2011 5 9 3 Chin J Nat Med May 2011 Vol. 9 No. 3 191

Chinese Journal of Natural Medicines 2011, 9(3): 01900192doi: 10.3724/SP.J.1009.2011.00190

ChineseJournal of Natural Medicines

Triterpenes and Steroids from Semi-mangrove Plant Hibiscus tiliaceus

WANG Zhong-Zhao1, LI Jun1, TANG Xv-Li2, LI Guo-Qiang1* 1School of Medicine and Pharmacy, Key Laboratory of Marine Drugs, Ministry of Education, Ocean University of China, Qingdao 266003, China; 2College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, China

Available online May 2011

[ABSTRACT] AIM: To study the chemical constituents of semi-mangrove plant Hibiscus tiliaceus. METHODS: The isolation and purification of compounds were performed by silica gel, Sephadex LH-20 and HPLC, and their structures were determined by com-parison of their physical and spectral data with the literatures. RESULTS: Ten compounds were isolated and identified as friedelin (1), pachysandiol (2), glutinol (3), lupeol (4), germanicol (5), stigmast-4-en-3-one (6), stigmast-4, 22-dien-3-one (7), ergosta-4, 6, 8 (14), 22-tetraen-3-one (8), -sitosterol (9), and stigmasterol (10). CONCLUSION: Compounds 35 and 8 were isolated from this plant for the first time. [KEY WORDS] Hibiscus tiliaceus; Triterpenes; Steroids

[CLC Number] R284.1 [Document code] A [Article ID] 1672-3651(2011)03-0191-03

1 Introduction The semi-mangrove plant Hibiscus tiliaceus belonging

to the genus Hibiscus (family Malvaceae) is widely distribu-ted in southeastern Asia, Philippines, Pacific Islands, the South Sea Islands and India. As a Chinese medicine, it is used as heat-clearing and detoxifying, stasis-dissipating and detumescence agents in folk [1]. A survey of literatures re-vealed that the constituents of the species H. tiliaceus were studied rarely [2-4]. Our preliminary examination of the stem and bark of H. tiliaceus collected from Hainan Island, South China, resulted in the isolation and characterization of ten compounds: friedelin (1), pachysandiol (2), glutinol (3), lu-peol (4), germanicol (5), stigmast-4-en-3-one (6), stigmast-4, 22-dien-3-one (7), ergosta-4, 6, 8 (14), 22-tetraen-3-one (8), -sitosterol (9) and stigmasterol (10) (Fig.1). Compounds 35 and 8 were obtained from this plant for the first time.

2 Apparatus and Reagents

Melting points (mp) were determined on an X4 micro-

[Received on] 31-Dec.-2010 [Research Funding] This project was supported by State Bureau of Oceanic Administration (No. 908-01-ST12) [*Corresponding author] LI Guo-Qiang: Prof., Tel: 0532-82032323, E-mail: liguoqiang@ouc.edu.cn These authors have no any conflict of interest to declare. Copyright 2011, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

melting apparatus and uncorrected. ESI-MS spectra were recorded on a Waters Q-TOF LC-MS-MS mass spectrometer. 1H and 13C NMR spectra were recorded on a JEOL JNMECP600 spectrometer with the residual CHCl3 (H 7.26, C 77.0) as an internal standard. Reversed-phase HPLC (Agilent 1100 series liquid chromatography equipped with a VWD detector and a semi-preparative ODS [5 m, 10 mm (i.d.) 25 cm] column), commercial Sigel (Qingdao Marine Chemical Group Co., 200-300 and 400600 mesh) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) were employed for separation and purifification.

3 Plant Material The stems and barks of H. tiliaceus were collected in

Hainan Island, South China, in July, 2006. The plant material was identified by Mr. ZHONG Cai-Rong, Administrative Bureau of Dongzhai National Nature Conservation, Haikou, Hainan province. A voucher specimen (060701) has been deposited at School of Medicine and Pharmacy, Ocean Uni-versity of China, Qingdao, China.

4 Extraction and Isolation The air-dried and powdered stems and barks of H. tili-

aceus (11.0 kg) were extracted with industrial methanol three times at room temperature. The extract was concentrated under reduced pressure to generate a dark residue (336.0 g), which was suspended in H2O and partitioned with petroleum ether, ethyl acetate and n-butanol, yielding 83.0, 40.0 and 45.0 g of extraction fractions, respectively. Combined petro-leum ether and ethyl acetate fraction based on TLC results.

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192 Chin J Nat Med May 2011 Vol. 9 No. 3 2011 5 9 3

Fig. 1 Structures of compounds 1-10

The petroleum ether and ethyl acetate fraction was subjected to column chromatography on silica gel eluted with petro-leum ether-acetone gradient (10 : 0-1 : 10) to give eight sub-fractions Frs.18. Frs.13 were further purified by repeated chromatographic techniques to yield compounds 1 (10 mg), 2 (50 mg), 3 (20 mg), 4 (6 mg), 5 (8.6 mg), 6 (10 mg), 7 (5 mg), 8 (6.7 mg), 9 (3.7 mg) and 10 (5.8 mg).

5 Identification

Compound 1 C30H50O, white needle, mp 261263 C. 1H NMR (600 MHz, CDCl3) : 2.39 (1H, ddd, J = 13.6, 5.1, 2.2 Hz, H-2a), 2.31 (1H, dd, J = 13.2, 7.3 Hz, H-2b), 2.25 (1H, q, J = 6.6 Hz, H-4), 1.97 (1H, m, H-1a), 1.75(1H, m, H-6a), 1.68 (1H, m, H-1b), 1.18 (3H, s, H-28), 1.05 (3H, s, H-27), 1.01 (3H, s, H-26), 1.00 (3H, s, H-29), 0.95 (3H, s, H-30), 0.88 (3H, d, J = 6.6 Hz, H-23), 0.87 (3H, s, H -25), 0.72 (3H, s, H-24). 13C NMR (150 MHz, CDCl3) : 213.4 (C-3), 59.5 (C-10), 58.3 (C-4), 53.2 (C-8), 42.9 (C-18), 42.2 (C-5), 41.6 (C-2), 41.4 (C-6), 39.8 (C-13), 39.3 (C-22), 38.4 (C-14), 37.5 (C-9), 36.1 (C-16), 35.7 (C-11), 35.4 (C-19), 35.1 (C-30), 32.8 (C-15), 32.5 (C-21), 32.2 (C-28), 31.9 (C-29), 30.6 (C-12), 30.1 (C-17), 28.3 (C-20), 22.4 (C-1), 20.4 (C-26 ), 18.7 (C-27), 18.3(C-7), 18.0 (C-25), 14.7 (C-24), 6.9 (C-23). Compound 1 was identified as friedelin by comparison of the physical and spectral data with the lit-erature [5].

Compound 2 C30H52O2, white needle, mp 290291 C. ESI-MS m/z 408 [M 2H2O]+. 1H NMR (600 MHz, CDCl3) :3.99 (1H, q, H-2), 3.54 (1H, t, H-3), 1.83 (1H, td, H-1b), 1.61 (1H, m, H-1a), 1.17 (3H, s, H-28), 1.01 (3H, s, H-26), 0.99 (6H, s, H-27, 29), 0.94 (3H, d, J = 7.2, H-23), 0.94 (3H, s, H-24), 0.94 (3H, s, H-30), 0.85 (3H, s, H-25). Compound 2 was identified as pachysandiol by comparison of the physical and spectral data with the literature [6].

Compound 3 C30H52O, white needle, mp 218219 C. 1H NMR (600 MHz, CDCl3) : 5.63 (1H, br d, J = 6.1 Hz, H-6), 3.47 (1H, t, J = 2.8, H-3), 1.16 (3H, s, H-24), 1.14 (3H, s, H-28), 1.09 (3H, s, H-23), 1.04 (3H, s, H-29), 1.00 (3H, s, H-26), 0.99 (3H, s, H-27), 0.95 (3H, s, H-30), 0.85 (3H, s,

H-25). 13C NMR (150 MHz, CDCl3) : 141.7 (C-5), 122.2 (C-6), 76.4 (C-3), 49.8 (C-10), 47.5 (C-18), 43.1 (C-8), 40.9 (C-14), 39.4 (C-4), 39.0 (C-22), 37.9 (C-13), 36.1 (C-16), 35.2 (C-19), 34.9 (C-9), 34.7 (C-11), 34.6(C-29), 33.2 (C-21), 32.5 (C-28), 32.2 (C-15), 32.1 (C-30), 30.4 (C-12), 30.2 (C-17), 29.0 (C-23), 28.3 (C-20), 27.9 (C-7), 25.6 (C-24), 23.7 (C-1), 19.7 (C-27), 18.5 (C-26), 18.3 (C-2), 16.3(C-25). Compound 3 was identified as glutinol by comparison of the physical and spectral data with the literature [7].

Compound 4 C30H50O, white needle, mp 214.0215.0 C. ESI-MS m/z 408 [M H2O]+. 1H NMR (600 MHz, CDCl3) : 4.69 (1H, br s, H-29a), 4.57 (1H, br s, H-29b), 3.18 (1H, dd, J = 11.6, 5.0 Hz, H-3), 2.37 (1H, m, H-19), 1.68 (3H, s, H-30), 1.03 (3H, s, H-26), 0.97 (3H, s, H-23), 0.94 (3H, s, H-27), 0.83 (3H, s, H-25), 0.79 (3H, s, H-28), 0.76 (3H, s, H-24). Compound 4 was identified as lupeol by comparison of the physical and spectral data with the literature [8].

Compound 5 C30H50O, white crystal, mp 175178 C. ESI-MS m/z 408 [M H20]+. 1H NMR (600 MHz, CDCl3) : 4.86 (1H, s, H-19), 3.20 (1H, dd, J = 11.5, 6.2 Hz, H-3), 1.08 (3H, s, H-30), 1.02 (3H, s, H-29), 0.97 (3H, s, H-28), 0.94 (6H, s, H-26, 27), 0.88 (3H, s, H-24), 0.77 (3H, s, H-25), 0.75 (3H, s, H-23). 13C NMR (150 MHz, CDCl3) : 142.8 (C-18), 129.7 (C-19), 79.0 (C-3), 55.5 (C-5), 51.2 (C-9), 43.3 (C-14), 40.8 (C-8), 38.9 (C-4, 13), 38.4 (C-1), 37.7 (C-16), 37.3 (C-22), 37.2 (C-10), 34.6 (C-7), 34.4 (C-17), 33.3 (C-21), 32.3 (C-20), 31.3 (C-29), 29.2 (C-30), 28.0 (C-23), 27.5 (C-15), 27.4 (C-2), 26.2 (C-12), 25.3 (C-28), 21.1 (C-11), 18.3 (C-6), 16.7 (C-26), 16.1 (C-25), 15.4 (C-24), 14.6 (C-27). Compound 5 was identified as germanicol by com-parison of the physical and spectral data with the literature [9].

Compound 6 C29H48O, white needle, mp 155.0157.0 C. ESI-MS m/z 412 [M]+. 1H NMR (600 MHz, CDCl3) : 5.72 (1H, s, H-4), 1.19 (3H, s, H-19), 0.92 (3H, d, J = 6.4 Hz, H-21), 0.85 (3H, t, J = 8.1 Hz, H-29), 0.83 (3H, d, J = 7.2 Hz, H-27), 0.81 (3H, d, J = 6.9 Hz, H-26), 0.71 (3H, s, H-18). 13C NMR (150 MHz, CDCl3) : 199.7 (C-3), 171.7 (C-5), 123.7 (C-4), 56.0 (C-17), 55.9 (C-14), 53.8 (C-9), 45.8 (C-24), 42.4 (C-13), 39.6 (C-12), 38.6 (C-10), 36.1 (C-20), 35.7 (C-1),

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2011 5 9 3 Chin J Nat Med May 2011 Vol. 9 No. 3 193

35.6 (C-8), 34.0 (C-22), 33.9 (C-2), 32.9 (C-6), 32.0 (C-7), 29.1 (C-25), 28.1 (C-16), 26.1 (C-23), 24.1 (C-15), 23.0 (C-28), 21.0 (C-11), 19.8 (C-26), 19.0 (C-27), 18.8 (C-21), 17.4 (C-19), 11.9 (C-29), 11.9 (C-18). Compound 6 was identified as stigmast-4-en-3-one by comparison of the physical and spectral data with the literature [10].

Compound 7 C29H46O, white needle, mp 115116 C. ESI-MS m/z 410 [M]+. 1H NMR (600 MHz, CDCl3) : 5. 72 (1H, s, H-4), 5.15 (1H, dd, J = 15.4, 8.3 Hz, H-22), 5.02 (1H, dd, J = 15.4, 8.8 Hz, H-23), 1.18 (3H, s, H-19), 1.01 (3H, d, J = 6.6 Hz, H-21), 0.85 (3H, d, J = 6.1 Hz, H-27), 0.80 (3H, t, J = 7.7 Hz, H-29), 0.80 (3H, d, J = 6.1 Hz, H-26), 0.72 (3H, s, H-18). Compound 7 was identified as stigmast-4, 22-dien-3- one by comparison of the physical and spectral data with the literature [11].

Compound 8 C28H40O, yellow needle, mp 110113 C. ESI-MS m/z 392 [M]+. 1H NMR (600 MHz, CDCl3) : 6.61 (1H, d, J = 9.5 Hz, H-7), 6.03 (1H, d, J = 9.5 Hz, H-6), 5.74 (1H, s, H-4), 5.24 (2H, m, H-2223), 1.06 (3H, d, J = 6.7 Hz, H-21), 1.00 (3H, s, H-19), 0.96 (3H, s, H-18), 0.93 (3H, d, J = 6.8 Hz, H-28), 0.85 (3H, d, J = 8.0 Hz, H-27), 0.83 (3H, d, J = 7.1 Hz, H-26). Compound 8 was identified as ergosta-4, 6, 8 (14), 22-tetraen-3-one by comparison of the physical and spectral data with the literature [12].

Compound 9 C29H50O, white needle, mp 139140 C. 1H NMR (600 MHz, CDCl3) : 5.35 (1H, d, J = 4.9 Hz, H-6), 3.52 (1H, m, H-3), 1.01 (3H, s, H-19), 0.92 (3H, d, J = 6.6 Hz, H-21), 0.85 (3H, t, J = 7.2 Hz, H-29), 0.83 (3H, d, J = 6.6 Hz, H-27), 0.81(3H, d, J = 6.1 Hz, H-26), 0.68 (3H, s, H-18). Compound 9 was identified as -sitosterol by comparison of the physical and spectral data with the literature [13].

Compound 10 C29H48O, white needle, mp 154156 C. 1H NMR (600 MHz, CDCl3) : 5.35 (1H, m, H-6), 5.16 (1H, dd, J = 15.4, 8.3 Hz, H-22), 5.02 (1H, dd, J = 15.4, 8.8 Hz, H-23), 3.52 (1H, m, H-3), 1.02 (3H, d, J = 6.6 Hz, H-21), 1.01 (3H, s, H-19), 0.85 (3H, d, J = 6.6 Hz, H-26), 0.80 (3H, t, J = 7.7 Hz, H-29), 0.80 (3H, d, J = 6.6 Hz, H-27), 0.70 (3H, s, H-18). Compound 10 was identified as stigmasterol by com-parison of the physical and spectral data with the literature [14].

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1, 1, 2, 1* 1, 266003; 2, 266100

(Hibiscus tiliaceus L.)Sephadex LH-20 10 , (1)(2)-(3)(4)(5)-4--3-(6)-4, 22--3-(7)-4, 6, 8 (14), 22--3-(8)-(9)(10) 3~5 8

; ;

908 (No. 908-01-ST12)