Send Orders for Reprints to [email protected] Current Bioactive Compounds 2018, 14, 000-000 1 RESEARCH ARTICLE Antimicrobial and Resistance Modifying Activities of Cerevisterol Isolated from Species

Theresa Appiah1, Christian Agyare1,*, Yinggang Luo2, Vivian Etsiapa Boamah1 and Yaw Duah Boakye1

1Microbiology Section, Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana; 2Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, China

Abstract: Background: The development of resistance by pathogenic microorganisms has renewed the worldwide search for novel antimicrobial agents. Mushrooms are of recent interest because a wide vari- ety of biologically active compounds have been isolated from them. This study isolated antimicrobial compound from two wood decaying mushrooms, Trametes gibbosa and Trametes elegans, and deter- mined the resistance modifying activities of the isolated compound. Methods: Bioactivity guided isolation of active principles from the methanol extract of T. gibbosa and T. elegans was performed using column and preparative high-performance liquid chromatography. The

A R T I C L E H I S T O R Y structures of isolated compounds were elucidated using nuclear magnetic resonance spectroscopy. Broth micro-dilution assay was used to determine the antimicrobial and resistance modifying activities of the Received: March 25, 2018 Revised: May 24, 2018 isolated compounds against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmo- Accepted: July 12, 2018 nella typhi, Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Can-

DOI: dida albicans, Aspergillus niger, Aspergillus flavus and Aspergillus tamarii. 10.2174/1573407214666180813101146 Results: Bioactivity guided isolation lead to the isolation of cerevisterol (ergosta-7, 22E-diene-35, 6- triol) from both T. gibbosa and T. elegans. The isolated cerevisterol inhibited the growth of S. typhi, S. aureus and A. niger with MICs of 25 g/mL each and 50 g/mL against E. faecalis. The MBCs of cerevisterol against S. typhi S. aureus, E. faecalis and A. niger were 50, 100, 200 and 100 g/mL, respec- tively. The sub-inhibitory concentration (3 g/mL) of cerevisterol modified the activity of erythromycin, ampicillin, ciprofloxacin, tetracycline and amoxicillin either by potentiating or reducing their activities.

Conclusion: Cerevisterol possesses both antimicrobial and resistance modifying activities. Keywords: Trametes gibbosa, Trametes elegans, cerevisterol, antibiotic resistance modifying activities.

1. INTRODUCTION from them [9, 10]. For instance, the peptide plectasin has been isolated from the mushroom, Pseudoplectania nigrella and The increasing rate of resistance by pathogenic microor- found to show antimicrobial activity [11]. Ganomycin A and ganisms has generated a global resurgence of interest in the B isolated from Ganoderma pfeifferi, showed activity against search for novel antimicrobial compounds [1, 2, 3]. Infec- Bacillus subtilis, Micrococcus flavus and Staphylococcus tions caused by drug-resistant microorganisms result in a aureus [12]. Cerevisterol isolated from Myrothecium sp. Z16, significant increase in mortality, morbidity, and prolonged exhibited activity against Candida albicans, Tricophyton treatments [4]. Antibiotic resistance increases health care rubrum and Aspergillus niger [13]. Again, Zhou et al. [14] utilization and, shifts antibiotic use towards more costly sec- reported that cerevisterol exhibited antibacterial activity of ond-line agents [5]. Antibiotics have been pivotal in treating 128 and 256 g/mL against Bacillus subtilis and Staphylococ- and preventing common infections, but their overuse and cus aureus, respectively, and antifungal activity of 256 g/mL misuse have contributed to an alarming increase in antibiotic against Penicillium chrysogenum. resistance worldwide [6]. This has necessitated the constant search for new and effective antimicrobial agents [7, 8]. Trametes gibbosa (Pers.) Fr. and Trametes elegans (Spreng. Fr.) Fr. belong to the family [15]. T. Mushrooms have been of interest recently due to a num- gibbosa and T. elegans are saprobic , which cause ber of biologically active compounds that have been isolated white rot of wood [16]. Though the Trametes are best known for their medicinal properties, much research has not been *Address correspondence to this author at the Microbiology Section, carried out on the medicinal properties, especially the antimi- Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical crobial activities of T. gibbosa and T. elegans. Awala and Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana; Tel: +233-246369803; E-mail: [email protected] Oyetayo [17] reported the antiradical activity of T. elegans. T.

1573-4072/18 $58.00+.00 © 2018 Bentham Science Publishers 2 Current Bioactive Compounds 2018, Vol. 14, No. 00 Appiah et al. elegans is also well known for its ability to degrade lignin by petroleum ether/ (Xilong Chemical Co. Ltd, [18]. Although Aina et al. [19] reported the antibacterial activ- Guangdong, China) mixture in the following ratios; 90:10, ity of T. elegans, studies on the medicinal properties of T. 80:20, 70:30, 60:40, 50:50, 40:60, 70:30, 80:20 and 90:10 %. elegans are limited. However, methanol extract of T. gibbosa Approximately 170 and 324 aliquots of 100 mL each were has been found to exhibit antibacterial [20, 21], antioxidant collected for T. elegans and T. gibbosa respectively. TLC [22] and HIV-1 reverse transcriptase activity in vitro [23]. analysis using mobile phase of various mixtures of petro- leum ether and ethyl acetate, resulted in the bulking of the Extracts or isolated compounds with antibiotic resistance eluents into eight fractions for T. gibbosa and seven fractions modifying activities have the potential of lowering the thera- for T. elegans. From the TLC Bioautography, fractions 4 and peutic dose of antibiotics and may lower the chances of de- 5 were bulked for both T. elegans and T. gibbosa. These velopment of drug resistance after prolonged use [24 25]. fractions were further re-chromatographed using silica gel There is a limited report on the antibiotic modifying activi- (Silica gel; 230 mesh; Qingdao Marine Chemical Plant, ties of compounds isolated from mushrooms, however, some Qingdao, China) and eluted with chloroform methanol (9:1) synergistic effects of mushroom extract have been reported to yield sub-fraction 5A for T. elegans and 5B for T. gib- [26-29]. The aim of this study was, therefore, to use bioac- bosa. tivity guided procedure to isolate and characterize antimicro- bial compound(s) from T. gibbosa and T. elegans, and de- 2.4. Preparative High-Performance Liquid Chromatog- termine the antibiotic resistance modifying activities of the raphy (pHPLC) of Fractions of T. gibbosa and T. elegans isolated compounds. Preparative high-performance liquid chromatography 2. METHODS (pHPLC) method of Ding et al. [31] was followed for purifi- cation and fraction collection. After determining a suitable 2.1. Collection of Mushroom Samples solvent for elution and suitable wavelength for UV detection, Fruiting bodies of Trametes gibbosa (Pers.) Fr. and HPLC separation and fraction collection were performed on Trametes elegans (Spreng. Fr.) Fr. were collected from farms a Beckman 110B solvent delivery module with a system and forests in Ayeduase (latitude 6°40'33 N, longitude Gold 166 variable wavelength detector, with column size 9.4 1°33'36 W, altitude 252 m) in the Ashanti Region, Ghana x 250 mm. A Beckman Altex 210A injector was used as the from June to November, 2014 from their natural habitats. injection port for HPLC separations and was controlled by These mushrooms were authenticated by Mr. A. K. Ape- system Gold 32-Karat software using an IBM 300PL PC. torgbor, a mycologist in the Department of Theoretical and Fractions 5A (10.50 mg) for T. elegans and 5B (12.08 mg) Applied Biology, Kwame Nkrumah University of Science for T. gibbosa were each dissolved in 3 mL methanol and a and Technology (KNUST), Kumasi, Ghana and voucher volume of 20 L injected into the column using 15 mL specimens (KNUST/HMI/2014/S005 and MeOH/H2O (30% v/v) gradient as the mobile phase for each KNUST/HMI/2014/S006, respectively) kept in the Herbar- run. The runtime was 15 min under a pump pressure of 15 ium of Department of Pharmacognosy, KNUST, Kumasi, MPa and flow rate of 3.0 mL/min at the wavelength of 208 Ghana. nm for fraction 5A, and 254 nm for 5B. Purified fractions were collected and evaporated to dryness using a rotary 2.2. Preparation of Methanol Extracts evaporator at 40oC under reduced pressure, which yielded compounds TE1 (5.05 mg) and TG1 (6.45 mg). The fruiting bodies of T. gibbosa and T. elegans were air dried and ground to fine powder using a laboratory mill ma- 2.5. Identification and Characterization of the Com- chine (Christy and Norris, Chelmsford, England, UK). Two pounds by Nuclear Magnetic Resonance (NMR) hundred grams of each sample was weighed and succes- sively extracted with 1L each of 70% v/v methanol. The One milligram (1 mg) each of isolated compounds, TE1 mixture was allowed to stand at room temperature (28oC) for and TG1 were dissolved in 0.5 mL deuterated pyridine. The three days with frequent agitation, homogenized and the su- solution was pipetted into clean 1.7 mm micro-cryoprobe pernatant filtered using Whatman filter paper No. 10 (Sigma- NMR tube for spectral analysis. The sample spectral of 1D Aldrich, Michigan, USA). The filtrates were concentrated in 1H NMR and 1D 13C NMR techniques were determined at a rotary evaporator (Rotavapor BÜCHI R-200 with heating 400.13 and 100.61 MHz, respectively, using Bruker Avance bath B-490, Büchi, Konstanz, Germany) at 40oC under re- 500 NMR spectrometer. Tetramethylsilane (TMS) was used duced pressure and lyophilized. The yield of the extract, re- as internal standard and coupling constants (J) measured in lated to the dried powdered mushroom materials of T. Hertz (Hz). Spectra from NMR experiments display chemi- gibbosa and T. elegans were 18.10 and 15.5% w/w, respec- cal shifts () in ppm downfield of tetramethylsilane (TMS). tively. The extracts were kept in airtight containers, labelled The 1H and 13C NMR data were processed using NMR Util- and stored in a desiccator until required for use [30]. ity Transfer Software (NUTS) 1D Version-20060331.

2.3. Fractionation of Methanol Extracts of T. gibbosa and 2.6. Culture Media and Reference Antibiotics T. elegans Using Column Chromatography Sabouraud dextrose agar, nutrient agar and broth were The crude methanol extracts of T. gibbosa and T. elegans purchased from Oxoid Limited, Basingstoke, United King- were each fractionated by silica gel column chromatography dom. Ciprofloxacin, ketoconazole, erythromycin, amoxicil- (Silica gel; 230 mesh; Qingdao Marine Chemical Plant, lin, ampicillin, ciprofloxacin and tetracycline were obtained Qingdao, China), and eluted with petroleum ether, followed from Sigma-Aldrich, Michigan, USA. Antimicrobial and Resistance Modifying Activities of Cerevisterol Isolated from Trametes Species Current Bioactive Compounds 2018, Vol. 14, No. 00 3

2.7. Test Organisms erence standards. The procedure was performed in independ- ent triplicates to validate the results. Pure cultures of Escherichia coli ATCC 25922, Pseudo- monas aeruginosa ATCC 4853, Klebsiella pneumoniae 2.9. Determination of Antibiotic Modifying Activity of ATCC 33495, Salmonella typhi ATCC 19430, Streptococcus Isolated Compounds from T. gibbosa and T. elegans pyogenes ATCC 19615 Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Bacillus subtilis The effect of sub-inhibitory concentration of the isolated NTCC 4853 and Candida albicans ATCC 10231 were ob- compounds from Trametes gibbosa and Trametes elegans on tained from the Microbiology Section, Department of Phar- the activity of some selected antibiotics was carried out, us- maceutics, Faculty of Pharmacy and Pharmaceutical Sci- ing the broth-dilution method [33]. The following antibiotics ences, Kwame Nkrumah University of Science and Technol- were used; erythromycin, amoxicillin, ampicillin, ciproflox- ogy (KNUST), Kumasi, Ghana. Pure cultures of Aspergillus acin and tetracycline. The MICs of the antibiotics were first niger ATCC 16888, Aspergillus flavus ATCC 200026 and determined using a concentration range from 1 to 1000 Aspergillus tamarii ATCC 1005 were obtained from De- μg/mL. Each well of the 96-well microtitre plate was filled partment of Animal and Crop Science, Faculty of Agricul- with 100 μL of double strength nutrient broth, appropriate ture, Kwame Nkrumah University of Science and Technol- volume of the antibiotics and 20 μL of 106 CFU/mL of the ogy (KNUST), Kumasi, Ghana. test organism. The plate was incubated for 24 h at 37C, after which 20 μL MTT was added and incubated at 37oC for 30 min. The appearance of purple colour indicated microbial 2.8. Determination of Antimicrobial Activity of Isolated growth while persistence yellow colour of the MTT indi- Compounds cated no microbial growth. MIC values were recorded as the The antimicrobial activity of isolated compounds was lowest concentration of the extract, which completely inhib- determined using the broth mico-dilution method [32]. Each ited microbial growth [34]. well of a 96-well microtitre plate was filled with 100 L In the presence of a sub-inhibitory concentration of 3 double strength nutrient broth. Stock solutions of extracts g/mL of the isolated compounds, the above procedure was within the range of 1 to 400 g/mL were prepared and 20 L repeated and MICs for the combined compounds and antibi- of inoculum (106 CFU/mL) was added to each well. The otics determined. plate was incubated at 37°C for 24 h. After incubation, 10 L of 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium 3. RESULTS bromide (MTT) (1.25 mg/mL) (Sigma Aldrich, Taufkirchen, Germany) was added to each well and the plate incubated 3.1. Isolate TG1/TE1 again at 37oC for 30 min. Ketoconazole at a concentration TG1 was isolated and purified from fraction 5B by HPLC ranging from 1.0 to 10.0 g/mL and ciprofloxacin at a in a yield of 6.45 mg (0.025% related to the dried methanol concentration range of 0.01 to 1.0 g/ mL were used as ref-

Table 1. The 1H NMR data measured for TG1 and TE1 (cerevisterol) and respective literature data.

TG1/TE1 Cerevisterol [37]

H  (ppm) Multiplet J (Hz)  (ppm) Multiplet J (Hz)

3 4.80 m 4.82 m

6 4.52 d 5.3 4.31 d 5.0

7 5.28 m 5.30 m

18 0.66 s 0.65 s

19 1.68 s 1.52 s

20

21 1.01 d 6.6 1.05 d 6.5

22 5.27 d 8.6 5.18 d 8.5

23 5.13 dd 7.5, 15.2 5.24 dd 7.0, 15.0

26 0.85 d 7.4 0.85 d 7.0

27 0.87 d 7.0 0.85 d 6.5

28 0.95 d 6.9 0.94 d 7.0

400.13 MHz, C5D5N 500 MHz, C5D5N s: singlet; d: doublet; m: multiplets; dd: doublet of doublets. 4 Current Bioactive Compounds 2018, Vol. 14, No. 00 Appiah et al.

Table 2. The 13C MNR data measured for TG1 and TE1 (cerevisterol) and respective literature data.

C TG1/TE1 Cerevisterol [37]

 (ppm)  (ppm)

1 31.8 32.4

2 31.5 33.0

3 67.4 67.3

4 40.9 41.8

5 76.5 75.9

6 74.0 74.0

7 121.4 120.2

8 141.6 141.3

9 43.6 43.5

10 38.1 37.8

11 21.5 22.2

12 39.5 39.6

13 43.5 43.5

14 55.4 55.0

15 23.8 23.2

16 27.5 28.2

17 56.5 55.9

18 13.3 12.3

19 17.0 18.6

20 40.2 40.6

21 20.2 21.2

22 135.2 135.9

23 132.5 131.8

24 42.9 42.8

25 33.1 33.1

26 20.8 19.6

27 20.8 19.9

28 19.0 17.6

100.62 MHz, C5D5N 125 MHz, C5D5N extract of T. gibbosa). It is a colourless needle-like crystal; it crystal; it does not fluoresce on TLC plates under UV light does not fluoresce on TLC plates under UV light ( = 254 ( = 254 and 356 nm), but it is easily detected following ex- and 356 nm), but it is easily detected following exposure to posure to anisaldehyde-sulphuric acid spray reagent (dark anisaldehyde-sulphuric acid spray reagent (dark spot) with Rf spot) with Rf value of 0.35 [mobile phase = chloroform: value of 0.35 [mobile phase = chloroform: methanol methanol (9.5:0.5)]. (9.5:0.5)]. The 1H-NMR spectra of both TE1 and TG1 in deuterated TE1 was also isolated and purified from fraction 5A by pyridine displayed two methyl singlets at H 0.66 and 1.68 HPLC in a yield of 5.05 mg (0.020% related to the dried ppm, four methyl doublets at H 0.85, (d, 7.4 Hz) 0.87 (d, methanol extract of T. elegans). It is a colourless needle-like 7.4 Hz), 0.95 (d, 6.8 Hz) and 1.01 (d, 6.6 Hz) ppm, three Antimicrobial and Resistance Modifying Activities of Cerevisterol Isolated from Trametes Species Current Bioactive Compounds 2018, Vol. 14, No. 00 5 olefinic protons at 5.27 (d 8.6 Hz), 5.13 (dd, 7.5, 15.2 Hz) against E. faecalis. The MBCs of cerevisterol against S. typhi, and 5.28 (m) ppm, two oxymethine protons at 4.52 (d, 5.3 S. aureus, E. faecalis and A. niger were 50, 100, 200 and 100 Hz) and 4.80 (m) ppm. The 13CNMR spectra showed the g /mL, respectively (Table 3). existence of 28 skeleton carbons: three sp3 carbons attached to oxygen atom, including two secondary carbons at C 67.4 3.3. Antibiotic modifying activity of TG1/TE2 and 74.0 ppm, and one tertiary at C 78.5 ppm, four olefinic (cerevisterol) from T. gibbosa and T. elegans carbons at C 121.4, 135.2, 132.5 and 141.6 ppm, six meth- The activity of erythromycin against S. aureus and S. yls, seven methylenes, six methines and two quaternary car- 1 13 typhi was potentiated four (4) and two (2) times, respectively bons. The H-NMR and CNMR of compounds TG1 and in the presence of sub-inhibitory concentration of TE1 together with the reported literature [35, 36, 37] were used to identify TG1 and TE1 as the steroid cerevisterol with cerevisterol. The sub-inhibitory concentration of cerevisterol : C H O (Fig. 1). enhanced the activity of ampicillin against S. aureus and 28 46 3 Enterococcus faecalis, two (2) and three (3) fold respec- 26 tively. The activity of amoxicillin against K. pneumoniae and 25 27 E. coli was also potentiated two (2) fold each in the presence of sub- inhibitory concentration of cerevisterol. However, 24 23 the sub-inhibitory concentrations of cerevisterol antagonized 21 the activities of tetracycline and ciprofloxacin against all test 28 18 22 organisms (Table 4). 20 12 17 H 11 4. DISCUSSION 19 13 16 The increasing level of antibiotic resistance by patho- 1 9 14 8 2 15 genic microorganisms has increased the search for new and 10 H H alternative compounds from natural products including 3 5 7 mushrooms [38, 39, 40]. In this study, a compound (Fig. 1)

HO 4 6 was isolated from both T. elegans and T. gibbosa and the OH structure elucidated using NMR data and confirmed by lit- OH erature data [41]. This lead to the identification of each com- Fig. (1). Molecular structure of Compound (TG1 and TE1) (Cerevis- pound (TE1 and TG1) as cerevisterol (ergosta-7,22E-diene- terol (ergosta-7,22E-diene-35,6-triol). 35,6-triol). Cerevisterol (TE1 and TG1) isolated from T. elegans and T. gibbosa is not unusual since other studies 3.2. Determination of Antimicrobial Activity of Isolated have found cerevisterol in fungi, and most especially mush- Compounds rooms [42, 43]. Though Leliebre-Lara et al. [43] reported the presence of cerevisterol in the genus Trametes (such as T. Cerevisterol inhibited the growth of S. typhi, S. aureus versicolor), this is being reported for the first time for T. and A. niger with MICs of 25 g /mL each, and 50 g /mL elegans and T. gibbosa. Cerevisterol has been reported to

Table 3. MIC and MBC of compound TG1/TE2 (Cerevisterol).

TG1/TE2 (Cerevisterol) Cipro Keto

Test Organisms MIC (g/mL) MBC (g/mL) MIC (g/mL) MIC (g/mL)

E. coli - -  0.16 nd

P. aeruginosa - -  0.31 nd

S. pyogenes - -  0.31 nd S. typhi 25 50 2.00 nd

S. aureus 25 100  0.31 nd

K. pneumonia - - 0.63 nd B. subtilis - - nd nd

E. faecalis 50 200 3.50 nd C. albicans - - 0.16  0.31 niger 25 100 nd 0.35 A. flavus - - nd 0.50 A. tamarii - - nd 2.00

- : MIC/MBC > highest concentration (400g/mL); Cipro: Ciprofloxacin; Keto: Ketoconazole; nd : not determined 6 Current Bioactive Compounds 2018, Vol. 14, No. 00 Appiah et al.

Table 4. Effect of sub-inhibitory concentration (3 g/mL) of cerevisterol on some antibiotics.

Antibiotic + PA EC SA BS SP KP EF ST Cerevisterol

Antibiotics MIC (μg/mL)

Erythromycin Only 100 100 50 200 50 100 100 50

+ Cere 400 400 12.5 400 200 100 1000 25

FP na na 4 na na na na 2

FR 4 4 na 4 4 na 10 na

Ciprofloxacin Only 3.125 1.56 3.125 1.56 3.125 3.125 3.125 3.125

+ Cere 400 50 > 1000 50 50 50 25 25

FP na na na na na na na na

FR 128 32 >320 32 16 16 8 8

Ampicillin Only 50 100 50 25 50 100 >1000 100

+ Cere 200 400 25 100 400 > 1000 400 200

FP na na 2 na na na 3 na

FR 4 4 na 4 8 10 na 2

Tetracycline Only 50 12.5 25 25 25 50 25 25

+ Cere 400 400 400 400 400 400 1000 400

FP na na na na na na na na

FR 8 32 16 16 16 8 40 16

Amoxicillin Only 100 200 100 100 200 100 400 100 + Cere 400 100 1000 400 400 50 400 400

FP na 2 na na na 2 na na

FR 4 na 10 4 2 na na 4

Cere: cerevisterol; Cipro: Ciprofloxacin; Amoxy: amoxicillin; Tetra: tetracycline; Erythro: erythromycin; Ampi: ampicillin; PA: Pseudomonas aeruginosa; EC: Escherichia coli; SA: Staphylococcus aureus; BS: Bacillus subtilis; SP: Streptococcus pyogenes; KP: Klebsiella pneumoniae; EF: Enterococcus faecalis; ST: Salmonella typhi; FP: fold potentiation; FR: fold reduction; - : fold potentiation or reduction not available. exhibit pharmacological activities such as anti-inflammatory the synergistic activity of erythromycin with cerevisterol [13] and antimicrobial [13, 14]. It has also been found to be against S. typhi and S. aureus (with fold potentiation of 2 cytotoxic to mouse P388 leukemia cells [44] and A549 hu- and 3, respectively), ampicillin against S. aureus and E. man alveolar epithelial cells grown in culture [13]. faecalis (with fold potentiation of 4 and 2, respectively), as well as amoxicillin against K. pneumoniae and E. coli (with In this study, cerevisterol (TE1 and TG1) inhibited the fold potentiation of 2 each) are being reported for the first growth of S. typhi, S. aureus and A. niger with MICs of 25 time. Cerevisterol (TE1 and TG1), however, antagonized g/mL each and 50 g/mL against E. faecalis (Table 3). It the in vitro activities of tetracycline and ciprofloxacin also exhibited broad-spectrum antimicrobial activity which is against the test organisms. There is a need to explore the similar to the report by Liu et al. [13] with respect to the effect of cerevisterol on the activities of other antimicrobial antifungal and antibacterial activities [14]. However, the agents. MICs of TG1/TE1 (cerevisterol) against S. aureus were lower (25 g/mL) compared to the MIC of 32 g/mL as re- CONCLUSION ported by Zhou et al. [14]. Cerevisterol (TE1 and TG1) isolated from both T. Although there is limited information on the synergistic elegans and T. gibbosa exhibited broad spectrum antimi- activity of standard antibiotics with compounds isolated crobial activity. Sub-inhibitory concentrations of cerevisterol from mushrooms, few reports on the synergistic antimicro- potentiated the activities of erythromycin, ampicillin and bial activity of mushrooms extracts in combination with amoxicillin. standard antibiotics have been reported [5, 45]. In addition Antimicrobial and Resistance Modifying Activities of Cerevisterol Isolated from Trametes Species Current Bioactive Compounds 2018, Vol. 14, No. 00 7

Appendix

26 25 27

24 H26 23 H27 21 H28

28 H18 18 22 H21 20 12 17 H 11 19 13 16 1 9 14 8 2 15 10 H H 3 5 7 HO 4 6 OH OH

H7 H19 Pyridine-d5 H23 H22 H3 H6

1H NMR spectra of cerevisterol (TG1 and TE1) (400.13 MHz, pyridine-d5).

45000 123.33 67 . 37 55 . 89 37 . 81 29 . 77 19 . 92 17 . 60 14 . 07 149.86 149.32 141.31 135.15 131.86 123.58 123.08 120.20 75 . 90 73 . 97 54 . 99 43 . 49 39 . 65 37 . 43 33 . 57 27 . 18 22 . 73 12 . 28 1. 2 1 149.59

40000

       

35000       

   30000

25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 f1 (ppm)

25000  

35 34 33 32 31 30 29 28 27 26 20000 f1 (ppm) P yridine -d5        

     15000

     10000 80 75 70 65 60 55 50 45 40 f1 (ppm)

5000 

0

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm) 13C NMR spectra of cerevisterol (TG1 and TE1) (100.62 MHz, pyridine-d5).

ETHICS APPROVAL AND CONSENT TO PARTICI- CONSENT FOR PUBLICATION PATE Not applicable. Not applicable. CONFLICT OF INTEREST HUMAN AND ANIMAL RIGHTS The authors declare no conflict of interest, financial or No Animals/Humans were used for studies that are the otherwise. basis of this research. 8 Current Bioactive Compounds 2018, Vol. 14, No. 00 Appiah et al.

ACKNOWLEDGEMENTS [17] Awala, S.I.; Oyetayo, V.O. Molecular identity and antimicrobial profile of Trametes species Collected from the Teaching and Re- We are grateful to Organization for Women in Science search Farm of the Federal University of Technology, Akure, Nige- for Developing Countries (OWSD) and the Mwalimu Nyer- ria. J. Advan. Med. Pharmaceut. Sci., 2015, 4(3), 2394-1111. ere African Union Scholarship for TA. Special appreciation [18] Lara, M.A.; Rodríquez-Malaver, A.J.; Rojas, O.J.; Holmquist, O.; González, A.M.; Bullón, J.P., N.; Araujo, E. Black liquor lignin bio- to the staff and postgraduate students in the research labora- degradation by Trametes elegans. Int. Biodeter. Biodegr., 2003, 52, tory of YL, Chengdu Institute of Biology, Chinese Academy 167-173. of Sciences, China for their support and technical assistance. [19] Aina, D.; Olawuyi, O.; Mensah-Agyei, G.; Laiya, A.; Adeoye-Isijola, M. 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