 2 Activity of ethanol extracts of D. fragrans (L.) Schott against 62 isolates of dermatophytes ethanol extracts of D. fragrans (mg/mL) MIC range

MIC

MFC range

MFC

Isolates (n)

Trichophyton rubrum (21)

0.059-1.890

(mean

(mean

±SD)

±SD)

0.819 ±0.656

0.118-3.780

1.029 ±0.891

FCZ (µg/mL) MIC

Trichophyton mentagrophytes

0.236-3.780

(14) Microsporum canis (11)

1.434

0.473-3.780

±0.919* 0.236-0.945

0.924

±1.042 0.473-3.780

±0.999 Microsporum gypseum (10)

0.236-0.473

0.898

2.059

1.353 ±1.277

0.473-0.945

±1.049

1.087 ±0.972

Trichophyton tonsurans (1)

0.945

0.945

1.890

1.890

Trichophyton violaceum (1)

0.945

0.945

1.890

1.890

0.473-0.945

0.827

0.473-1.890

1.063

Epidermophyton floccosum (4)

#

Candida parapsilosis (ATCC

1.000

22019) MIC: means value of the minimal inhibitory concentrations, MFC: means value of the minimal fungicidal concentrations, FCZ: fluconazole * compared to the MIC value of Trichophyton rubrum, P<0.05. #

compared to the MFC value of Trichophyton rubrum, P<0.05

Table 3 Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) values of antifungal drugs against dermatophyte species #

Isolates (n)*

DF (mg/mL)

miconazole (μg/mL)

MIC

MFC

MIC

MFC

CMCC(F)T1a

0.059

0.118

0.039

0.039

CMCC(F)T1b

0.473

0.473

0.156

0.156

CMCC(F)T1c

0.236

0.236

0.078

0.078

CMCC(F)T1d

0.473

0.945

0.313

0.313

CMCC(F)T1e

0.118

0.236

0.156

0.156

Standard isolates (23) Trichophyton rubrum (9)

CMCC(F)T1f

0.945

1.890

0.313

0.313

CMCC(F)T1g

0.473

0.473

0.313

0.156

CMCC(F)T1h

0.236

0.236

0.078

0.078

ATCC YA4438

0.473

1.890

0.625

0.313

CMCC(F)T5a

0.945

1.890

0.039

0.313

CMCC(F)T5b

1.890

3.780

0.313

0.625

CMCC(F)T5c

0.236

0.473

0.156

0.156

CMCC(F)T5d

0.473

1.890

0.313

0.313

CMCC(F)T5f

1.890

3.780

0.625

0.625

ATCC MYA4439

1.890

1.890

0.313

0.625

CMCC(F)M3c

0.945

0.945

0.313

0.313

CBS113489

0.945

3.780

0.313

0.313

CMCC(F)M3h

0.236

0.473

0.313

0.625

CMCC(F)M3d

0.236

0.473

0.313

0.625

CMCC(F)M2c

0.236

0.473

0.156

0.625

CMCC(F)M2e

0.473

0.945

0.078

0.156

0.945

1.890

0.313

0.625

0.945

1.890

0.313

0.625

3518

0.473

0.473

0.156

0.156

3579

1.890

1.890

1.250

1.250

3598

1.890

0.473

0.156

0.156

3622

1.890

1.890

1.250

1.250

3766

0.945

0.945

0.313

0.313

3781

0.473

0.473

0.156

0.156

3782

0.945

0.945

0.313

0.313

3862

0.473

0.945

0.156

0.156

3949

0.473

0.473

0.156

0.156

3950

1.890

1.890

0.625

0.625

3952

1.890

3.780

0.625

0.625

3955

0.473

0.945

0.156

0.313

3247

1.890

1.890

0.313

0.313

3460

3.780

3.780

0.625

0.625

3461

1.890

1.890

0.313

0.313

3491

1.890

1.890

0.625

0.625

3498

0.945

0.945

0.156

0.156

3521

0.945

0.945

0.078

0.078

Trichophyton mentagrophyte (6)

Microsporum canis (4)

Microsporum gypseum (2)

Trichophyton tonsurans (1) CMCC(F)T3e Trichophyton violaceum (1) CMCC(F)T4e Clinical isolates (39) Trichophyton rubrum (12)

Trichophyton mentagrophytes (8)

3746

0.473

1.890

0.078

0.078

3772

0.945

1.890

0.078

0.078

3218

3.780

3.780

0.625

0.625

3582

0.473

0.945

0.156

0.156

5812

0.945

0.945

0.156

0.156

5977

0.473

0.473

0.156

0.156

6177

0.945

0.945

0.313

0.313

6640

0.236

0.236

0.078

0.078

7852

0.945

1.890

0.313

0.625

4090

0.473

0.473

0.078

0.078

6211

0.473

0.945

0.156

0.156

6498

0.236

0.473

0.078

0.078

6503

0.945

0.945

0.625

0.625

6504

0.473

0.945

0.156

0.156

6513

0.945

0.945

1.250

1.250

6632

3.780

3.780

0.313

0.313

6690

0.945

0.945

0.625

0.625

3023

0.945

0.945

0.625

0.625

5703

0.945

1.890

1.250

1.250

6167

0.945

0.945

1.250

1.250

6236

0.473

0.473

0.625

0.625

Microsporum canis (7)

Microsporum gypseum (8)

Epidermophyton floccosum (4)

* The number of isolates #

Ethanol extracts of D. fragrans (L.) Schott

Table. 4 Mode of drug interaction against 2 dermatophytes on basis of the fractional inhibitory concentration (FIC) index test

MICa

MICc range

combinations Trichophyton

FICI

Mode of

range

interaction

1.0~1.5

indifferent

1.5~2.5

indifferent

DF with TBF

rubrum

DF

0.4730

0.2360~0.4730

CMCC(F)T1d

TBF

0.0113

0.0056~0.0113

DF

0.4730

0.2360~0.4730

MCZ

0.3125

0.3125~0.6250

DF with MCZ

Trichophyton

DF with TBF

mentagrophytes

DF

0.2360

0.2360~0.4730

1.25~2.

CMCC(F)T5c

TBF

0.0113

0.0028~0.0113

25

DF

0.2360

0.2360~0.4730

MCZ

0. 1562

0.0781~0.1562

indifferent

DF with MCZ 1.5~3.0

indifferent

MICa: minimal inhibitory concentrations (MIC) of drug alone; MICc: MIC in combination; FICI = [MICA in combination/MICA]/ [MICB in combination/MICB]; 0.5
Appendix 1 Full names of the abbreviations in this paper Abbreviations

full name

ATCC

American type culture collection

CFU

colony-forming units

CLSI

Clinical and Laboratory Standards Institute

D. fragrans

Dryopteris fragrans (L.) Schott

FCZ

fluconazole

FIC

fractional inhibitory concentration

HPLC-ESI-QTOF-MS/MS

high-performance liquid chromatography coupled with electrospray ionization and quadruple time-of-flight mass spectrometry

MCZ

miconazole

MFC

minimal fungicidal concentrations

MIC

minimum inhibitory concentrations

min

minute

MOPS

3-Morpholinopropanesulfoinc Acid

RPMI

Roswell Park Memorial Institute

SDA

Sabouraud dextrose agar

TBF

terbinafine

T. mentagrophyte

Trichophyton mentagrophytes

T. rubrum

Trichophyton rubrum

UPLC

ultra-performance liquid chromatography

YEPD

yeast extract peptone dextrose medium