玉柏石松三萜成分研究

玉柏石松三萜成分的研究

药学学报 Acta Pharmaceutica Sinica 2009, 44 (8): 891?894 · 891 ·

Triterpenoid from Lycopodium obscurum L.

DENG Tie-zhong, AI Yong, CHEN Yu, YANG Guang-zhong*

(Laboratory for Natural Product Chemistry, College of Pharmacy, South Central University for Nationalities, Wuhan 430074, China)

Abstract: To study the triterpenoid constituents from air-dried whole plants of Lycopodium obscurum L., the constituents were isolated by normal-phase and reverse-phase silica gel column chromatorgraphy from the EtOAc extract. Their structures were elucidated on the basis of spectral analysis. Five triterpenoids were purified and identified as 3β, 19α-dihydroxy-20β-acetate-serrat-14-en-21β-ol (1), serratenediol (2), α-onocerin (3), 26-nor-8-oxo-α-onocerin (4), (3β, 8β, 14α, 21α)-26, 27-dinoronocerane-3, 8, 14, 21-tetrol (5). Compound 1 is a new serratane-type triterpene and compound 5 is isolated from this plant for the first time.

Key words: Lycopodium; Lycopodium obscurum L.; serratane triterpenoid

CLC number: R284.1 Document code: A Article ID: 0513-4870 (2009) 08-0891-04

玉柏石松三萜成分研究

邓铁忠, 艾 勇, 陈 玉, 杨光忠*

(中南民族大学药学院天然产物化学研究室, 湖北 武汉 430074)

摘要: 为了研究玉柏石松全草的三萜类成分, 运用正相和反相硅胶柱色谱技术对玉柏石松的乙酸乙酯提 取物进行分离纯化, 并运用波谱技术对其结构进行鉴定。共分离得到5个三萜类化合物, 其结构分别鉴定为3β, 19α-dihydroxy-20β-acetate-serrat-14-en-21β-ol (1), serratenediol (2), α-onocerin (3), 26-nor-8-oxo-α-onocerin (4), (3β, 8β, 14α, 21α)-26, 27-dinoronocerane-3, 8, 14, 21-tetrol (5)。其中化合物1为新的石松型三萜化合物, 化合物5为首次从该植物中分离得到。

关键词: 石松属; 玉柏石松; 石松三萜

Lycopodium obscurum L. is widely used as tradi-tional Chinese herbal medicine for the treatment of arthritic pain, quadriplegia, dysmenorrhea, and contu-sion[1]. Previous phytochemical studies of this plant had showed that serratane-type triterpenoids[2] or derivatives of its biogenetic precursor α-onocerin[3] were well-known characteristic constituents. Serratane is an unique family of pentacyclic triterpenoids possessing seven tertiary methyls and a central seven-member C-ring, usually with a C=C bond between C (14) and C (15) and O-functionalities at C (3) and C (21)[4]. Pharmacological studies have shown that serratane-type

Received 2009-02-16.

Project supported by National Natural Science Foundation of China

(No.30670215).

*

Corresponding author Tel / Fax: 86-27-67841196,

E-mail: yanggz888@http://wendang.chazidian.com

triterpenoids have pharmacological action, including cancer chemopreventive activity[5] and inhibitory effects against Candida albicans secreted aspartic proteases[6]. As part of research projects on the study of ethnomedi-cinal plants, we have investigated L. obscurum and reported herein the isolation and structure elucidation of a new serratane-type triterpenoid together with four known compounds: serratenediol (2), α-onocerin (3), 26-nor-8-oxo-α-onocerin (4), and (3β, 8β, 14α, 21α)-26, 27-dinoronocerane-3, 8, 14, 21-tetrol (5) (Figure 1).

Compound 1 was obtained as white amorphous powder, its molecular formula was deduced as C32H52O5 by HR-EI-MS (found [M]+ m/z 516.380 7, calcd. 516.381 4), requiring seven degrees of unsatura-tion. The NMR spectral showed the presence of an acetyl group at δC 171.4, δC 21.3, and δH 1.93 (3H, s), which was supported by the ion fragment at m/z

玉柏石松三萜成分的研究

· 892 · 药学学报 Acta Pharmaceutica Sinica 2009, 44 (8): 891?894

Figure 1 Structures of compound 1?5

456.359 2 [M-CH3COOH]+. With the exception of an

acetyl group signal, its 1H NMR (Table 1) showed seven tertiary methyls (δ 0.83 (3H, s), 0.87 (3H, s), 1.06 (3H, s), 1.17 (3H, s), 1.20 (3H, s), 1.24 (3H, s) and 1.26 (3H, s)), three hydroxy groups (δ 6.30 (1H, d, J = 4.8 Hz), 6.70 (1H, d, J = 4.2 Hz), 5.80 (1H, s)), and four oxygenated methines (δ 3.52 (1H, dd, J = 14.8, 8.4 Hz), 4.08 (1H, br s), 4.74 (1H, dd, J = 10.0, 4.8 Hz), 5.71

(1H, dd, J = 10.0, 2.4 Hz)), and an olefinic proton δ 5.54 (1H, br s). The 13C NMR (Table 1) and DEPT spectrum of 1 displayed 32 carbon signals, including a C=C bond at δC 139.8 (s), δC 122.8 (d), four oxygenated methines, seven methyls, eight methylenes, four methines, as well as five quarternary carbons. All the above data suggested that 1 belongs to a serratane-type triterpenoid with an acetyl group as ester substituent[7]. Comparison of the 1H and 13C NMR spectroscopic data of 1 with those of the known com-pounds serratenediol[8], showed that the two compounds were very similar. Compound 1 and serratenediol were mainly different in the ring E. With the help of 1

H-1H COSY and HSQC spectrum, the partial structure -CH(O)-CH(O)-CH(O)- was deduced in ring E. Thus, two CH2 (19) and CH2 (20) signals in serratenediol were replaced by two oxygenated CH in 1 which was supported by HMBC correlations H-19/C-13 (δC 58.9), C-18 (δC 43.1), C-20 (δC 76.7), and H-20/ C-19 (δC 74.6), and H-21/C-17 (δC 43.3), C-19 (δC 74.6), C-20 (δC 76.7), C-22 (δC 38.5). The ester unit was positioned at C-20 on the basis of HMBC correlation H-20/δC 171.4. The relative configuration of 1 was derived from the ROESY spectrum. The β-orientation of the 3-OH was deduced from the correlations of H-3/ H3-23, H-5. The ROSEY correlations H-19/H-13 and H-13/

H3-26 suggested that 19-OH was α-oriented. The 20-OAc and 21-OH was deduced to be in β-position which was based on the correlations H3-29/H-20, H-21 and H-20/H-21 (Figure 2). Therefore, compound 1 was elucidated as 3β, 19α-dihydroxy-20β-acetate-serrat- 14-en-21β-ol.

Table 1 1H NMR, 13C NMR and HMBC data of compound 1 (in C5D5N)

Position 1

H NMR

13

C NMR HMBC

1 0.95 (1H, m) 38.8

1.71 (1H, m) 2 1.88 (2H, m)

28.1

3 3.52 (1H, dd, J = 14.8, 77.8 C (2), C (4), C (23), C (24) 8.4 Hz) 4

39.4

5 0.85 (1H, m) 55.8 C (4), C (10) 6 1.54 (2H, m) 19.2

7 1.43 (2H, m) 45.3 C (5), C (8) 8

37.2

9 0.81 (1H, m) 62.8 C (8), C (10) 10

38.1 11 1.85 (2H, m)

27.5

12 1.91 (1H, m)

26.9 C (9), C (13), C (14) 3.45 (1H, t, J = 7.8 Hz) 13 2.48 (1H, br d, J = 58.9

11.4 Hz) 14

139.8 15 5.54 (1H, br s) 122.8 16 2.10 (2H, m) 24.7

17 2.40 (1H, m) 43.3 C (13), C (16), C (18), C (22), C (29) 18

43.1

19 4.74 (1H, dd, J = 10.0, 74.6 C (13), C (18), C (20) 4.8 Hz)

20 5.71 (1H, dd, J = 10.0, 76.7 C (19), C (31) 2.4 Hz)

21 4.08 (1H, br s) 77.2 C (17), C (19), C (20), C (22) 22

38.5

23 1.20 (3H, s) 28.5 C (3), C (4), C (5), C (24) 24 1.06 (3H, s) 16.2 C (3), C (4), C (5), C (23) 25 0.83 (3H, s) 15.9 C (1), C (5), C (9), C (10) 26 0.87 (3H, s) 20.0 C (7), C (8), C (9), C (27) 27 1.88 (1H, s) 56.3 C (7), C (8), C (9), C (13), 2.45 (1H, m) C (14), C (15), C (26) 28 1.26 (3H, s) 9.8 C (13), C (18), C (17), C (19) 29 1.17 (3H, s) 21.3 C (17), C (21), C (22), C (30) 30 1.24 (3H, s) 28.4 C (17), C (21), C (22), C (29) 31

171.4 32 1.93 (3H, s)

21.3 C (31) OH 6.70 (1H, d, J = 4.2 Hz) OH 6.30 (1H, d, J = 4.8 Hz) OH

5.80 (1H, d, br s)

玉柏石松三萜成分的研究

DENG Tie-zhong, et al: Triterpenoid from Lycopodium obscurum L.

· 893 ·

Figure 2 Key ROESY of compound 1

Table 2 13C NMR data of compound 2?5

Position 2a 3a 4a 5b 1 38.0 37.4 37.7 36.3 2 28.8 28.6 29.1 27.8 3 78.3 77.9 78.1 79.7 4 40.0 39.6 40.0 39.0 5 56.4 54.8 55.3 56.0 6 19.8 24.4 24.7 17.6 7 45.9 38.5 42.8 41.1 8 37.8 149.1 211.9 67.7 9 63.3 57.7 65.1 56.1 10 39.4 39.6 42.9 38.7 11 25.9 23.0 22.0 23.5 12 27.9 23.0 24.2 23.5 13 58.0 57.7 58.0 56.1 14 139.1 149.1 148.8 67.2 15 123.1 38.5 39.0 41.1 16 25.0 24.4 24.5 17.6 17 50.4 54.8 53.9 56.6 18 38.8 39.6 40.0 38.7 19 36.8 37.4 37.9 36.3 20 29.1 28.6 29.1 27.8 21 78.6 77.9 78.4 79.7 22 39.8 39.6 40.1 39.0 23 28.7 28.8 29.2 29.0 24 16.5 16.2 16.6 16.6 25 16.8 14.7 15.2 16.3 26 20.5 106.7 27 56.9 106.7 108.3 28 14.2 14.7 15.3 16.3 29 15.9 16.2 16.4 16.6 30

29.0

28.8

29.2

29.0

a

in C5D5N; bin CD3OD

Experimental

General experimental procedures NMR spectra were run on Bruker AM-300 and 600 spectrometers with TMS as internal standard. EI-MS and HR-EI-MS were measured with a Finnigan MAT 95 instrument. Thin-layer chromatography (TLC) was performed on silica gel 60 GF254, and column chromatography was carried using silica gel (200?300 mesh) from Qingdao Haiyang Chemical Group Co., China and C18 reversed- phase silica gel from YMC CO., LTD., Japan.

Plant material The whole plant of Lycopodium obscurum L. was collected from Jianshi county, Hubei province, China and identified by Professor WAN Ding-rong, College of Pharmacy, South Central University for Nationalities.

Extraction and isolation The air-dried whole plant of Lycopodium obscurum L. (12.3 kg) was powdered and then extracted with MeOH three times at room temperature. The MeOH extract (1.65 kg) was suspended in 3% tartaric acid/H2O (pH 3) and then par-titioned with EtOAc. The EtOAc extract (890 g) was suspended in 90% H2O/MeOH and then successively partitioned with petroleum ether (PE), EtOAc and n-BuOH. The EtOAc extract (324 g) was subjected to CC (silica gel, PE:acetone 9∶1, 8∶2, 7∶3, 1∶1, 3∶7, 0∶1, v/v) to give 9 fractions (Fr.1?Fr.9). Fr.5 was subjected to CC (silica gel, CHCl3-acetone 1∶0 → 1∶1) to give 5 subfractions (Fr.5.1?Fr.5.5). Fr.5.4 was subjected to CC (ODS, H2O-MeOH 9∶1 → 1∶9) to give compound 1 (4 mg). Compound 3 (40 mg) was recrystallized from Fr.5.2. Fr.5.3 was subjected to CC (silica gel, cyclohexane-EtOAc 9∶1 → 1∶1) to give 4 subfractions (Fr.5.3.1?Fr.5.3.4), Fr.5.3.3 was subjected to CC (ODS, H2O-MeOH 7∶3 → 3∶7) to give compound 4 (10 mg). Fr.2 was subjected to CC (silica gel, cyclohexane-acetone 99∶1 → 7∶3) to give 5 subfractions (Fr.2.1?Fr.2.5). Fr.2.3 was subjected to CC (silica gel, cyclohexane-EtOAc 98∶2 → 9∶1) to obtain compound 2 (29 mg). Fr.7 was subjected to CC (ODS, H2O-MeOH 7∶3 → 1∶0) to give 7 subfractions (Fr.7.1?Fr.7.7). Fr.7.4 was subjected to CC (silica gel, CHCl3-EtOAc 9∶1 → 1∶1) to give 4 subfractions (Fr.7.4.1?Fr.7.4.4). Fr.7.4.2 was subjected to CC (ODS, H2O-MeOH 7∶3 → 3∶7) to give compound 5 (10 mg).

Identification of compounds 1?5

Compound 1: white amorphous powder, C32H52O5, EI-MS (70 eV) m/z 516 [M]+ (32), 456 (19), 438 (20), 294 (23), 207(95), 190 (100), 189 (74), 135 (42), 121(51), 107(42); HR-EI-MS m/z 516.380 7 (calcd. for C32H52O5, 516.381 4). 1H NMR and 13C NMR data were listed in Table 1.

Compound 2: white amorphous powder, C30H50O2, 1

H NMR (600 MHz, C5D5N) δ: 0.81 (3H, s, CH3), 0.89 (3H, s, CH3), 0.96 (3H, s, CH3), 1.08 (3H, s, CH3), 1.13

玉柏石松三萜成分的研究

· 894 · 药学学报 Acta Pharmaceutica Sinica 2009, 44 (8): 891?894

(3H, s, CH3), 1.21 (3H, s, CH3), 1.24 (3H, s, CH3), 3.54 (1H, dd, J = 10.2, 5.4 Hz, H-3), 3.49 (1H, dd, J = 8.4, 7.8 Hz, H-21), 5.49 (1H, s, H-15), 5.85(1H, br s, OH), 5.95 (1H, br s, OH); 13

C NMR data were listed in Table 2. These data were identical to the literature values of serratenediol[8].

Compound 3: white amorphous powder, C30H50O2,

1

H NMR (600 MHz, C5D5N) δ: 0.78 (6H, s, H3-25, 28),

1.05 (6H, s, H3-24, 30), 1.23 (6H, s, H3-23, 29), 3.52

(2H, dd, J = 11.4, 4.8 Hz, H-3, 21), 4.80 (2H, s, H2-26,

27), 5.05 (2H, s, H2-26, 27), 5.90 (2H, s, 3, 21-OH); 13C NMR data were listed in Table 2. These data coincide with the literature values of α-onocerin[3].

Compound 4: white amorphous powder, C29H48O3,

1H NMR (600 MHz, C5D5N) δ: 0.73 (3H, s, CH3), 0.74

(3H, s, CH3), 0.98 (3H, s, CH3), 1.00 (3H, s, CH3), 1.20

(3H, s, CH3), 1.25 (3H, s, CH3), 3.50 (2H, m, H-3, 21),

5.07 (1H, s, H2-27), 5.29 (1H, s, H2-27); 13C NMR data

were listed in Table 2. These data agree with the literature values of 26-nor-8-oxo-α-onocerin[3].

Compound 5: white amorphous powder, C28H50O4,

1

H NMR (300 MHz, CD3OD) δ: 0.79 (6H, s, 2×CH3),

0.97 (6H, s, 2×CH3), 0.98 (6H, s, 2×CH3), 3.12 (2H, dd,

J = 11.7, 6.0 Hz, H-3, 21), 3.94 (2H, br s, H-8, 21); 13

C NMR data were listed in Table 2. These data correspond to the literature values of (3β, 8β, 14α, 21α)-26, 27-dinoronocerane-3, 8, 14, 21-tetrol[9].

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