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).
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).