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 Trametes 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 Polyporaceae [15]. T. Mushrooms have been of interest recently due to a num- gibbosa and T. elegans are saprobic polypores, 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/ethyl acetate (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