Chemical constituents of Chirita drakei Burtt collected in Ha Long bay, Quang Ninh province, Viet Nam

Vietnam Journal of Chemistry, International Edition, 55(2): 202-206, 2017 DOI: 10.15625/2525-2321.2017-00444 202 Chemical constituents of Chirita drakei Burtt collected in Ha Long bay, Quang Ninh province, Viet Nam Part 1. Compounds isolated from the n-hexane and ethyl acetate extracts Nguyen Thi Hoang Anh 1,2* , Nguyen Van Tuan 2,3 , Tran Duc Quan 1 , Dao Duc Thien 1 , Nguyen Thanh Tam 1 , Giang Thi Kim Lien 4 , Trinh Thi Thuy 1 , Tran Van Sung 1 1 Institute of Chemistry, Vietnam Academy of Science and Technology 2 Graduate University of Science and Technology, Vietnam Academy of Science and Technology 3 Asean College, Van Lam, Hung Yen, Vietnam 4 University of Da Nang Received 14 March 2017; Accepted for publication 11 April 2017 Abstract Two triterpenes, an anthraquinone, two lignans and a phenolic compound were isolated from the n-hexane and ethyl acetate extracts of the aerial part of Chirita drakei Burtt collected in islands, on mountain slopes of Ha Long bay, Quang Ninh province. Their structures have been elucidated by mass, NMR spectroscopy and comparison with published data. There are no report on the chemical constituents of Chirita drakei before our study. Keywords. Chirita drakei, triterpene, anthraquinone, lignin. 1. INTRODUCTION Ha long bay is one of seven new world’s natural wonders, which contains the full value of geology, geomorphology and biodiversity. A set of different plant species were found here, including mangrove plants or species grow in coastal sand of islands, on mountain slopes, on mountain peaks or in store. Until now, 17 endemic plant species have been detected in Ha long bay, for example Hedyotis lecomtei, Allophylus leviscens, Chirita gemella, etc. [1]. Chirita is the oldest genus of the Gesneriaceae family, distributed in Malaysia, Southeast Asia and south of China. Some Chirita species are used in folk medicine as poultice, hemostasis agents or for treatment of hypertension,... [2]. Until now there are only few studies on chemical and biological activities of the Chirita species. However, these investigations revealed interesting results. For example, in the folk medicine of China, Chirita eburnea Hance is known as reagent to heal coughing up blood, immunodeficiency diseases. Its chemical composition contains 4 new -dunnion derivatives, a new anthraquinone together with 7 known anthraquinones. The ethyl acetate fraction of ethanol extract together with two new compounds: (R)-7,8- dihydroxy- -dunnion, (R)-7-methoxy-6,8- dihydroxy- -dunnione revealed free radical scavenging activity with IC50 value of 5.2 µg/mL, 8.4 µM, and 3.6 µM, respectively while of ascorbate 6.8 µM [3]. Chirita fimbrisepala is used in folk medicine for treatment of inflammatory such as hepatitis, enteritis. One of four isolated compounds from the roots of this plant is a new flavone glycoside, named mahuangchiside [4]. Therefore, in order to contribute to the understanding, research, evaluation, protection, maintenance, and exploitation of the genetic resources of rare plant species growing in the Ha Long Bay, the chemical composition Chirita drakei Burtt species were investigated. This paper deals with the isolation and structural elucidation of 6 compounds from the n- hexane and ethyl acetate extracts of C. drakei’s aerial parts. All isolated compounds, except 3, were obtained for the first time from Chirita genus. 2. EXPERIMENTAL 2.1. Equipments and methods IR: Impact 410, Nicolet, Germany; ESI-MS: LC- MSD-Trap-SL, Varian, USA, NMR: Bruker Avance 500, Germany with TMS as internal reference (for 1 H) and solvent signal (for 13 C). CC used silica gel VJC, 55(2), 2017 Nguyen Thi Hoang Anh et al. 203 60G, size 0.043-0.063 mm (Merck), TLC: precoated silica gel G60F254 plates (Merck), spots were detected by spraying with vanillin 1 % in conc. H2SO4 and heating at 110 o C. 2.2. Plant material The aerial parts of Chirita drakei were collected in the islands of Ha Long Bay, Quang Ninh province, Vietnam in October 2013. A voucher specimen (VHH.HL 10.2013.1) is deposited in Institute of Chemistry, VAST, Hanoi, Vietnam. The scientific name was identified by Dr. Tran Thi Phuong Anh, Viet Nam National Museum of Nature, VAST, Hanoi, Vietnam. 2.3. Extraction and isolation of the compounds The aerial parts of C. drakei (1.7 kg) were extracted with MeOH:H2O (9:1) (four times) at room temperature. The methanol extract was concentrated under vacuum and then aqueous solution was extracted with n-hexane, EtOAc and n-BuOH, successively. The solvent was evaporated in vacuum to afford n-hexane (10.0 g), EtOAc (10.8 g) and n-BuOH (55.0 g) extracts. The n-hexane extract (10.0 g) was separated by chromatography on silica gel, eluting gradient with n-hexane: EtOAc to furnish 10 fractions (H1-H10), which were combined according to TLC monitoring. The third fraction H3 (250 mg) was further purified over silica gel column with n-hexane:EtOAc (from 9:1 to 8:2) as eluting solvent to yield 7 mg of compound 1. The tenth fraction H10 (40 mg) was chromatographed on silica gel with n-hexane:EtOAc (7:3) to give 11 mg compound 2. The EtOAc extract (10.8 g) was given on silica gel column, eluting gradient with CH2Cl2:MeOH (from 100:0 to 0:100) to furnish 12 fractions (E1- E12). The fifth fraction E5 (350 mg) was further purified over silica gel column with n-hexane:EtOAc (2:1) as eluent to yield 5 mg of compound 3 and 15 mg of 4. The seventh fraction E7 (350 mg) was given over silica gel column, eluted with n-hexane:EtOAc (6:4) to furnish 11 mg of compound 5. Compound 6 (15 mg) was obtained by repeated silica gel column chromatography of fraction E10 with CH2Cl2:MeOH (10:1). Besides, -sitosterol and -sitosterol glucoside were isolated from fraction E3 and E12, respectively, by crystallization method. 24-methylen-lanost-8-en-3 -ol (1) Amorphous white powder. ESI-MS: m/z: 441 [M+H] + , C31H53O. 1 H-NMR (500 MHz, CD3OD), H (ppm), J (Hz): 4.71, 4.66 (each br s, 2H-24), 3.24 (dd, J = 4.0; 11.5, H-3), 0.69, 0.81, 0.88, 0.98, 1.00 (each s, 3H, CH3- 18, 19, 28, 29, 30), 0.92 (d, J = 6.5, CH3-21), 1.02, 1.03 (each d, J = 6.5, CH3-26, 27). 13 C-NMR (125 MHz, CD3OD) C (ppm): 35.59 (C-1), 27.86 (C-2), 79.01 (C-3), 38.90 (C-4), 50.39 (C-5), 18.26 (C-6), 28.21 (C-7), 134.41 (C-8, C-9), 37.03 (C-10), 21.01 (C-11), 26.51 (C-12), 44.51 (C- 13), 49.82 (C-14), 31.00 (C-15), 30.85 (C-16), 50.41 (C-17), 15.76 (C-18), 18.72 (C-19), 36.49 (C-20), 19.15 (C-21), 35.59 (C-22), 31.29 (C-23), 156.94 (C-24), 34.99 (C-25), 22.00 (C-26), 21.87 (C-27), 15.43 (C-28), 27.97 (C-29), 24.27 (C-30), 105.93 (C-24 1 ). 3 ,24-dihydroxy-urs-12-ene-28-oic acid (2) Amorphous white powder. ESI-MS: m/z 473 [M+H] + , 471 [M-H] - (C30H48O4). 1 H-NMR (500 MHz, CD3OD) H: 3.79 (br s, H- 3), 5.26 (br s, H-12), 2.22 (d, J = 11.5 Hz, H-18), 3.70, 3.41 (each d, J = 11.5 Hz, 2H-24). 13 C-NMR (125 MHz, CD3OD) C: 34.38 (C-1), 71.30 (C-3), 126.91 (C-12), 139.59 (C-13), 54.39 (C-18), 66.32 (C-24), 181.63 (C-28), 17.67 (C-29), 21.57 (C-30). Digiferruginol (3) Orange-yellow powder. ESI-MS: m/z 253 [M- H] - C15H10O4. 1 H- and 13 C-NMR, see table 1. Epoxyconiferyl alcohol (4) Amorphous brown-black powder. ESI-MS: m/z 195 [M-H] - (C10H12O4). 1 H- and 13 C-NMR, see table 1. Lariciresinol (5) White powder. [ ]D 25 (MeOH, c 0.1) = +32 0 . ESI-MS: m/z 383 [M+Na] + C20H24O6Na. 1 H- and 13 C-NMR, see table 1. Isolariciresinol (6) White powder. [ ]D 25 (MeOH, c 0.1) = +41 0 . ESI-MS: m/z 359 [M-H] - C20H23O6. 1 H- and 13 C- NMR, see table 1. 3. RESULTS AND DISCUSSION Six compounds were isolated from n-hexane and ethyl acetate extracts of the aerial parts of C. drakei by repeated column chromatography with the appropriate solvent systems. Their structures were identified as 24-methylen-lanost-8-en-3 -ol (1), 3 ,24-dihydroxy-urs-12-en-28-oic acid (2), digiferruginol (3), epoxyconiferyl alcohol (4), VJC, 55(2), 2017 Chemical constituents of Chirita drakei 204 lariciresinol (5), isolariciresinol (6) by the analysis of their 1D, 2D-NMR, ESI-MS spectra and compared with published data. Compound 1 showed the pseudo molecular ion peak at m/z 441 [M+H] + in the positive ESI-MS spectrum, according to the molecular formula C31H52O of this compound. Its 1 H-NMR spectrum revealed signals of 8 methyl groups [ H 0.69, 0.81, 0.88, 0.98, 1.00 (each 3H, s), 0.92 (d, J = 6.5 Hz), 1.02, 1.03 (each d, J = 6.5 Hz)], one hydroxy-methin group at H 3.24 (dd, J = 4.0 & 11.5 Hz), one >C=CH2 group at H 4.71, 4.66 (each 1H, br s), and aliphatic protons resonanced in the range of 1.10- 2.30 ppm. Besides the carbon signals, which are suitable with 1 H-NMR data, the 13 C-NMR spectrum indicated the presence of totally substituted double bond at C 134.41 (x2C). These two carbons are the characteristic signals of double bond at C8-C9 in lanosten-type triterpene. Consequently, the structure of 1 was identified as 24-methylen-lanost-8-en-3 -ol from the analysis mentioned above and compared with reported data [5, 6]. It was isolated for the first time from Neolitsea sericea species, Lauraceae family. Compound 2 has molecular formula of C30H48O4 based on the NMR and positive ESI-MS data (m/z 473 [M+H] + ). The spectroscopic data suggested that 2 belong to the urs-12-en-28-oic acid series. This suggestion was confirmed by 1 H-NMR signals at H 5.26 (br s, H-12), 2.22 (d, J = 11.5 Hz, H-18), 3.79 br s (H-3) together with 6 methyl groups [ H 0.84, 0.95, 1.05, 1.17 (each 3H, s), 0.92 (d, J = 6.5 Hz), 0.99 (br s)] and 13 C-NMR signals at C 71.30 (C-3), 126.91 (C-12), 139.59 (C-13) and 181.63 (C-28). Besides, its NMR spectra showed a hydroxy- methylen group [( H 3.70, 3.41 (each d, J = 11.5 Hz), C 66.32)]. The HMBC correlations between protons at H 3.70, 3.41 and carbons at C 22.81 (C- 23), 44.0 (C-4), 71.30 (C-3) confirmed the position of the second hydroxyl group at C-24. The α configuration of hydroxyl group at C-3 was identified by the chemical shift and multiplicity peak of hydroxyl-methine proton (3.79 ppm, brs). Thus, the structure of 2 was determined as 3 ,24- dihydroxy-urs-12-en-28-oic acid by comparison with spectroscopic data in the literature [7]. Compound 3 gave the pseudo-molecular ion peak at m/z 253 [M-H] - in the negative ESI-MS spectrum, according to the molecular formula C15H10O4 for this compound. Its 1 H- and 13 C-NMR of 3 showed an anthraquinone type, including six aromatic protons of two aromatic rings. The substituents in 3 were determined as two carbonyl groups at C 182.30, 188.98, a hydroxyl group at C 159.7 and a hydroxymethyl group at H 4.87 (s, 2H); C 59.13. The spectroscopic data of 3 were indentical to those of 1-hydroxy-2- hydroxymethylanthraquinone (digiferruginol) [8]. The molecular formula of 4 was deduced as C10H12O4 from the negative ESI-MS spectrum (m/z 195 [M-H] - ) and NMR data. Its 1 H- NMR spectrum revealed a typical pattern of a 1,3,4-trisubstituted benzene ring at H 6.89 (d, J = 2.0 Hz), 6.88 (d, J = 8.2 Hz) and 6.81(dd, J = 2.0 & 8.2 Hz), one aromatic methoxy group at H 3.89 (s, 3H) and one phenolic hydroxyl group at H 5.72. A hydroxy-methylene group attached to C-2 ’ was revealed by the signals at H 3.88 (dd, J = 3.5 & 9.0 Hz), 4.24 (dd, J = 6.5 & 9.0 Hz) and C 71.64. In summary, the structure of 4 was elucidated as epoxyconiferyl alcohol by comparison with spectroscopic data in the literature [9]. Compound 5 indicated the pseudo-molecular ion peak at m/z 383 [M+Na] + in the positive ESI-MS spectrum, according to the molecular formula C20H24O6 of this compound. Analysis of the 1 H-, 13 C- NMR and DEPT spectral data indicated 5 to be a tetrahydrofuran-type lignan, showing six aromatic protons in a pair of ABX system at δH 6.85 (d, J = 2.0 Hz), 6.86 (d, J = 8.0 Hz), 6.80 (dd, J = 2.0 & 8.0 Hz) and 6.68 (2H, m), 6.83 (d, J = 8.0 Hz), two oxy- methylene groups at H 3.73-3.78 m, 3.92 m/ C 60.88) and 3.74 (dd, J = 6.2 & 8.5 Hz), 4.04 (dd, J = 6.5 & 8.5 Hz)/ C 72.88, two phenolic hydroxyl groups at H 5.65, 5.56, two methoxy groups (δH 3.87, 3.86/δC 55.92), an oxy-methine group at H 4.78 (d, J = 7.0 Hz)/ C 82.83 and four aliphatic protons. Finally, 5 was established as (+) lariciresinol by comparison with spectroscopic data and [ ]D value in the literatures [10]. Moreover, the structure of 5 was also confirmed by 2D-NMR methods. Compound 6 has the same molecular formula of C20H24O6 as of 5 based on ESI-MS (m/z 359 [M-H] - ) and NMR data. Its NMR spectra were very similar to those of 5 with two exceptions. The first is the disappearance of one aromatic proton in 1 H-NMR of 6. The second, an oxy-methine group in 5 was replaced by methine group at H 3.82 (d, J = 10.0 Hz)/ C 48.06 in NMR spectra of 6. Combination of the above spectroscopic analysis and comparison with spectroscopic data and [ ]D value in [11] led to conclusion that 6 is (+) isolariciresinol. The reassignment of signals CH-2, CH-3 and CH-5', CH-6' in the literature based on the corrections between H-2 (6.67 ppm) and C-7 (33.57 ppm), H-5 (6.21 ppm) and C-7’ (48.06 ppm), H-5’ (6.76 ppm) and C-1’ (138.62 ppm), H-6’ (6.63 ppm) and C-7’ VJC, 55(2), 2017 Nguyen Thi Hoang Anh et al. 205 Table 1: 1 H- and 13 C-NMR (500 and 125 MHz) data of compounds 3-5 (CDCl3) and 6 (CD3OD) C 1 H 13 C 3 4 5 6 3 4 5 6 1 - - - - 159.70 C 132.88 C 134.79 C 129.03 C 2 - 6.89 d (2.0) 6.85 d (2.0) 6.67 s 137.99 C 108.66 CH 108.36 CH 112.41 CH 3 7.77 d (7.8) - - - 134.09 CH 146.74 C 146.65 C 147.20 C 4 7.85 d (7.8) - - - 119.34 CH 145.26 C 145.04 C 145.27 C 5 8.30 m 6.88 d (8.2) 6.86 d (8.0) 6.21 s 127.30 + CH 114.31 CH 114.20 CH 117.35 CH 5a - - - - 133.21*C - - - 6 7.82 m 6.81 dd (2.0 & 8.2) 6.80 dd (1.5 & 8.0) - 134.14 # CH 118.94 CH 118.75 CH 134.17 C 7 7.82 m - 4.78 d (7.0) 2.79 d (7.5) 134.69 # CH - 82.83 CH 33.57 CH2 8 8.30 m - 2.40 m 2.02 m 126.84 + CH - 52.58 CH 40.03 CH 8a - - - - 133.73*C - - - 9 - - 3.73-3.78 m 3.92 m 3.71 m (2H) 188.98 C - 60.88 CH2 65.98 CH2 9a - - - - 115.20 C - - - 10 - - - - 182.30 C - - - 10a - - - - 131.85 C - - - 1’ 4.87 s 3.88 dd (3.5 & 9.0) 4.24 dd (6.5 & 9.0) - - 59.13 CH2 71.64 CH2 132.28 C 138.62 C 2’ - 3.10 m 6.68 m 6.70 d (1.5) - 54.13 CH 111.24 CH 113.82 CH 3’ - 4.74 d (4.5) - - - 85.86 CH 146.55 C 149.01 C 4’ - - - - - - 144.01 C 145.94 C 5’ - - 6.83 d (8.0) 6.76 d (8.0) - - 114.44 CH 115.99 CH 6’ - - 6.68 m 6.63 dd (1.5 & 8.0) - - 121.97 CH 123.20 CH 7’ - - 2.54 dd (10.5 & 13.0) 2.90 dd (5.0 & 13.5) 3.82 d (10.0) - - 33.30 CH2 48.06 CH 8’ - - 2.72 m 1.79 t (10.0) - - 42.41 CH 48.02 CH 9’ - - 3.74 dd (6.2 & 8.5) 4.04 dd (6.5 & 8.5) 3.42 dd (4.0 & 11.0) 3.67 d (4.0) - - 72.88 CH2 62.27 CH2 OMe - 3.89 s 3.87 s 3.79 s - 55.94 CH3 55.92 56.36 CH3 OMe - - 3.86 s 3.82 s - - 55.92 56.41 CH3 CH2OH 2.35 brs - - - - - - - OH-1 13.01 s - - - - - - - OH-4 - 5.72 s 5.65 s - - - - - OH-4’ - - 5.56 s - - - - - (48.06 ppm) in the HMBC spectrum. (+) lariciresinol and (+) isolariciresinol were isolated before from medicinal plant Fagraea racemosa [10]. The structure of compounds 1-6. VJC, 55(2), 2017 Chemical constituents of Chirita drakei 206 1: 24-methylen-lanost-8-en-3 -ol 2: 3 ,24-dihydroxy-urs-12-en-28-oic acid 3: Digiferruginol 4: Epoxyconiferyl alcohol 5: (+)-Lariciresinol 6: (+)-Isolariciresinol Acknowledgments. We would like to thank the National Foundation for Science and Technology Development of Vietnam (NAFOSTED) for financial support (Reference code 104.01.2013.59). REFERENCES 1. Nguyen Tien Hiep, Kiew R. New and interesting plants from Ha Long Bay, Vietnam, Gardens’ Bulletin (Singapore), 52, 185-202 (2000). 2. Yao Su, Jun-Long Bi, Yue-Hu Wang, Ying Tan, Jun Yang, Hong-Xin Liu, Wei Gu, Ge-Fen Yin and Chun-Lin Long. Chemical Constituents from Chirita longgangensis var. hongyao with Inhibitory Activity against Porcine Respiratory and Reproductive Syndrome Virus, J. Braz. Chem. Soc., 23, 1925 (2012). 3. Xiang-Hai Cai, Xiao-Dong Luo, Jun Zhou, Xiao- Jiang Hao. Quinones from Chirita eburnean, J. Nat. Prod., 68, 797-799 (2005). 4. Li Dong Zhou, Jing Guang Yu, Jia Guo. Mahuangchiside, a New Flavone Glycoside from Chirita fimbrisepala, Chinese Chemical Letters, 11(2), 131-132 (2000). 5. Akihisa T., Moto E., Shimizu N., Tamura T., Matsumoto T. 24-Methylenelanost-8-enol and (24E)- and (24Z)-24-Ethylidenelanost-8-enols in Neolitsea sericea, J. Jpn. Oil Chem. Soc, 35, 731 (1986). 6. Sharma M. C., Ohira T., Yatagai M. Lanostane triterpenes from the bark of Neolitsea sericea, Phytochemistry, 37, 201 (1994). 7. Deepak M., Handa Sukhdev S. 3 ,24-Dihydroxy-urs- 12-en-28-oic acid from Verbena officinalis, Phytochemistry, 49, 269 (1998). 8. Pong Chang, Kuo-Hsiung Lee. Cytotoxic antileukemic anthraquinones from Morinda parvifolia, Phytochemistry, 23(8), 1733-1736 (1984). 9. Kostova, I., Dinchev, D., Mikhova, B., Iossifova, T. Epoxyconiferyl alcohol from Fraxinus oxycarpa bark, Phytochemistry, 38, 801 (1995). 10. Okuyama, E., Suzumura, K., Yamazaki, M. Pharmacologically Active Components of Todopon Puok (Fagraea racemosa), a Medicinal Plant from Borneo, Chem. Pharm. Bull, 43, 2200 (1995). 11. Erdemoglu N., Sener B., Ozcan Y., Ide S. Structural and spectroscopic characteristics of two new dibenzylbutane type lignans from Taxus baccata L., J., Mol. Struc., 655, 459 (2003). Corresponding author: Nguyen Thi Hoang Anh Institute of Chemistry Vietnam Academy of Science and Technology No. 18, Hoang Quoc Viet, Cau Giay District, Hanoi E-mail: hoanganhvhh@gmail.com.