The triterpenoid and steroid from the fruiting body of ganoderma applanatum (pers.) pat. in Viet Nam - Hoang Van Trung

Vietnam Journal of Science and Technology 56 (5) (2018) 550-556 DOI: 10.15625/2525-2518/56/5/12588 THE TRITERPENOID AND STEROID FROM THE FRUITING BODY OF Ganoderma applanatum (Pers.) Pat. IN VIET NAM Hoang Van Trung 1 , Nguyen Tan Thanh 1 , Nguyen Ngoc Tuan 2 , Doan Manh Dung 3 , Tran Dinh Thang 1, * 1 School of Chemistry, Biology and Environment, Vinh University, Vinh City 2 Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City, Ho Chi Minh City 3 Hue University of Science- Hue University, 77 Nguyen Hue, Hue City * Email: thangtd@vinhuni.edu.vn Received: 25 May 2018; Accepted for publication: 5 September 2018 Abstract. Five compounds, ergosterol (1), 5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol (2); ergosta-7,22-dien-3β-ol (3); lanosta-7,9(11),24-triene-3,26-diol (4) and 3β-hydroxy-5α-lanosta- 7,9,24(E)-trien-26-oic acid (5) were isolated from fruiting body of Ganoderma applanatum (Pers.) Pat. (Ganodermataceae). The structures of the isolated compounds were established by spectroscopic methods (MS, 1 H-NMR, 13 C-NMR, DEPT). Keywords: Ganoderma applanatum, Ganodermataceae, triterpenoid, steroid, lanosta-7,9(11),24- triene-3,26-diol. Classification numbers: 1.1.1; 1.1.6. 1. INTRODUCTION “Linh chi” - the Vietnamese name for Ganoderma can be used in the prevention and treatment of various types of disease. Various compounds have been isolated from the fruiting bodies, spores, gills, and mycelia of many Ganoderma mushrooms. Ganoderma contains many bioactive natural components, including triterpenes, steroid, polysaccharides, proteins, and unsaturated fatty acids [1-5]. It has been identified as new potent lead structures for the development of novel pharmaceuticals against infectious diseases, cancer, and other diseases. The majority of biological activities are its immunomodulatory and antitumor activities [1–8]. The majority of triterpenes and steroid of Ganoderma exhibit a wide range of biological activities, including antitumor, immunomodulatory, anti-HIV-1, antioxidant, antimicrobial antihypertensive, antiangiogenic, antiandrogenic, and antihepatitis B activities [9-12]. Ganoderma applanatum (Ganodermataceae) that has been widely used as a folk medicine for the treatment and prevention of various diseases [13]. Herein, we report the isolation of five compounds (ergosterol (1), 5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol (2); ergosta-7,22-dien- 3β-ol (3); lanosta-7,9(11),24-triene-3,26-diol (4) and 3β-hydroxy-5α-lanosta-7,9,24(E)-trien-26- Hoang Van Trung, Nguyen Tan Thanh, Nguyen Ngoc Tuan,Tran Dinh Thang 568 oic acid (5)) from fruiting body of Ganoderma applanatum (Pers.) Pat. (Ganodermataceae). The structures of these compounds were elucidated using a combination of 1D and 2D NMR techniques ( 1 H-, 13 C-NMR, HSQC and HMBC). 2. EXPERIMENTAL 2.1. General Melting points were recorded on MP55 Melting Point System; Optical rotations were measured using an AUTOPOL VI Automatic Polarimeter; 1D-NMR and spectra were obtained on the Bruker AV-III 500 NMR spectrometer; The electrospray ionization mass spectrometry (ESI-MS); Column chromatography was performed on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, E. Merck); Thin layer chromatography was conducted on precoated Kieselgel 60 F 254 plates (Merck); the compounds were visualized by spraying with 10 % (v/v) H2SO4 followed by heating at 110 °C for 10 min. 2.2. Fungal material In August 2015, the basidiomycetes of Ganoderma applanatum (Ganodermataceae) were collected at the Puhuong of Nghe An Province, Vietnam. It was identified by Prof. Dr. Ngo Anh, Department of Biology, Hue University. A voucher specimen (Vinh-TSWu 20150815) was deposited at the herbarium of the School of Chemistry, Biology and Environment, Vinh University. 2.3. Extraction and isolation The dried basidiomycetes (3.0 kg) of Ganoderma applanatum were extracted with MeOH to give a deep brown crude (128 g) after concentration in vacuo. The crude extract was suspended in water and subjected to a liquid/liquid partition using ethyl acetate and butanol to afford ethyl acetate (32 g), butanol (33 g) and water soluble (55 g) fractions. The crude ethyl acetate were applied to silica gel column chromatography using gradient of hexane and acetone increasing polarity (100:0 to 2:1) to afford minor fraction s, and were monitored by TLC to combine into ten major fractions (Frs. G1-G10). Purification of fraction G1 (1.2 g) by the silica gel column chromatography eluting with a mixture of hexane and acetone (15:1) afforded compound 1 (123 mg). G3 (2.6 g) was subjected to the silica gel column chromatography eluting with a hexane and ethyl acetate solvent mixture (15:1) to yield compound 4 (38 mg). G4 (2.5 g) was subjected to the silica gel column chromatography eluting with a mixture of hexane and acetone (9:1) to produce compound 2 (10 mg) and 3 (31 mg). G6 (2.9 g) was subjected to the silica gel column chromatography eluting with chloroform and methanol solvent mixture (10:1) and further recrystallization of the minor fraction to afford compound 5 (30 mg). Compound 1, ergosterol: white powder, m.p. 166-167 o C; EI-MS m/z 396 [M] + ; 1 H-NMR (500MHz, CDCl3, ppm): 5.49 (1H, m, H-7), 5.35 (1H, m, H-6), 5.28 (1H, dd, J = 15.5, 7.5 Hz, H-22), 5.25 (1H, dd, J = 15.5, 7.0 Hz, H-23), 3.48 (1H, m, H-3), 1.05 (3H, d, J = 7.0 Hz, H-28), 0.96 (3H, s, H-19), 0.93 (3H, d, J = 7.0 Hz, H-27), 0.85 (3H, d, J = 6.5 Hz, H-26), 0.82 (3H, d, J = 6.5 Hz, H-21), 0.61 (3H, s, H-18); 13 C-NMR (125MHz, CDCl3, ppm): 11.5 (C-18), 15.8 (C- 19), 17.0 (C-28), 19.1 (C-21), 19.4 (C-26), 20.4 (C-27), 20.6 (C-11), 22.2 (C-15), 27.4 (C-16), 31.5 (C-2), 32.2 (C-25), 37.7 (C-10), 38.2 (C-12), 39.0 (C-1), 40.0 (C-20), 40.4 (C-4), 41.8 (C- 24), 42.2 (C-13), 45.5 (C-9), 53.6 (C-14), 55.0 (C-17), 68.3 (C-3), 115.8 (C-7), 118.2 (C-6), 131.2 (C-22), 135.0 (C-23), 139.8 (C-8), 140.3 (C-5). The triterpenoid and steroid from the fruiting body of Ganoderma applanatum (Pers.) Pat. in VN 569 Compound 2, 5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol: white powder, m.p.: 177-178 oC; EI-MS m/z 428 [M] + ; 1 H-NMR (500MHz, CDCl3, ppm): 6.44 (1H, d, J = 8.5 Hz, H-7), 6.23 (1H, d, J = 8.5 Hz, H-6), 5.28 (1H, m, H-22), 5.18 (1H, m, H-23), 3.58 (1H, m, H-3), 1.05 (3H, d, J = 6.5 Hz, H-21), 0.96 (3H, d, J = 7.0 Hz, H-28), 0.95 (3H, s, H-19), 0.89 (3H, d, J = 6.5 Hz, H-27), 0.87 (3H, s, H-18), 0.87 (3H, d, J = 6.5 Hz, H-26); 13 C-NMR (125MHz, CDCl3, ppm): 12.5 (C-18), 17.2 (C-28), 17.9 (C-19), 19.4 (C-26), 19.7 (C-27), 20.2 (C-21), 21.7 (C-11), 22.8 (C-15), 28.2 (C-16), 29.9 (C-2), 32.4 (C-25), 34.5 (C-1), 36.5 (C-10), 36.9 (C-4), 38.7 (C-12), 40.1 (C-20), 42.0 (C-24), 44.0 (C-13), 50.9 (C-9), 51.2 (C-14), 55.4 (C-17), 64.6 (C-3), 78.4 (C- 8), 81.4 (C-5), 130.1 (C-7), 131.5 (C-23), 135.2 (C-6), 135.6 (C-22). Compound 3, ergosta-7,22-dien-3β-ol: colorless needles; [α]D 20 -5 (c = 0.85, CHCl3); m.p. 185.5-187 °C ; EI-MS (rel. int.): m/z 398([M] + , 4), 217(8), 255(9), 107(15), 69(24), 43(100); IR (KBr) νmax: 3341, 2951, 2928, 2870, 1663, 1458, 1377, 1044, 970 cm -1 ; 1 H-NMR (CDCl3, 500 MHz) (δ ppm): 5.22 (1H, dd, J =15.0, 7.5 Hz, H-22), 5.16 (1H, m, H-7), 5.19 (1H, dd, J =15.0, 8.0 Hz, H-23), 3.59 (1H, m, H-3), 1.01 (3H, d, J = 7.0 Hz, H-21), 0.91 (3H, d, J = 7.0 Hz, H-28), 0.83 (3H, d, J = 7.5 Hz, H-27), 0.80 (3H, d, J = 8.0 Hz, H-26), 0.79 (3H, s, H-19), 0.55 (3H, s, H-18); 13 C-NMR (CDCl3, 125 MHz) (δ ppm):139.6 (C-8), 135.7 (C-22), 131.9 (C-23), 117.5 (C- 7), 71.1 (C-3), 56.0 (C-17), 55.1 (C-14), 49.5 (C-9), 43.3 (C-13), 42.8 (C-24), 40.5 (C-20), 40.3 (C-5), 39.5 (C-12), 38.0 (C-4), 37.2 (C-1), 34.2 (C-10), 33.1 (C-25), 31.5 (C-2), 29.7 (C-6), 28.1 (C-16), 22.9 (C-15), 21.6 (C-11), 21.1 (C-21), 20.0 (C-27), 19.7 (C-26), 17.6 (C-28), 13.0 (C- 19), 12.1 (C-18). Compound 4, lanosta-7,9(11),24-triene-3,26-diol: white powder, m.p. 171-172 0 C. EI-MS m/z 440 [M] + ; EI-MS m/z 412 [M-H2O] + (13), 397(6), 394(19), 383(10), 379(21), 376(15), 269(15), 251(57), 69(100); 1 H-NMR (500 MHz, CDCl3) (δ ppm): 5.46 (1H, s, H-7); 5.32 (1H, d, J = 13.5 Hz, H-11); 5.32 (1H, d, J = 13.5 Hz, H-24); 3.76(2H, d, J = 4.0 Hz, H-26); 3.00 (1H, d, J = 3.5Hz, H-3); 2.15(1H, s); 2.08 (1H, s, H-12); 2.08 (3H, s, H-19); 2.08(3H, s, H-29); 1.54 (1H, s, H-17); 1.54 (3H, s, H-27); 1.54 (3H, s, H-28);1.36 (2H, m, H-15); 1.36 (2H, m, H-1); 1.27 (2H, m, H-2); 1.27 (2H, m, H-20); 1.05 (2H, d, J = 8.5Hz, H-22); 0.98 (1H, d, J = 8.5Hz, H- 5); 0.91 (2H, s, H-6); 0.91 (2H, s, H-16); 0.91 (3H, s, H-21); 0.84 (2H, s, H-23); 0.77 (3H, s, H- 30); 0.53 (3H, s, H-18); 13 C-NMR (125 MHz, CDCl3) (δ ppm): 142.7(C-8); 146.0 (C-9); 134.4(C-25); 127.0(C-24); 120.2(C-7); 116.3(C-11); 79.0(C-3); 69.1(C-26); 50.9(C-17); 50.3(C- 14); 49.2(C-5); 43.8(C-13); 37.9(C-12); 38.7(C-4); 35.9(C-22); 35.7(C-1); 37.4(C-10); 36.1(C- 20); 31.5(C-15) ;27.9(C-16); 27.8(C-2); 25.6(C-28); 22.8(C-19); 24.6(C-23); 28.2(C-29); 23.0(C-6); 15.8(C-30); 15.7(C-18); 18.4(C-21); 13.7(C-27). Compound 5, 3β-hydroxy-5α-lanosta-7,9,24(E)-trien-26-oic acid: colorless needles; m.p. 243-244 o C; ESI-MS m/z 453 [M-H] - ; 1 H-NMR (500 MHz, CDCl3, δ ppm): 6.75 (1H, t, J = 7.0 Hz, H-24); 5.42 (1H, s, H-7); 5.26 (1H, s, H-11); 3.18 (1H, dd, J = 4.7, 11.2 Hz, H-3); 1.76 (3H, s, H-27); 0.93 (3H, s, H-19); 0.92 (3H, s, H-29); 0.87 (3H, d, J = 5.6 Hz, H-21); 0.81 (6H, s, H- 18, H-28); 0.50 (3H, s, H-30); 13 C-NMR (125 MHz, CDCl3) (δ ppm): 170.9 (C-26); 146.0 (C- 9); 144.0 (C-24); 142.6 (C-8); 127.2 (C-25); 120.3 (C-7); 116.2 (C-11); 78.8 (C-3); 50.9 (C-17); 50.3 (C-14); 48.7 (C-5); 43.8 (C-13); 38.7 (C-4); 37.8 (C-12); 37.4 (C-10); 36.2 (C-20); 35.8 (C- 1); 34.9 (C-22); 31.5 (C-16); 28.1 (C-29); 27.9 (C-2); 27.5 (C-15); 25.8 (C-23); 25.5 (C-28); 23.0 (C-6); 22.7 (C-19); 18.3 (C-21); 15.7 (C-18); 15.8 (C-30); 12.1 (C-27). 3. RESULTS AND DISCUSSION The dried fruiting bodies of Ganoderma applanatum were powdered and refluxed with methanol, and the resulted extracts were partitioned with ethyl acetate and butanol to afford Hoang Van Trung, Nguyen Tan Thanh, Nguyen Ngoc Tuan,Tran Dinh Thang 570 ethyl acetate and butanol fractions successively. The ethyl acetate layer was subjected into purification by a repeated column chromatography to result in five compounds ergosterol (1), 5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol (2), ergosta-7,22-dien-3β-ol (3), lanosta- 7,9(11),24-triene-3,26-diol (4), 3β-hydroxy-5α-lanosta-7,9,24(E)-trien-26-oic acid (5) (Figure 1), respectively. Compound 1 was obtained as white powder. The molecular formula of 1 was established through the EI-MS analysis. The EI-MS showed a pseudomolecular ion peak at m/z 396 [M] + (suggesting to a molecular formula of C28H44O), indicating seven indices of hydrogen deficiency. The 1 H-NMR exhibited the signals of one oxygenated methine proton [ H 3.48 (H- 3)], six methyl groups [ H 1.05 (H-28), 0.96 (H-19), 0.93 (H-27), 0.85 (H-26), 0.82 (H-21), and 0.61 (H-18)], and four olefinic protons [ H 5.28 (1H, dd, J = 15.5, 7.5 Hz, H-22), 5.25 (1H, dd, J = 15.5, 7.0 Hz, H-23), 5.35 (1H, m, H-6), and 5.49 (1H, m, H-7)]. Moreover, the 13 C-NMR spectrum displayed the signals of 28 carbons, including six olefinic carbons [ C 115.8 (C-7); 118.2(C-6); 131.2(C-22); 135.0 (C-23); 139.8(C-8) and 140.3(C-5)] and one oxygenated carbon [ C 68.3 (C-3)]. According to the UV, IR, EI-MS, 1 H-, 13 C-NMR, DEPT spectral analysis and comparison of the spectral data between 1 and ergosterol, the structure of 1 was identified as ergosterol [14,15]. Compound 2 was obtained as white powder. The 1 H-NMR displayed the presence of one oxygenated methine proton [ H 3.58 (H-3)], six methyl groups [ H 1.05 (H-21), 0.96 (H-28), 0.95 (H-19), 0.89 (H-27), 0.87 (H-18), 0.87 (H-26)], and four olefinic protons [ H 6.23 (H-6), 6.44 (H-7), 5.28 (H-22) and 5.18 (H-23)]. The 13 C-NMR and DEPT spectrum of 2 displayed signals for six methyl carbons, seven methylens, eleven methines, and four quaternary carbons. In addition, the 13 C-NMR spectrum of compound 2 showed signals consistent with the presence of three oxygenated carbons [δC 81.4 (C-5); 78.4 (C-8); 64.6 (C-3)] and four olefnic carbons [ C 135.6 (C-22) and 131.5 (C-23); 135.2 (C-6) and 130.1 (C-7)], which were similar to those observed for compound 1, except for the absence of one C=C double bond. Notably, these spectroscopic data were consistent with those reported in the literature for a known compound 5α,8 -epidioxyergosta-6,22-dien-3 -ol [16]. Compound 3 was obtained as optically active white powder. The 1 H-NMR spectrum of 3 showed clear signals for six methyl groups [ H 0.55 (3H, s), 0.79 (3H, s), 0.80 (3H, d, J = 8,0 Hz), 0.83 (3H, d, J = 7,5), 0.91 (3H, d, J = 7.0 Hz) và 1.01 (3H, d, J = 7.0 Hz)], a carbinol proton [ H 3.59 (1H, m)] and three olefinic protons [ H 5.22 (1H, dd, J = 15.0, 7.5 Hz), 5.19 (1H, dd, J = 15.0, 8.0 Hz) and 5.16 (1H, m)]. Additionally, the 13 C-NMR displayed signals of 28 carbons, including 4 olefinic carbons ( C 117.5, 139.6, 131.9 and 135.7) and one oxygenated carbon ( C 71.1). Based on the above evidence, the chemical structure of 3 was established as ergosta-7,22-dien-3-ol [17,18]. It was reported from Coriolus sanguineus, Fomes sp., Polyporus sp... Compound 4 was obtained as white powder. The molecular formula of 4 was established through the EI-MS analysis. The EI-MS showed a pseudomolecular ion peak at m/z 440 [M] + , combination with 1 H- and 13 C-NMR spectral data allowed to propose the molecular formula of C30H48O2. The 1 H-NMR spectrum showed signals due to three olefinic protons [ H 5.46 (1H, s, H-7); 5.32 (1H, d, J = 13.5Hz, H-11); 5.32(1H, d, J = 13.5 Hz, H-24)], and one oxygenated methine proton [ H 3.00 (1H, d, J = 3.5Hz, H-3)]. Additionally, its 1 H-NMR showed signals due to protons for six methyl groups [ H 2.08 (3H, s, H-29); 2.08 (3H, s, H-19); 1.54 (3H, s, H-27); 0.91 (3H, s, H-21); 0.77 (3H, s, H-30); 0.53 (3H, s, H-18)]. The 13C-NMR and DEPT spectra of 4 exhibited 30 carbon signals, corresponding to a triterpenoid (including 7 methyl, 6 methine, The triterpenoid and steroid from the fruiting body of Ganoderma applanatum (Pers.) Pat. in VN 571 10 methylene and 7 nonprotonated carbons). Its 13 C-NMR showed the typical signals of olefinic carbons [ C 142.7(C-8); 146.0 (C-9); 134.4(C-25); 127.0(C-24); 120.2(C-7); 116.3(C-11)]. Compounds 4 was identified as lanosta-7,9(11),24-triene-3,26-diol by comparison of its physical and spectroscopic properties with those reported in the literature [8]. This compound has been isolated from Ganoderma lucidum [9]. Compound 5 was obtained as white powder. The EI-MS of 5 showed a pseudomolecular ion peak at m/z 454 [M] + , combination with 1 H- and 13 C-NMR spectral data allowed to propose the molecular formula of C30H46O3. In its 1 H-NMR spectrum, there were proton signals characteristic of three olefinic protons [ H 5.47 (1H, d, H-7); 6.91 (1H, t, H-24) and 5.32 (1H, d, H-11)]. In the downfield region, one oxygenated proton at δH 3.26 (1H, d, H-3) suggested the C- 3 hydroxylation. Moreover, the 1 H-NMR spectrum displayed signals of seven methyl groups [ H 1.00 (H-29); 0.98 (H-19); 0.88 (H-28); 0.88 (H-30); 0.57 (H-18), 1.84 (H-27), 0.92 (H-21)]. There were proton signals characteristic of the triterpenoid basic skeleton (lanostane). The 13 C- NMR and DEPT spectrum of 5 displayed characteristic signals for 30 carbons, including: seven methyl, eight methylens, seven methines and seven quaternary carbons. In its 13 C-NMR spectrum, there were signals characteristic of olefinic carbons [ C 146.0 (C-9); 143.0 (C-8); 146.0 (C-25); 127.0 (C-26); 120.3 (C-7); 116.2 (C-11)], seven carbon methyl groups [ C 16.0 (C-30); 28.1 (C-29); 26.0 (C-28); 22.7 (C-19); 18.3 (C-21); 16.0 (C-18); 12.0 (C-27)]. Notably, these spectroscopic data were consistent with those reported in the literature for a known compound 3β -hydroxy- 5α-lanosta 7,9,24 (E)-trien-26-oic acid. The compound 5 defined as 3β- hydroxy-5α-lanosta 7,9,24 (E)-trien-26-oic acid. This compound has been isolated from Ganoderma lucidum [19]. (1) Ergosterol (2) 5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol (3) Ergosta-7,22-dien-3β-ol (4) lanosta-7,9(11),24-triene-3,26-diol (5) 3β-hydroxy-5α-lanosta-7,9,24(E)-trien-26-oic acid 4. CONCLUSION Finally, we have succeeded in studying chemical composition of the Ganoderma applanatum (Pers.) Pat. and resulted in the identification of five compounds, including ergosterol (1), 5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol (2); ergosta-7,22-dien-3β-ol (3); lanosta-7,9(11),24-triene-3,26-diol (4) and 3β-hydroxy-5α-lanosta-7,9,24(E)-trien-26-oic acid (5). The structure elucidation of the five compounds were determined on the basis of MS and NMR spectrometric methods. Hoang Van Trung, Nguyen Tan Thanh, Nguyen Ngoc Tuan,Tran Dinh Thang 572 REFERENCES 1. Isaka M., Chinthanom P., Kongthong S., Srichomthong K. and Choeklin R. - Lanostane triterpenes from cultures of the basidiomycete Ganoderma orbiforme BCC 22324. Phytochemistry 87 (2013) 133–139. 2. Yuen J. W. M. and Gohel M. D. I. - Anticancer effects of Ganoderma lucidum: A review of scientific evidence, Nutr. Cancer 53 (2005) 11–17. 3. El-Mekkawy S., Meselhy M. 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