Mushroom species verification
AP fruiting bodies were collected from a Chang Daeng mushroom farm in Prapradaeng, Samutprakarn, Thailand. The identity of the mushroom was confirmed by the DNA sequence similarity of the internal transcribed spacer (ITS) region of ribosomal RNA [15]. DNA was extracted using the method reported by Luangsuphabool et al. [16]. The extracted DNA was submitted to Bioneer sequencing service (Bioneer Corporation, Korea) [17] for polymerase chain reaction (PCR) amplification using primer pair ITS1/ITS4, then analyse the DNA sequence. The mushroom species was identified by comparing nucleotide sequences database on GenBank.
Crude extracts preparation
The sun-dried A. polytricha (AP) was grounded into a powder. Macerations were performed in sequential steps to provide the three crude extracts. AP powder (1 kg) was first macerated twice with hexane (10 L) in an incubator shaker at 225 rpm, at room temperature for 72 h. The combined hexane layers were filtered, and the solvent was evaporated under reduced pressure at 40 °C to give a crude hexane extract (APH) as a thick yellow paste (3.90 g). The dry residue from step 1 was extracted twice with ethanol (10 L) under the same conditions to give the crude ethanol extract (APE) as a dark purple thick paste (4.74 g). Lastly, the dry residue from step 2 was extracted with water (10 L) with stirring at 4 °C for 72 h. The combined water extracts were filtered, and water was removed by freeze-drying to give crude water extract (APW) as a thick brown paste (19.88 g).
HIV-1 protease inhibitor assay
AP extracts (1 mg/ml) were evaluated using HIV-1 Protease Inhibitor Screening Kit (Fluorometric) (Biovision Incorporated, CA, USA). Pepstatin (1 mM) and DMSO (1%, v/v) were used as a positive control and solvent control, respectively. The assay was performed according to the manufacturer’s instruction. Then the HIV-1 protease fluorescent substrate was added and measured fluorescence (Excitation/Emission = 330/450 nm) in a kinetic-mode for 90 min at 37 °C using PerkinElmer EnSpire plate reader.
Cell culture
3 T3-L1 cells, normal mouse fibroblasts were maintained in DMEM supplemented with 10% (v/v) BCS at 37 °C in a humidified incubator with 5% of CO2.
Cytotoxicity assay
All extracts and their identified compounds were tested for toxicity on 3 T3-L1 cells by MTT method. The extracts were dissolved in 0.1% (v/v) of dimethyl sulfoxide (DMSO) at varying concentrations (0.03–1.00 mg/mL). The 3 T3-L1 cells (5 × 103 cells, 100 μl) were seeded in each well of 96-well plate overnight to let the cell settle on the plate. Then the cells were treated with the compounds (100 μl) for 24, 48 and 72 h. The DMSO at 0.1% (v/v) and the untreated cell conditions were used as vehicle control and normal control, respectively. At the end of each incubation periods, MTT reagent (20 μl) was added. After 3 hours of incubation, the supernatant was removed then the water-insoluble formazan was dissolved in DMSO (150 μl). The absorbance of the converted dye was measured at a wavelength of 570 nm. The results were reported in CC50 values, calculated by standard curve analysis of four parameters logistic in Sigma plot 12 software.
Statistical analysis
All experiments were performed in triplicates for each condition. The results were presented as the mean with the standard error of the mean (mean ± SEM) of three independent experiments. Statistic significant was analysed using one-way ANOVA following Dunnett’s test by SPSS 16.0 software. The P values less than 0.05 were considered statistically significant.
Purification of APH fraction
APH crude extract was analysed using TLC silica plate (silica gel 60 F254, Merck), using hexane/ethyl acetate (80:20, v/v, analytical grade) as mobile phases to reveal four separate spots, referring as fraction 1 (F1), fraction 2 (F2), fraction 3 (F3) and fraction (F4).
The APH crude extract (650.2 mg) was dissolved in ethyl acetate to induce the crystallisation of F4 from the mixture. The crystals (15.7 mg) were filtered and dried. The mother liquor was collected, and the solvent was removed to give the thick oil (634.5 mg) which was further purified using flash silica gel 60 column chromatography (hexane/ethyl acetate, 80:20, v/v) to give three fractions, F1 + F2 (392.0 mg), F3 (118.0 mg) and F4 (116.0 mg).
Preparative liquid chromatography (prep LC)
The pre-crystallisation APH crude mixture (450.0 mg) was purified by Reveleris® prep purification system with Reveleris® silica flash cartridge (24 g) using hexane/ethyl acetate (80:20, v/v) as a mobile phase at flow rate of 32 ml/min to give F1 + F2 (168.2 mg), F1 + F2 + F3 (142.3 mg), F3 (52.7 mg) and F4 (75.4 mg). The mixture of F1 + F2 (168.2 mg) was further purified by Reveleris® prep system using hexane/ethyl acetate (95:5, v/v) as a mobile phase to give F1 (149.9 mg) and F2 (2.2 mg). The mixture of F1 + F2 + F3 (20 mg) was purified by TLC plate silica gel 60 F254, using hexane/ethyl acetate (80:20, v/v) as a mobile phase. The band of F2 (1.2 mg) was purely isolated.
Compound identification
Isolated compounds were structurally elucidated using NMR, FTIR, HRMS and GC/MS techniques. The 1H (500 MHz), 13C (125 MHz) and 2D NMR: COSY and DEPT NMR spectra were recorded on a 500 MHz Agilent spectrometer in deuterated chloroform (CDCl3). The FTIR analyses were performed using Nicolet 6700 FTIR spectrometer (Thermo scientific). High-resolution mass spectra were obtained using an Agilent 6510 Q-TOF Mass Spectrometer (ESI). The GC/MS analyses were performed using Agilent 6870/5973n MS (EI) spectrometer. The peaks were identified using the mass spectral library available in the software.
Fraction 1 (F1)
Rf value (TLC): 0.88 (ethyl acetate/hexane, 20:80). HRMS (ESI): m/z 884.5865 [M]+ (calculated for C57H104O6 = 884.7833). FTIR (cm − 1): 2921.41, 2852.12 (C-H) and 1742.29 (C=O). 1H NMR δH (ppm): 5.349 (m, CH=CH), 5.264 (m, CH), 4.286 (dd, CH2), 4.151 (dd, CH2), 2.770 (t, =CH-CH2-CH=), 2.315 (t, CH2CO2), 2.042 (m, CH2CH=CH), 1.607 (m, CH2CH2CO2), 1.254 (m, CH2) and 0.881 (t, CH3). 13C NMR (CDCl3, 125 MHz) δC (ppm): 173.306, 173.261, 172.859 (C), 130.240, 130.039, 130.023, 129.697, 128.077, 127.910 (CH), 68.891 (CH), 62.106, 62.104 (CH2), 34.212, 34.068, 34.038, 31.940, 31.917, 31.537, 29.781, 29.720, 29.716, 29.675, 29.641, 29.625, 29.622, 29.542, 29.496, 29.375, 29.360, 29.345, 29.330, 29.288, 29.212, 29.193, 29.147, 29.136, 29.098, 29.064, 27.239, 27.213, 27.211, 27.190, 25.642, 24.898, 24.879, 24.849, 22.702, 22.694, 22.584 (CH2), 14.128, 14.082 (CH3).
Fraction 2 (F2)
Rf value (TLC): 0.61 (ethyl acetate/hexane, 20:80). HRMS (ESI): m/z 862.6072 [M]+ (calculated for C55H106O6 = 862.7989). FTIR (cm − 1): 2960.97, 2913.33, 2849.18 (C-H) and 1735.55 (C=O). 1H NMR δH (ppm): 5.349 (m, CH=CH), 5.264 (m, CH), 4.282 (dd, CH2), 4.148 (dd, CH2), 2.769 (t, =CH-CH2-CH=), 2.323 (t, CH2CO2), 2.040 (m, CH2CH=CH), 1.598 (m, CH2CH2CO2), 1.253 (m, CH2) and 0.880 (t, CH3).
Fraction 3 (F3)
Rf value (TLC): 0.44 (ethyl acetate/hexane, 20:80). HRMS (ESI): m/z 281.2468 [M + H]+ (calculated for C18H33O2 = 281.2481). FTIR (cm − 1): 2955.28, 2914.71, 2847.62 (C-H), 1699.69 (C=O), 1471.38, 1462.96 and 1429.69 (C=C). 1H NMR δH (ppm) 5.344 (m, CH=CH), 2.771 (t, =CH-CH2-CH=), 2.345 (t, CH2CO2), 2.042 (m, CH2CH=CH), 1.631 (m, CH2CH2CO2), 1.255 (m, CH2) and 0.880 (t, CH3).
Fraction 4 (F4)
Rf value (TLC): 0.27 (ethyl acetate/hexane, 20:80). HRMS (ESI): m/z 395.3303 [M-H]+ (calculated for C28H43O = 395.3314). GCMS (EI) m/z 396 [M]+. FTIR spectra (cm− 1) 3414.02 (O-H); 2952.17, 2928.38 and 2868.82 (C-H) and 1655.20 (C=C). 1H NMR spectra δH (ppm): 5.575 (dd, 1H), 5.385 (m, 1H), 5.205 (m, 1H), 3.639 (m, 2H), 2.459 (ddd, 2H), 2.284 (t, 2H); the position of this signal varied from 1.250–2.080 ppm in the other saturated methylene and methine protons (total 18H); 1.044 (d, 3H), 0.948 (s, 3H), 0.925 (d, 3H), 0.833 (t, 6H) and 0.632 (s, 3H). 13C NMR δC (ppm): 141.351, 139.769 (C), 135.551, 131.962, 119.573, 116.273, 70.457, 55.728, 54.555, 46.244 (CH), 42.830 (C), 42.815 (CH), 40.797 (CH2), 40.418 (CH), 39.082, 38.373 (CH2), 37.026 (C), 33.085 (CH), 31.997, 28.283, 22.991, 21.110 (CH2), 21.098, 19.949, 19.642, 17.601, 16.281 and 12.047 (CH3).
Base hydrolysis of fraction 1 (F1)
Methanolic sodium hydroxide solution (0.5 M, 5 mL) was added to F1 (110 mg). The mixture was heated with stirring at 90 °C for 10 min. The reaction was cooled in an ice bath, then methanol (5 mL) was added. The resulting mixture was heated at 90 °C for 10 min. Upon cooling, the pH of the reaction mixture was adjusted to zero by adding 1 M of HCl (10 mL). The acidic solution was extracted with dichloromethane (3 × 15 mL). The combined extracts were dried over anhydrous sodium sulfate and filtered. The dichloromethane was removed under reduced pressure to give a crude product (107 mg), which was analysed by GC/MS.