Lentinus tigrinus, also known as tiger sawgill or Kabuteng tigre is an edible saprobic mushroom of the family Polyporaceae which grows on fallen logs in the forest.1-2 It is considered one of the most common rotting basidiomycetes yet very pleasing to the eye because of its color and appearance.3 A study reported that the pileus of L. tigrinus contained higher amounts of protein (25.9%), fat (2.1%), ash (7.4%) and energetic value (142.1 kcal/100 g) than the stipe, while the stipe contained higher amounts of total carbohydrates (67.7%) than the pileus. The lyophilized hot water extract of the fruiting body of L. tigrinus (100 and 250 mg/kg) significantly lowered the glucose level of diabetic mice by 26.9%. The ethanolic extract of the fruiting body and secondary mycelia of L. tigrinus exhibited high antibacterial activities against Staphylococcus aureus.1 Another study reported that a 59 kDa laccase which was isolated from the broth of mycelial culture of the mushroom L. tigrinus exhibited an inhibitory activity against HIV-1 reverse transcriptase (IC50 = 2.4 µM).4 Furthermore, L. tigrinus acetonitrile extract exhibited a 39.2% radical scavenging activity with an EC50 value of 637.75 mg/L, contained 451 mg ascorbic acid equivalent/g sample total phenolics, and showed antibacterial activity against Staphylococcus aureus with a 9.48 mm diameter zone of inhibition.5 In addition, L. tigrinus also exhibited hyper accumulation potentials with the ability to concentrate varying amounts of lead, cadmium and chromium (9.21, 1.52 and 4.29 ppm, respectively).3,6 In another study, L. tigrinus n-hexane extract was reported to contain flavonoid (3.67 μg/mg) and exhibited antibacterial activity.7
We report herein the isolation of cerevisterol (1), stellasterol (2) and ergosterol (3) from the dichloromethane extract of the fruiting bodies of L. tigrinus (Figure 1). To the best of our knowledge, this is the first report on the isolation of 1-3 from the fruiting bodies of L. tigrinus.
MATERIALS AND METHODS
General Experimental Procedure
The NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl3 at 600 MHz for 1H NMR and 150 MHz for 13C NMR spectra. Column chromatography was performed with silica gel 60 (70-230 mesh). Thin layer chromatography was performed with plastic backed plates coated with silica gel F254. The plates were visualized by spraying with vanillin/H2SO4 solution followed by warming.
Fruiting bodies of L. tigrinus was initially detached from the rotten stumps and logs found within the vicinity of Mt. Makiling, Laguna, Philippines from June to July 2017. The collected specimens were cleaned, placed in a paper bag and was brought to the laboratory for analysis. Authentication of the identity of the collected L. tigrinus was done by one of the authors (MEDC) using available published literature and pictorial guides.
Isolation of the Sterols from L. tigrinus
The fruiting bodies of Lentinus tigrinus (140 g) were freeze-dried (30.14 g), then ground in a blender, soaked in CH2Cl2 for three days, and filtered. The filtrate was concentrated under vacuum to afford a crude extract (0.426 g) which was chromatographed by gradient elution using increasing proportions of acetone in CH2Cl2 at 10% increment by volume. The CH2Cl2 fraction was rechromatographed using 15% EtOAc in petroleum ether to afford a mixture of 2 and 3 (77.6 mg) after washing with petroleum ether. The 70% acetone in CH2Cl2 fraction was rechromatographed using CH3CN:Et2O:CH2Cl2 (2.5:2.5:5, v/v) to provide 1 (3.9 mg) after washing with petroleum ether.
Cerevisterol (1): 1H-NMR (600 MHz, CDCl3): δ 5.33 (d, J = 5.4 Hz, H-7), 5.20 (dd, J = 7.2, 15 Hz, H-23), 5.16 (dd, J = 7.2, 15 Hz, H-22), 4.06 (m, H-3), 3.60 (m, H-6), 1.07 (s, H-19), 1.00 (d, J = 6.6 Hz, H-21), 0.90 (d, J = 7.2 Hz, H-28), 0.82 (d, J = 6.6 Hz, H-27), 0.80 (d, J = 6.6 Hz, H-26), 0.58 (s, H-18); 13C-NMR (150 MHz, CDC13): δ 32.96 (C-1), 30.857 (C-2), 67.73 (C-3), 39.22 (C-4), 75.97 (C-5), 73.66 (C-6), 117.53 (C-7), 144.00 (C-8), 43.47 (C-9), 37.14 (C-10), 22.04 (C-11), 39.22 (C-12), 43.76 (C-13), 54.75 (C-14), 22.88 (C-15), 27.90 (C-16), 55.99 (C-17), 12.33 (C-18), 18.82 (C-19), 40.39 (C-28), 19.64 (C-21), 135.36 (C-22), 132.18 (C-23), 42.81 (C-24), 33.07 (C-25), 19.94 (C-26), 21.11 (C-27), 17.58 (C-28).
Stellasterol (2): 1H-NMR (600 MHz, CDCl3): δ 0.52 (s, H-18), 0.80 (d, J = 6.6 Hz, H-26), 0.82 (d, J = 7.2 Hz, H-27), 0.77 (s, H-19), 0.90 (d, J = 6.6 Hz, H-28), 0.99 (d, J = 6.6 Hz, H-21), 3.58 (m, H-3), 5.13 (br t, J = 2.4 Hz, H-7), 5.20 (dd, J = 7.8, 15.0 Hz, H-23), 5.17 (dd, J = 7.8, 15.0 Hz, H-22); 13C-NMR (150 MHz, CDCl3): δ 37.13 (C-1), 31.46 (C-2), 71.06 (C-3), 37.97 (C-4), 40.46 (C-5), 29.63 (C-6), 117.45 (C-7), 139.56 (C-8), 49.45 (C-9), 34.21 (C-10), 21.53 (C-11), 39.44 (C-12), 43.30 (C-13), 55.10 (C-14), 22.92 (C-15), 28.09 (C-16), 55.96 (C-17), 12.08 (C-18), 13.02 (C-19), 40.26 (C-20), 21.10 (C-21), 135.66 (C-22), 131.88 (C-23), 42.81 (C-24), 33.08 (C-25), 19.63 (C-26), 19.94 (C-27), 17.59 (C-28).
Ergosterol (3): 1H-NMR (600 MHz, CDC13): δ 5.55 (dd, J = 2.4, 5.4, Hz, H-6), 5.36 (dd, J = 2.4, 5.4 Hz, H-7), 5.20 (dd, J = 7.8, 15.0 Hz, H-23), 5.17 (dd, J = 7.8, 15.0 Hz, H-22), 3.61 (m, H-3), 1.01 (d, J = 6.6 Hz, H-21), 0.92 (s, H-19), 0.90 (d, J = 6.6 Hz, H-28), 0.82 ( d, J = 7.2 Hz, H-26), 0.80 (d, J = 6.6 Hz, H-27), 0.61 (s, H-18). 13C-NMR (150 MHz, CDC13): δ 38.36 (C-1), 31.98 (C-2), 70.46 (C-3), 40.78 (C-4), 139.77 (C-5), 119.58 (C-6), 116.28 (C-7), 141.35 (C-8), 46.25 (C-9), 37.02 (C-10), 21.10, 21.09 (C-11, C-21), 39.08 (C-12), 42.82, 42.81 (C-13, C-24), 54.55 (C-14), 22.99 (C-15), 28.27 (C-16), 55.73 (C-17), 12.04 (C-18), 16.27 (C-19), 40.40 (C-20), 135.56 (C-22), 131.97 (C-23), 33.08 (C-25), 19.63 (C-26), 19.94 (C-27), 17.59 (C-28).
RESULTS AND DISCUSSION
Silica gel chromatography of the dichloromethane extract of the fruiting bodies of Lentinus tigrinus yielded cerevisterol (1), stellasterol (2) and ergosterol (3). The NMR data of 1 are in accordance with the data reported in the literature for cerevisterol;8-9 2 for stellasterol;10 and 3 for ergosterol.8
Although bioassays were not conducted on the isolated compounds, there were previous studies that reported on their biological activities. Cerevisterol (1) and ergosta4,6,8(14),22-tetraen-3-one which were isolated from P. tuber-regium were tested for anti-inflammatory effects on RAW 264.7 macrophages. Both sterols inhibited the production of NO, TNF-α, and PGE2 in LPS-treated RAW 264.7 cells. These compounds also inhibited the expression of the iNOS and COX2 proteins in a dose-dependent manner and repressed the expression of iNOS, COX2, TNF-α, and SOCS3 mRNAs.11 Stellasterol (2) showed antibiotic activity against Gram positive bacteria.12 Another study reported that cell cycle arrest against the human cancer cell lines, MCF-7 and SH-SY5Y was exhibited by stellasterol.13 Furthermore, 2 showed anti-inflammatory activity against iNOS, CHOP and IKB-α expression.14 A study reported that ergosterol (3) provided significant protection against the promotion of bladder tumor induced by many types of promoters in the environment.15 Moreover, the ergosterol content of brown and white button mushrooms correlated with their antioxidant activities.16 In another study, 1 was reported to have the capability to inhibit lipid peroxidation.17
The dichloromethane extract of the fruiting bodies of L. tigrinus afforded cerevisterol (1), and a mixture of stellasterol (2) and ergosterol (3). To the best of our knowledge, this is the first report on the isolation of 1-3 from the fruiting bodies of L. tigrinus. Earlier studies on L. tigrinus extracts showed antibacterial properties. This may be partly attributed to stellasterol (2) which was reported to exhibit antibacterial properties. Furthermore, an earlier study on L. tigrinus extract indicated antioxidant properties. Ergosterol (3) which was reported to exhibit antioxidant properties may contribute to this bioactivity. Additionally, 1 and 2 were reported to possess anti-inflammatory activity, while 2 and 3 showed anticancer properties.