Sesquiterpene lactones from Saussurea involucrata

Fitoterapia 82 (2011) 983–987

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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

Sesquiterpene lactones from Saussurea involucrata Wan Xiao a, Xian Li a, Ning Li a, Makabili Bolati b, Xinjun Wang b, Xiaoguang Jia b,⁎, Yuqing Zhao a, c,⁎⁎ a

Shenyang Pharmaceutical University, Shenyang 110016, People's Republic of China Xinjiang Institute of Chinese Materia Medica and Ethnodrug Urumqi 830002, People's Republic of China Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People's Republic of China b c

a r t i c l e

i n f o

Article history: Received 28 April 2011 Accepted in revised form 18 May 2011 Available online 27 May 2011 Keywords: Saussurea involucrate Guaiane-type sesquiterpene lactones CD technique Anti-inflammatory activities Cytotoxic activities

a b s t r a c t Bioassay-directed separation of the ethyl acetate extract from the aerial parts of Saussurea involucrata (Kar. et Kir. ex Maxim) led to the isolation of three new sesquiterpene lactones sausinlactones A (1), B (2), C (3) and six known ones (4–9). The structures were established by spectroscopic data (UV, IR, HRESIMS, 1D and 2D NMR). CD technique was also employed to determine the absolute configurations of new compounds 1–3. The anti-inflammatory activities of compounds 1–9 and antitumor activities of new compounds 1–3 were tested. The results presented that compounds 5 and 6 were responsible for anti-inflammatory activities, and compounds 1 and 2 showed significant cytotoxic activities against A549 cells. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Saussurea involucrata (Kar. et Kir. ex Maxim), a rare Chinese medicinal herb grown in Xinjiang Uygur Autonomous Region of China, has been used for the cure of rheumatic arthritis and gynopathy [1]. Coumarins, flavonoids [2], and sesquiterpene lactones [3,4] have been reported from this plant. Previously, flavonoids [5] were regarded as the principal components responsible for the anti-inflammatory effects of S. involucrata. Bioassay-directed separation of the ethyl acetate extract from the aerial parts of S. involucrata, nine sesquiterpene lactones (Fig. 1), including three new compounds (1–3) and six known ones (4–9), were obtained. In general, the ring junctions in guaiane-type sesquiterpene lactones are cis at C ⁎ Correspondence to: Xinjiang Institute of Chinese Materia Medica and Ethnodrug Urumqi 830002, People's Republic of China. ⁎⁎ Correspondence to: Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People's Republic of China. Tel.: + 86 24 23986521; fax: + 86 24 23986522. E-mail address: [email protected] (Y. Zhao). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.05.015

(1)–C (5) [3,4], whereas the three new sesquiterpene lactones described in this paper possess trans fusions at C (1)–C (5). Anti-proliferation and nitric oxide inhibition assays on RAW 264.7 cells showed that the major guaiane lactones such as compounds 5 and 6 were responsible for the antiinflammatory activities of the plant. Guaiane-type sesquiterpene lactones are known to possess antitumor activities [6,7]. Compounds 1–3 were tested in vitro for their cytotoxic activities against the human cancer cell lines Hep-3B, A549, and U87. Compounds 1 and 2 showed significant cytotoxic activities against A549 cells. 2. Experimental 2.1. Generals Column chromatography (cc): silica gel (SiO2: 200–300 mesh, Qingdao Marine Chemical Group, Co.); macroporous resin D101 (Hebei, Co.); Sephadex LH-20 (Pharmacia, Co.). Prep. HPLC (Beijing CXTH3000 system): P3000 pump, UV3000 spectrophotometric detector at 203 nm, Daisogel C 18 reversed-phase column (10 μm, 30 × 250 nm). UV

984

W. Xiao et al. / Fitoterapia 82 (2011) 983–987 14 H 3

HO

2 4

H 1

10

5 6

15

9 8 7

H

HO

HO

OH

H

11 O 12

H

H O

O

13

O

O

1

O

2

3

H

HOH2C H

R1

OH

R2 H H

O

O O O 4 R1=OH, R2 =OH 5 R1=H, R 2 =OH 6 R1=H, R 2 =O- -Glc 7 R1=H, R 2 =O- 6'-O-acetyl- -Glc 8 R1=O- -Glc, R2=H

9

Fig. 1. Structures of compounds 1–9.

Spectra: Shimadzu UV-2201 spectrophotometer; MeOH soln.; in λmax (logε). IR Spectra: Bruker IFS-55 spectrophotometer. Optical rotations: Perkin-Elmer polarimeter. 1H and 13C NMR Spectra: Bruker AV-600 and ARX-300 spectrometer; δ in ppm rel. to Me4Si as internal standard, J in Hz. HR-TOF-MS: Bruker micro TOF-Q mass spectrometer; in m/z (rel.%).

and 2 (40 mg, tR = 20 min). F2–3 was also separated by reversed-phase preparative HPLC (YMC-pack ODS, 250 × 10 mm, 65% MeOH) to afford 3 (30 mg, tR = 40 min). F4 [CH2Cl2: MeOH (100:5)] to afford 4(10 mg) and 9(15 mg). F6 [CH2Cl2: MeOH (100:8)] to afford 6 (200 mg), 8 (13 mg), and 7 (20 mg).

2.2. Plant material

2.3.1. Sausinlactones A-(1S, 3S, 5S, 6S, 7S, 11S)-3-hydroxyl-11, 13-dihydrodehydrocostuslactone (1) Brown oil; [α] 20 D + 85.5°(c = 0.36,MeOH); IR (KBr) νmax3460, 1756, 1635 cm − 1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 249.1481 [M + H] + (calcd. for C15H20O3, 249.1485).

The aerial parts of S. involucrata (Kar. et Kir. ex Maxim) were collected from Xinjiang Province, China, in Aug. 2008. The plant material was identified by Prof. Xiaoguang Jia (Xinjiang Institute of Chinese Material Medica and Ethnodrug). A voucher specimen (No. 200800823) is deposited in School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. 2.3. Extraction and isolation Dried aerial parts of S. involucrata (4.5 kg) were extracted three times with 95% EtOH under reflux for 3 h. The organic solvent was concentrated to afford a crude extract. The extract was suspended in H2O and then partitioned successively with petroleum ether, ethyl acetate and n-BuOH. Then the anti-inflammatory activities of the total extract, petroleum ether Fr., ethyl acetate Fr. and n-BuOH Fr. were investigated. The results indicated that the ethyl acetate Fr. possessed good anti-inflammatory activity. The ethyl acetate Fr. (60.0 g) was subjected to a silica gel column chromatography, using a gradient of MeOH in CH2Cl2, to give ten fractions (F1–F10). F1, F2 and F6 possessed good anti-inflammatory activities. F1 [CH2Cl2: MeOH (100:2)] to afford 5 (70 mg). F2 [CH2Cl2: MeOH(100:3)] was separated into eight fractions (F2–1–F2–8) by HPLC (ODS, 60% MeOH). F2–3 was purified by reversed-phase preparative HPLC (YMC-pack ODS, 250 × 10 mm, 60% MeOH) to afford 1 (25 mg, tR = 18 min)

2.3.2. Sausinlactones B-(1S, 3S, 5S, 6S, 7S, 11R)-3-hydroxyl-11, 13-dihydrodehydrocostuslactone (2) Colorless needles, mp 135–137 °C; [α] 20 D + 125.9° (c = 0.13,MeOH); IR (KBr) νmax 3460, 1756, 1635 cm − 1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 249.1478 [M + H] +( calcd. for C15H20O3, 249.1485). 2.3.3. Sausinlactones C-(1S, 3S, 5S, 6S, 7S, 8S, 11S)-3-hydroxyl11,13-dihydrodehydrocostuslactone (3) Brown oil; [α] 20D + 51.5°(c = 0.33, MeOH); IR (KBr) νmax 3465, 3409, 1756, 1635 cm − 1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 265.1428 [M + H] + (calcd. for C15H20O4, 265.1434). 2.4. Bioassay procedure In vitro anti-inflammatory activities were evaluated by monitoring the proliferation of RAW264.7 cells and the Nitric Oxide (NO) levels. RAW264.7 cells were cultured to the log phase, MTT method tested the effect of different concentrations (25, 50, 100, and 200 μmol/L) of sample on proliferation of cells in response to LPS. Griess kit measured the effect of

W. Xiao et al. / Fitoterapia 82 (2011) 983–987 Table 1 1 H and 13C NMR data for compounds 1, 2, and 3 (Pyridine-d5, 1H NMR 300MHZ, No.

1

43.3(CH) 39.3(CH2)

3 4 5 6 7 8

72.9(CH) 155.2(C) 49.4(CH) 84.1(CH) 50.3(CH) 32.7(CH2)

9

37.0(CH2)

10 11 12 13 14

149.5(C) 42.0(CH) 178.5(C) 13.4(CH3) 108.7(CH2)

15

112.8(CH2)

a

C NMR 75MHZ, δppm, J in Hz).a

2 δH( J in Hz)

δC 1 2

13

985

3

δC

2.79 m 2.00 m 2.39 m 4.78 t (8.1)

δH( J in Hz)

43.2(CH) 39.2(CH2) 72.9(CH) 155.3(C) 49.7(CH) 84.2(CH) 45.4(CH) 28.9(CH2)

2.80 m 4.08 t (9.8) 1.80dd (9.8, 10.4) 1.12 m 1.90 m 2.40 m 1.85 m

149.5(C) 39.4(CH) 179.6(C) 11.5(CH3) 108.7(CH2)

1.15 d (7.0) 5.67 br.s 5.53 br.s 5.08 s 4.98 s

43.5(CH) 39.4(CH2) 72.8(CH) 155.3(C) 50.3(CH) 80.1(CH) 56.3(CH) 75.4(CH2)

2.81 m 4.20 t (9.3) 2.23 m 1.21 ddd (4.5,12.3) 1.63 m 1.85ddd(4.5, 12.3) 2.40 m

36.8(CH2)

2.26 m

δC

2.80 m 1.95 m 2.38 m 4.78 t (8.1)

47.8(CH2) 145.5(C) 42.0(CH) 179.3(C) 16.8(CH3) 108.8(CH2)

2.65 m 1.02 d (7.8) 5.69 br.s 5.56 br.s 5.06 s 4.88 s

112.7(CH2)

114.7(CH2)

δH( J in Hz) 2.91 m 2.42 m 2.01 m 4.81 t (7.8) 2.90 m 4.20 t (9.6) 2.37 m 3.90 m 2.39 m 2.94 m 2.81 1.67 d (7.2) 5.68 br.s 5.58 br.s 5.18 s 4.82 s

The assignments were based on 1H-1H COSY, HMQC, and HMBC experiments.

H-11, H-5/H-3, H-5/H-7α, revealed the β-orientation of H-1 and the hydroxyl group at C-3, and the α-orientation of the 11-methyl function at C-11. In the CD spectrum of 1, a strong positive Cotton effect was presented at 220 nm [11], therefore, the structure and absolute configuration of 1 were elucidated, and 1 was named (1S, 3S, 5S, 6S, 7S, 11S)-3hydroxyl-11, 13-dihydrodehydrocostuslactone (sausinlactones A). Compound 2 was obtained as colorless needle (MeOH). Its molecular formula was determined as C15H20O3 by HRESIMS (m/z 249.1478, [M + H] +). The 1H and 13C NMR spectroscopic data of 2 were very close to those of 1. The major differences between the 1H and 13C NMR data of these two compounds were the chemical shifts of C-7, C-8, C-11, C-13. The NOE correlations (Fig. 3) of H-1/H-6β, H-1/H-9β, H-1/H-8β, H-5/ H-3, H-5/H-7α, H-6/11-Me, without the correlation between H-6 and H-11, implied that the methyl at C-11 in β configuration. The CD spectrum showed a positive Cotton effect at 220 nm, therefore, the structure and absolute configuration of 2. Thus, compound 2 was identified as (1S, 3S, 5S, 6S, 7S, 11R)-3-hydroxyl-11, 13-dihydrodehydrocostuslactone (sausinlactones B). Compound 3 was obtained as brown oil (MeOH). Its molecular formula was determined as C15H20O4 by HRESIMS (m/z 265.1428, [M + H] +). The 1H NMR spectrum showed

those concentrations of sample on NO secretion of RAW 264.7 cells in response to LPS [8,9]. Cytotoxic assays of compounds 1–3 against the Hep-3B, A549, and U87 cell lines were carried out using the MTT method [10], as described previously. 3. Results and discussion Compound 1 was obtained as brown oil (MeOH). Its molecular formula was determined as C15H20O3 by HRESIMS (m/z 249.1481, [M + H] +). The 1H NMR spectrum showed one methyl group at δ 1.15 (3H, d, J = 7.2 Hz, H-13), 6,7lactone group at δ 4.08 (1H, t, J = 9.8 Hz, H-6), 1.80 (1H, dd, J = 9.8, 10.4 Hz, H-7), four olefinic protons at δ 5.08 and δ 4.98 (2H, each s), 5.67 and 5.53 (2H, each brs). The 13C NMR spectrum showed 15 carbon signals, consisting of four olefinic carbon signals at δ 108.4, 112.8, 149.5, 155.2, a carbonyl carbon signal at δ 178.5, and two oxygenated carbon signals at δ 72.9 and δ 84.1, one methyl signal at δ 13.4. The data were closely comparable to those of 11,13α-dihydrozaluzanin C. By analyzing the HMQC, HMBC (Fig. 2), and 1H– 1H COSY spectra, compound 1 was elucidated as a guaiane-type sesquiterpene lactone. The relative configuration of 1 was determined by the NOE correlations observed in the NOESY spectrum. The NOE correlations (Fig. 3) of H-1/H-6β, H-1/H-9β, H-1/H-8β, H-6β/

H

H

H HO

HO

HO H

H O

H

CH 3

H

H O

H

O 1

OH H

H CH 3

O 2 Fig. 2. The important HMBC correlations of 1, 2, and 3.

O H O 3

CH 3

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W. Xiao et al. / Fitoterapia 82 (2011) 983–987

1

2

Fig. 3. Selected NOESY correlations of compounds 1 and 2.

80

*

5

6

7

8

9

43.8 40.5 73.9 156.4 50.7 80.8 56.3 75.4 49.4 146.0 42.0 179.1 16.9 113.8 111.8

47.4 32.4 30.2 151.3 52.7 80.3 55.5 75.3 47.0 144.6 41.8 179.4 16.2 109.9 114.6

46.8 31.9 29.5 152.2 52.3 79.3 53.3 83.0 44.0 145.2 40.2 179.0 16.2 108.8 114.1 103.9 73.9 77.3 70.2 76.8 61.3

47.6 32.5 30.1 152.4 53.1 80.8 54.4 84.5 45.2 145.8 41.5 179.2 16.9 109.4 114.3 105.4 75.2 78.8 71.5 78.8 64.7 170.7 20.8

42.6 36.9 79.5 150.5 49.2 83.1 41.1 31.6 35.9 149.6 48.3 178.4 13.0 112.7 110.0 103.0 73.7 76.9 70.3 79.5 61.3

48.5 24.9 29.8 151.8 51.8 80.9 42.8 24.4 28.6 75.0 47.0 178.5 13.1 68.3 108.3

The assignments were based on HMQC, HMBC experiments.

* *

40

* 20

C NMR 75MHZ, δppm, J in

4

* *

60

13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 1″-C=O 2″-CH3

ethyl acetate Fr compound 5 compound 6 rolipram

100

0

No.

a

A

* *

*

*

* *

*

*

**

25

50

100

200

Concentrations(µM)

B

Concentration of nitrite (nmol/L)

Table 2 13 C NMR data for compounds 4–9 (Pyridine-d5, Hz).a

The known compounds 4–9 were identified as 3α, 8αdihydroxyl-11βH-11, 13- dihydrodehydrocostuslactone (4), 8α-hydroxyl-11βH-11,13-dihydrodehydrocostuslactone (5), 11β,13-dihydrodehydrocostuslactone-8-O-β-D-glucoside (6), 11β,13-dihydrodehydrocostuslactone-8-O-[6′-O-acetyl-βD-glucoside] (7), 11α,13 -dihydroglucozaluzanin C (8), japonicolactone (9) on the basis of the NMR data (Table 2) [12–15].

Inhibition (%)

6,7-lactone group at δ 4.08 (1H, t, J = 9.8 Hz, H-6), 1.80 (1H, dd, J = 9.8, 10.4 Hz, H-7), four exomethylene protons at δ 5.08 and δ 4.98 (2H, each s), δ 5.67 and δ 5.53 (2H, each brs). The 13 C NMR spectrum showed 15 carbon signals, consisting of four olefinic carbon signals at δ 108.8, 114.7, 145.5, 155.3, a carbonyl carbon signal at δ 178.5, and three oxygenated carbon signals at δ 72.8, 75.4, and δ 80.1. The 1H and 13C NMR spectroscopic data as well as the observed 1H– 1H COSY, HMQC, HMBC (Fig. 2), The NOE correlations suggested that compound 3 was a guaiane-type sesquiterpene lactone. The NOE correlations of H-1/H-6β, H-1/H-9β, H-1/H-8, H-6β/H11, H-5/H-3, H-5/H-7α, revealed the β-orientation of H-1 and the hydroxyl group at C-3, and the α-orientation of the hydroxyl group at C-3 and the 11-methyl function at C-11.The CD spectrum showed a positive Cotton effect at 220 nm, therefore, the structure and absolute configuration of 3 were elucidated, and 3 was named (1S, 3S, 5S, 6S, 7S, 8S, 11S)-3hydroxyl-11, 13-dihydrodehydrocostuslactone (sausinlactones C).

30

ethyl acetate Fr

#

25

compound 5 compound 6

# #

20

* *

15

* *

**

*

10

*

5 0

* * * *

control

LPS

25

50

100

200

LPS+sample(µM)

Fig. 4. Anti-inflammatory effects of compounds 5 and 6. (A)Effects of 5 and 6 on the proliferation of RAW 264.7 cells. (B) Effects of 5 and 6 on the NO production of RAW 264.7 cells. RAW264.7 cells were cultured at the log phase. MTT method tested the effect of different concentrations (25, 50, 100, and 200 μmol/L) of sample on proliferation of cells in response to LPS. Griess kit measured the effect of those concentrations of sample on NO secretion of RAW 264.7 cells in response to LPS. Each value indicates the mean ± SEM (n = 3), #P b 0.01, compared with control, *P b 0.01, compared with LPS.

W. Xiao et al. / Fitoterapia 82 (2011) 983–987

Then, the anti-inflammatory activities of compounds 5 and 6 were investigated. Compound 6 showed anti-proliferation activities of RAW 264.7 cell with IC50 ± SD values of 98.01 ± 2.11 μM. The results (Fig. 4) showed that 25 ~200 μmol/L of 5 and 6 inhibited the proliferation of RAW 264.7 cells in response to LPS, and inhibited NO secretion of RAW 264.7 cells in response to LPS in a dose dependent manner. So compounds 5 and 6 can inhibit the proliferation and NO secretion of RAW 264.7 cell in response to LPS obviously. Three new compounds were evaluated for their cytotoxic activity against three tumor cell lines, namely, Hep-3B, A549, and U87. Compounds 1 and 2 showed significant cytotoxic activities against A549 cells with IC50 ± SD values of 0.01 ± 0.12, 2.89 ± 0.11 μM. Acknowledgments The project was financially supported by the Technical Innovation Foundation of Xinjiang Provincial Economic and Trade Commission (No. 2007001), E&T Modern Center for Natural Products of Liaoning Province of China (No. 2008402021) and the National Key Technologies R & D Program of China during the 11th Five-Year Plan Period (2009ZX09501-011). Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.fitote.2011.05.015.

987

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