Chinese Journal of Natural Medicines 2014, 12(4): 0297−0299

Chinese Journal of Natural Medicines

A new diterpene from the stems of Trigonostemon heterophyllus LI Yi-Xing, ZUO Wen-Jian#, MEI Wen-Li, CHEN Hui-Qin, DAI Hao-Fu* 1

Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China; 2 Haikou Experimental Station, Chinese Academy of Tropical Agricultural Sciences, Haikou 570102, China Available online Apr. 2014

[ABSTRACT] AIM: To investigate the chemical constituents in the stems of Trigonostemon heterophyllus. METHOD: The chemical constituents were isolated by column chromatography on silica gel, Rp-18, and Sephadex LH-20, and their structures were elucidated on the basis of spectroscopic analysis. RESULTS: Three compounds were isolated and identified as a new diterpene, trigonoheterene B (1), together with two known compounds, trigonostemone (2) and trigonochinene B (3). CONCLUSION: Compound 1 is new. Compounds 2 and 3 showed antibacterial activities. [KEY WORDS] Trigonostemon heterophyllus; Trigonoheterene B; Diterpene; Antibacterial activities

[CLC Number] R284.1

[Document code] A

[Article ID] 2095-6975(2014)04-0297-03

Introduction The genus Trigonostemon Blume (Euphorbiaceae), comprising approximately fifty species, grows mainly in the tropical and subtropical regions of Asia [1]. Trigonostemon reidioides Craib has been used in Thai medicine as an antidote, expectorant, and laxative agent. Previous chemical investigations on this genus afforded a number of structurally interesting diterpenoids [2-15], some of which showed a variety of biological activities, including cytotoxic [5] and Met tyrosine kinase inhibitory [7] activities. In an earlier study, a new diterpenoid, trigonoheterene and a new naphthoquinone, trigonoheterone [16] were isolated from Trigonostemon heterophyllus Merr. from this laboratory. Yue et al reported five new daphnane diterpenoids, a new 3,

4-seco-cleistan-thane dinorditerpenoid, and a new prenylated bisabolane sesquiterpenoid [13] from the same species. In a continuation, a new diterpenoid, trigonoheterene B (1) (Fig. 1), together with trigonostemone (2) and trigonochinene B (3) were isolated from this plant. Here, the isolation and structural elucidation of trigonoheterene B, as well as the antibacterial activity of the three compounds are described.

Fig. 1 Structure of trigonoheterene B

Results and Discussion [Received on] 25-Oct.-2012 [Research funding] This project was supported by the Special Fund for Agro-scientific Research in the Public Interest (No. 201303117) and Major Technology Project of Hainan Province (ZDZX2013023-1, ZDZX2013008-4). [*Corresponding author] DAI Hao-Fu: Prof., Tel/Fax: 86-89866961869, E-mail: [email protected] # Co-first author. These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

Trigonoheterene B (1) was obtained as a red oil. A molecular formula of C22H32O6 was assigned for 1 on the basis of its negative-ion HRESI-MS peak (m/z 391.2137 [M − H]−), in combination with an analysis of the 13C NMR spectrum (DEPT), corresponding to seven degrees of unsaturation. The IR spectrum showed an absorption band at 1 728 cm−1, ascribable to carbonyl groups. The 1H NMR spectrum (Table 1) showed three methyl singlets at δ 1.10, 1.26, and 1.77, two

– 297 –

LI Yi-Xing, et al. / Chin J Nat Med, 2014, 12(4): 297−299

methoxyl singlets at δ 3.63 and 3.66, an ABX terminal vinyl system signals at δ 5.08 (dd, J = 16.4, 2.0 Hz, 1H), 5.12 (dd, J = 10.4, 2.0 Hz, 1H), and 6.05 (dd, J = 16.4, 10.4 Hz, 1H), and an ABX3 system signals of an isopropenyl group at δ 1.77 (s, 3H), 4.73 (s, 1H), and 4.95 (s, 1H). The 13C NMR spectrum (DEPT) resolved 22 carbon resonances attributed to one conjugated carboxylic carbonyl, two ester carbonyls, three sp2 quaternary carbons, one sp2 methine, two sp2 methylenes, two quaternary sp3 carbons, one sp3 methine, five sp3 methylenes, and five methyls, including the two methoxy groups. These NMR data were quite similar to those of trigoheteric acid methyl ester [13], indicating the occurrence of the same diterpene skeleton in both compounds. The differences were the disappearance of the aromatic ring in trigoheteric acid methyl ester, and the substitution of a carboxylic group, a methoxycarbonyl, a methyl, an ABX terminal vinyl system, a quaternary sp3 carbon, and an sp3 methylene. The HMBC correlations (Fig. 2) from Ha-16, Hb-16, and Ha-15 to C-13, and from H3-17 to C-13 and C-15 placed the terminal vinyl system and the methyl group at C-13. The fragment CH 3 OO-C(12)- C(13)CH2(14)-C(8)-CH2(7) was confirmed by the HMBC correlations of H3-CH3O (δ 3.66) with C-12, and of H2-14 with C-12, C-13, C-8 and C-7. Furthermore, according to the molecular Table 1 1H (400 MHz) and 13C (100 MHz) data of compound 1 (in CD3OD, J in Hz) No. 1

13

C NMR

1

H NMR

34.5

1.84 (1H, m), 1.65 (1H, m)

2

30.5

2.53 (1H, m), 2.39 (1H, m)

3

176.2

─

4

147.4

─

5

46.8

2.23 (1H, dd, J = 12.0, 2.8)

6

25.3

1.72 (1H, m), 1.57 (1H, m)

7

31.2

2.02 (1H, m), 1.96 (1H, m)

8

136.1

─

9

139.3

─

10

41.4

─

11

173.3

─

12

177.8

─

13

49.9

─

14

45.9

2.57 (2H, m)

15

143.5

6.05 (1H, dd, J = 16.4, 10.4)

16

114.3

5.12 (1H, dd, J = 10.4, 2.0), 5.08 (1H, dd, J = 16.4, 2.0)

17

21.1

1.26 (3H, s)

18

23.3

1.77 (3H, s)

19

115.5

4.73 (1H, s), 4.95 (1H, s)

20

24.3

1.10 (3H, s)

3-OCH3

52.0

3.63 (3H, s)

12-OCH3

52.7

3.66 (3H, s)

Fig. 2 Key HMBC correlations (H→C) of trigonoheterene B

formula, the remaining carboxylic carbonyl (δ 173.3) was tentatively assigned to the sp2 quaternary carbon of C-9 (δ 139.3), which did not correlate with any protons in the HMBC spectrum. The relative configuration of 1 was deduced from the analysis of its ROESY correlations. The ROESY interaction from H2-1 to H-5 showed that the methyl propionate group and H-5 were at the same side and that CH3-20 was on the other side. When CH3-20 is in an α-orientation, H-5 should be in a β-orientation. Based on the above evidence, the structure of 1 was established, and named trigonoheterene B. Trigonoheterene B (1): Red oil; [α] 22 −250 (c 0.02, D MeOH); UV (MeOH) λmax (log ε) 194 (5.00), 209 (4.42), 221 (4.32) nm; IR (KBr) vmax 2 951, 1 728, 1 650, 1 372, 668 cm−1; 1 H- and 13C NMR data, see Table 1; HR-ESI-MS calcd. for － C22H31O6 [M − H] 391.212 0, found 391.213 7. The identity of compounds 2 and 3 was established as trigonostemone (2) [17] and trigonochinene B (3) [18], respectively, by comparison of their spectroscopic data with those in the literature. These compounds were tested for in vitro antibacterial activty against Staphylococcus aureus by the filter paper disc agar diffusion method. Compounds 2 and 3 showed antibacterial activities, diameters of inhibition zones of which were 15.2 and 13.5 mm, respectively, while compound 1 was completely inactive. The MICs of compounds 2 and 3 against S. aureus were 2.5 and 5.0 mg·mL−1, respectively using kanamycin as a positive control.

Experimental Apparatus and reagents The NMR spectra were recorded on a Bruker AV-400 spectrometer (400 MHz for 1H and 100 MHz for 13C), using TMS as an internal standard. The HRESI-MS spectra were measured with an API QSTAR Pulsar mass spectrometer. The IR spectra were obtained on a Nicolet 380 FT-IR instrument from KBr pellets. The UV spectra were measured on a Shimadzu UV-2550 spectrometer. The optical rotation was recorded using a Rudolph Autopol III polarimeter (USA). Column chromatography was performed with silica gel 200−300 mesh (Qingdao Marine Chemical Industry Factory, Qingdao, China), Sephadex LH-20 (Merck, Germany) and Rp-18 (Merck, Germany). TLC was performed with silica gel GF254 (Qingdao Marine Chemical Industry Factory, Qingdao, China). Plant material The stems of Trigonostemon heterophyllus were collected

– 298 –

LI Yi-Xing, et al. / Chin J Nat Med, 2014, 12(4): 297−299

in Baoting county, Hainan province, China in March 2009, and authenticated by Assoc. Prof. DAI Zheng-Fu of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, where a voucher specimen (No. A20090303) was deposited. Extraction and isolation The stems of T. heterophyllus (dry weight 15.0 kg) were extracted three times with 95% EtOH at room temperature. The extract was evaporated under reduced pressure and the residue was suspended in H2O and partitioned with petroleum ether, EtOAc, and n-BuOH, respectively. The EtOAc fraction (58.0 g) was separated into 7 fractions on a silica gel column using a step gradient elution of CHCl3−MeOH (50 : 1 to 0 : 1, V/V). Fr. 1 (4.08 g) was submitted to Sephadex LH-20, eluting with CHCl3−MeOH (1 : 1, V/V) to afford four fractions (1.1−1.4). Fr. 1.3 (2.41 g) was subjected to CC, eluting with petroleum ether−EtOAc (12 : 1 to 2 : 1, 200−300 mesh) to give seven fractions (1.3.1−1.3.7). Fraction 1.3.6 was applied to Sephadex LH-20, eluting with CHCl3−MeOH (1 : 1, V/V) to give 2 (11.5 mg). Fr. 2 (3.00 g) was subjected to a Sephadex LH-20 column, eluting with CHCl3−MeOH (1 : 1, V/V) to afford five fractions (2.1−2.5). Fraction 2.3 was applied to silica gel CC, eluting with petroleum ether−EtOAc (7 : 1, V/V), then Rp-18 with MeOH−H2O (7 : 3, V/V) as eluent, to give 3 (1.7 mg). Fr. 3 (2.68 g) was subjected to CC, eluting with petroleum ether−EtOAc (25 : 1 to 2 : 1, V/V) to give nine fractions (3.1−3.9). Fr. 3.3 (0.08 g) was submitted to Sephadex LH-20, eluting with CHCl3− MeOH (1 : 1, V/V) to afford five fractions (3.3.1−3.3.5). Fr. 3.3.2 (10.3 mg) was purified by CC, with petroleum ether−EtOAc (18 : 1, V/V) as eluent to give 1 (3.2 mg). Antimicrobial assay All compounds were tested for in vitro antibacterial activity against Staphyllococcus aureus (CMCC(B) 26003 strain), obtained from the National Institutes for Food and Drug Control, by the filter paper disc agar diffusion method [19]. The media nutrient agar was used to culture the bacteria. The sterile agar media was poured into petri-plates to a uniform depth of 5 mm and was allowed to solidify. The bacterial suspensions were streaked over the surface of the media using a sterile cotton swab. Samples (50 μL, 20 mg·mL−1) of the compounds were impregnated on sterile filter paper discs of 6 mm size, respectively. These discs were then aseptically applied to the surface of the agar plates at well-spaced intervals. Control discs impregnated with of acetone (50 μL) and kanamycin sulfate (50 μL, 0.64 mg·mL−1) were used as controls for the test discs in the experiment. The plates were incubated at 36 °C for 24 h. Determination of the MIC values was performed by dilution of the four compounds. Experiments were done in triplicate, and the results were obtained as mean values of the three measurements.

References [1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13] [14] [15]

[16]

[17] [18]

Delectis florae reipublicae popularis sinicae agendae academiae sinicae edita. Flora Reipublica Popularis Sinicae [M]. Beijing: Science Press, 1997, 44: 162. Tempeam A, Thasana N, Dawornkricharut A, et al. In vitro cytotoxicity of some Thai medicinal plants and daphnane diterpenoids from Trigonostemon redioides [J]. Mahidol Univ J Pharm Sci, 2002, 29(3-4): 25-31. Jayasuriya H, Zink DL, Borris RP, et al. Rediocides B-E, potent insecticides from Trigonostemon reidioides [J]. J Nat Prod, 2004, 67(2): 228-231. Tempeam A, Thasana N, Pavaro C, et al. A new cytotoxic daphnane diterpenoid, rediocide G, from Trigonostemon reidioides [J]. Chem Pharm Bull, 2005, 53(10): 1321-1323. Soonthornchareonnon N, Sakayarojkul M, Isaka M, et al. Acaricidal daphnane diterpenoids from Trigonostemon reidioides (Kurz) Craib roots [J]. Chem Pharm Bull, 2005, 53 (2): 241-243. Chen HD, He XF, Ai J, et al. Trigochilides A and B, two highly modified daphnane-type diterpenoids from Trigonostemon chinensis [J]. Org Lett, 2009, 11(18): 4080-4083. Chen HD, Yang SP, He XF, et al. Trigochinins A-C: three new daphnane-type diterpenoides from Trigonostemon chinensis [J]. Org Lett. 2010, 12(6): 1168-1171. Chen HD, Yang SP, He XF, et al. Trigochinins D-I: six new daphnane-type diterpenoids from Trigonostemon chinensis [J]. Tetrahedron, 2010, 66: 5065-5070. Zhang L, Luo RH, Wang F, et al. Daphnane diterpenoids isolated from Trigonostemon thyrsoideum as HIV-1 antivirals [J]. Phytochemistry, 2010, 71: 1879-1883. Zhang L, Luo RH, Wang F, et al. Highly functionalized daphnane diterpenoids from Trigonostemon thyrsoideum [J]. Org Lett, 2010, 12(1): 152-152. Lin BD, Han ML, Ji YC, et al. Trigoxyphlins A-G: diterpenes from Trigonostemon xyphophylloides [J]. J Nat Prod, 2010, 73: 1301-1305. Li SF, Di YT, Li SL, et al. Trigonosins A-F: daphnane diterpenoids from Trigonostemon thyrsoideum [J]. J Nat Prod, 2011, 74(3): 464-469. Dong SH, Liu HB, Xu CH, et al. Constituents of Trigonostemon heterophyllus [J]. J Nat Prod, 2011, 74(12): 2576-2581. Yin S, Su ZS, Zhou ZW, et al. Antimicrobial diterpenes from Trigonostemon chinensis [J] J Nat Prod, 2008, 71: 1414-1417. Li YX, Mei WL, Zuo WJ, et al. Two new compounds from Trigonostemon heterophyllus [J]. Phytochem Lett, 2012, 5(1): 41-44. Kokpol U, Thebpatiphat S, Boonyaratavej S, et al. Structure of trigonostemone, a new phenanthrenone from the Thai plant Trigonostemon reidioides [J]. J Nat Prod, 1990, 53(5): 1148-1151. Yin S, Su ZS, Zhou ZW, et al. Antimicrobial diterpenes from Trigonostemon chinensis [J]. J Nat Prod, 2008, 71: 1414-1417. Xu SY, Bian RL, Chen X. Methods of Pharmacology Experiments [M]. Beijing: People’ s Sanitation Press, 2003: 1651.

Cite this article as: LI Yi-Xing, ZUO Wen-Jian, MEI Wen-Li, CHEN Hui-Qin, DAI Hao-Fu. A new diterpene from the stems of Trigonostemon heterophyllus [J]. Chinese Journal of Natural Medicines, 2014, 12(4): 297-299

– 299 –