Diterpenoids from the wood of Podocarpus neriifolitus - Nguyễn Hoàng Sa

Vietnam Journal of Chemistry, International Edition, 54(4): 488-490, 2016 DOI: 10.15625/0866-7144.2016-00352 488 Diterpenoids from the wood of Podocarpus neriifolitus Nguyen Hoang Sa 1 , Nguyen Thanh Tam 2 , Nguyen Thi Hoang Anh 2 , Dao Duc Thien 2 , Tran Duc Quan 2 , Dinh Thi Phong 3 , Le Quoc Thang 4 , Tran Van Sung 2 , Trinh Thi Thuy 2* 1 Nha Trang College of Education, 01 Nguyen Chanh road, Nhatrang, Khanhhoa 2 Institute of Chemistry, Vietnam Academy of Science and Technology (VAST), 3 Vietnam National Museum of Nature, VAST 4 Hue University’s College of Educations, 34 Le Loi, Hue City Received 22 July 2016; Accepted for publication 12 August 2016 Abstract Using combined chromatographic methods, three known diterpenoids, inumakiol D (1), totarol (2) and totarol-19- carboxylic acid (3) along with β-sitosterol and β-sitosterol glucoside were isolated from the ethyl acetate extract of the wood of Podocarpus neriifolius collected in Lam Dong province, Vietnam. Their structures were determined by MS, 1D-, 2D-NMR data analysis and comparison with published references. This is the first report of compounds 1-3 from this plant. Keywords. Podocarpus neriifolius, diterpenoid, inumakiol D, totarol, totarol-19-carboxylic. 1. INTRODUCTION Podocarpus neriifolius D. Don - “Thông tre lá dài”- (Podocarpaceae) is tree with straight and round trunk, growing up to 20 - 25m high. It was sparsely distributed in primary forest areas in northern Vietnam as Nghean, Hatinh, Yenbai, Tuyenquang provinces ... and found at an altitude of 2300 m in the Bidoup Nui Ba National Park, Lamdong province. A decoction of the leaves of this plant is used in Vietnamese traditional medicine for the treatment of rheumatism and joint pain [1]. There have been some reports on the presence of flavonoids, diterpenoids in this plant [2-4]. In our research on the chemical constituents of Pinales species, three diterpenoids, inumakiol D (1), totarol (2) and totarol-19-carboxylic acid (3) and along with β-sitosterol and β-sitosterol glucoside were isolated from the ethyl acetate extract of the wood of P. neriifolius. 2. EXPERIMENTAL 2.1. General 1 H-NMR (500 MHz) and 13 C-NMR (125 MHz) were taken on a Bruker Avance AM500 spectrometer using TMS as internal standard for 1 H and solvent signal for 13 C. ESI-MS was taken on an Agilent 1100 LC-MSD Trap spectrometer. Merck TLC aluminum sheets with silica gel 60 F254 (layer thickness 0.2 mm) were used. Column chromatography (CC) was carried out on silica gel Merck 60 (0.040-0.063 mm) and Sephadex LH-20. 2.2. Plant Material Podocarpus neriifolius was collected in the Bidoup Nui Ba National Park in Lam Dong province, Vietnam in January, 2013 and identified by Dr. Nguyen Tien Hiep. A voucher specimen is deposited in the Vietnam National Museum of Nature Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam. 2.3. Extraction and Isolation The dried and powdered wood of Podocarpus neriifolius (1.4 kg) was extracted with methanol: water (95:5 w/w) for 24 h, three times. After concentration under reduced pressure, the crude extract was suspended in water and sequentially partitioned with n-hexane, ethyl acetate and n-butanol. The organic solvents were evaporated to VJC, 54(4) 2016 Trinh Thi Thuy, et al. 489 yield the corresponding extracts (0.45 g, 15.0 g and 13.2 g), respectively. The ethyl acetate residue (15.0 g) was subjected to silica gel column, eluting with solvent system n- hexane-CH2Cl2-MeOH (increasing amounts of CH2Cl2 from 70 to 100 % and MeOH from 0 to 35%) to yield 18 fractions (F1-F18). The fraction F3 (350 mg) was rechromatographed over a flash silica gel column using n-hexane-EtOAc (gradient from 99:1 to 85:15) as eluant to give compound 2 (33 mg). The fraction F9 (350 mg) was rechromatographed on silica gel column, eluted with hexane-EtOAc (8:2) to give 3 (15 mg). The fraction F14 (325 mg) was further separated by column chromatograph on silica gel eluted with CH2Cl2- MeOH (gradient from 100:1 to 80:20) to give seven subfractions (F14.1-F14.7). The subfraction F14.3 was purified on Sephadex LH-20 column eluting with MeOH to afford compound 1 (9 mg). The fraction F7 (419 mg) was rechromatographed on silica gel column, eluted with hexane-EtOAc (9:1) to give β-sitosterol (25 mg). The fraction F18 was repeatedly separated on Sephadex LH-20 column, eluted with MeOH to yield β-sitosterol glucoside (12 mg). NMR spectral data of β-sitosterol and β-sitosterol glucoside are in good agreement with those of published data [5, 6]. Inumakiol D (1): Yellow solid. ESI-MS m/z 355.3 [M + Na] + . 1 H-NMR (500 MHz, CDCl3 & CD3OD): δH 6.99 (1H, d, J = 8.5 Hz), 6.68 (1H, d, J = 8.5 Hz), 4.99 (1H, br s), 3.53-3.50 (1H, m), 1.42 (3H, d, J = 7.0 Hz), 1.37 (3H, d, J = 7.0 Hz), 1.31 (3H, s), 1.07 (3H, s). 13 C-NMR (125 MHz, CDCl3 & CD3OD), see table 1. Totarol (2): Colorless solid. ESI-MS m/z 287.5 [M+H] + . 1 H-NMR (500 MHz, CDCl3): δH 6.98 (1H, d, J = 8.5 Hz), 6.49 (1H, d, J = 8.5 Hz), 4.51 (1H, s), 3.30- 3.27 (1H, m), 2.93 (1H, dd, J = 17.0, 6.5 Hz), 1.35 (1H, d, J = 7.0 Hz), 1.33 (1H, d, J = 7.0 Hz), 1.17 (3H, s), 0.94 (3H, s), 0.91 (3H, s). 13 C-NMR (125 MHz, CDCl3), see table 1. Totarol-19-carboxylic acid (3): 1 H-NMR (500 MHz, CDCl3): δH 6.99 (1H, d, J = 8.5 Hz), 6.52 (1H, d, J = 8.5 Hz), 3.31 – 3.25 (1H, m), 2.95 (1H, dd, J = 16.5, 4.5 Hz), 1.35 (1H, d, J = 7.0 Hz), 1.34 (1H, d, J = 7.0 Hz), 1.33 (1H, s), 1.12 (1H, s). 13 C-NMR (125 MHz, CDCl3), see table 1. 3. RESULTS AND DISCUSSION Compound 1 was obtained as yellow solid. The positive ESI-MS of 1 gave a molecular ion peak at m/z 355.3 [M+Na] + , corresponding to the molecular formula C20H28O4. The 1 H- and 13 C-NMR spectra of 1 (table 1) showed the presence of an isopropyl group [δC 28.10 (d), 20.64 (q), 20.68 (q) and δH 3.53-3.50 (m), 1.42 (d), 1.37 (d)], an aromatic ring [δC 154.36 (s), 140.35 (s), 134.24 (s), 133.39 (s), 124.21 (d), 117.13 (d)], two methyl carbons [δC 28.57 (q) and 22.51 (q)] attached to quaternary carbons, and a carboxyl group [δC 181.13 (s)]. These data and the molecular formula suggested that 1 was a diterpenoid. The HMBC correlations observed between H-12 (δH 6.68), H-16 (δH 1.37), H-17 (δH 1.42) and C-14 (δC 133.39), between H-15 (δH 3.53-3.50) and C-8 (δC 134.24); and between H-11 (δH 6.99), H-12 (δH 6.68), H-15 (δH 3.53-3.50) and C-13 (δC 154.36) indicated that the isopropyl group was attached at C- 14 and the phenolic hydroxyl group was at C-13. Other HMBC correlations noted between H-5 (δH 1.97) and C-7 (δC 65.45) and between H-7 (δH 4.99) and C-5 (δC 45.38), C-8 (δC 134.24), C-9 (δC 140.35) showed that the hydroxyl group was located at C-7. From above spectral data, the structure of 1 was determined as inumakiol D. The 13 C-NMR data (in pyridine-d5) of 1 were in good agreement with those of inumakiol D in [7]. This compound was found for the first time in Podocarpus macrophyllus [7]. 1: R 1 = COOH R 2 = OH 2: R 1 = CH3 R 2 = H 3: R 1 = COOH R 2 = H Figure 1: The structure of compounds 1-3 isolated from Podocarpus neriifolius wood 1 H- and 13 C-NMR spectral data of compound 2 were similar with those of 1 except for the presence of an additional methyl group and the absence of one carbonyl and one secondary hydroxyl. Its 1 H- and 13 C-NMR spectra exhibited the presence of an VJC, 54(4) 2016 Diterpenoids from the wood of 490 isopropyl group [δC 27.15 (d), 20.34 (q), 20.34 (q) and δH 3.33-3.27 (m), 1.35 (d), 1.33 (d)], an aromatic ring [δC 151.97 (s), 143.19 (s), 134.01 (s), 130.97 (s), 122.97 (d), 114.27 (d)], three methyl carbons [δC33.23 (q), 25.17 (q) and 21.57 (q)] attached to quaternary carbons. In addition, it showed five methylene carbons (δC 41.56, 39.58, 28.75, 19.48, 19.34), a methine carbon (δC 49.55) and two quaternary carbons (δC 37.68, 33.25). 1 H- and 13 C- NMR data of 2 are identical with those of totarol in previous report [8]. So, compound 2 was determined as totarol. Table 1: 13 C-NMR spectral data of compounds 1 (in CDCl3 + CD3OD) and 2-3 (in CDCl3) Position 1 2 (CDCl3) 3 (CDCl3) 1 40.09 41.56 40.11 2 20.44 19.47 20.07 3 37.72 39.58 37.22 4 43.5 33.25 43.75 5 45.38 49.55 52.08 6 31.15 19.34 21.09 7 65.45 28.75 30.01 8 134.24 134.01 134.26 9 140.35 143.19 140.98 10 38.92 37.68 38.52 11 124.21 122.97 124.14 12 117.13 114.27 114.58 13 154.36 151.97 152.05 14 133.39 130.97 130.89 15 28.10 27.15 27.26 16 20.64 20.34 20.31 17 20.68 20.34 20.41 18 28.57 33.23 28.62 19 181.13 21.57 183.91 20 22.51 25.17 23.20 1D-NMR spectra of compound 3 are also similar to those of compound 1 except for the absence of a hydroxyl group at C-7. Thus, 3 was elucidated as totarol-19-carboxylic acid by comparison with reported data [9]. This compound was found in some Podocarpus species [7, 9]. Acknowledgment. We are indebted to the National Foundation for Science & Technology Development (NAFOSTED) of Vietnam for financial support of this research (code: 104.01-2013.62). REFERENCES 1. Pham Hoang Ho. Flora of Vietnam, Youth Publishing House, Ho Chi Minh City, 1, 226 (2000). 2. Syed Hailer Mehdi Rizvi, Wasiur Rahman, Masayoshi Okigawa and Nobusuke Kawano. Biflavones from Podocarpus neriifolius, Phytochemistry, 13, 1990 (1974). 3. Syed Haider Mehdi Rizvi and Wasiur Rahman. 7,4’- dimethylaromadendrin and its glucoside from Podocarpus neriifolius, Phytochemistry, 13, 2879 (1974). 4. K. Shrestha, A. H. Banskota, S. Kodata, S. P. Shrivastava, G. Strobel and M. B. Gewali. An antiproliferative norditerpene dilactone, Nagilactone C, from Podocarpus neriifolius, Phytomedicine, 8(6), 489-491 (2001). 5. Shumaia Parvin, Md. Abdul Kader, Md. Abdul Muhit, Md. Ekramul Haque, Md. Ashik Mosaddik and Mir Imam Ibne Wahed. Triterpenoids and phytosteroids from stem bark of Crataeva nurvala buch ham, Journal of Applied Pharmaceutical Science, 1(9), 47-50 (2011). 6. Hisashi Kojima, Noriko Sato, Akiko Hatano, Haruo Ogura. Sterol glucosides from Prunella vulgaris, Phytochemistry, 7, 2351-2355 (1990). 7. Kimihiko Sato, Keito Sugawara, Hirono Takeuchi, Hyun-Sun Park, Toshiyuki Akiyama,Tetsuo Koyama, Yutaka Aoyagi, Koichi Takeya, Takanori Tsugane, and Susumu Shimura. Antibacterial Novel Phenolic Diterpenes from Podocarpus macrophyllus D. Don., Chem. Pharm. Bull., 56(12), 1691-1697 (2008). 8. Takahiro Miyake, Hideo Kigoshi and Hiroyuki Akita. Chemoenzymatic synthesis of (+)-totarol, (+)- podototarin, (+)-sempervirol, and (+)-jolkinolides E and D, Tetrahedron: Asymmetry, 18, 2915-2922 (2007). 9. Jayr De Paiva Campello, Sebastiao Ferreira Fonseca, Ching-Jer Chang, Ernest Wenker. Terpens of Podocarpus lambertius, Phytochemistry, 14, 243-248 (1975). Corresponding author: Trinh Thi Thuy Institute of Chemistry Vietnam Academy of Science and Technology 18, Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam E-mail: thuy@ich.vast.vn; Tel. 0084-4-32121149; Fax. 0084-4-8361283.