Pentacyclic triterpenes from the resin of Liquidambar formosana

Fitoterapia 82 (2011) 927–931

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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

Pentacyclic triterpenes from the resin of Liquidambar formosana Nian-Yun Yang a, Jian-Hua Chen b, Gui-Sheng Zhou a, Yu-Ping Tang a, Jin-Ao Duan a,⁎, Li-Juan Tian c, Xun-Hong Liu d a b c d

Jiangsu Key Laboratory for TCM Formulae Research, Nanjing University of Traditional Chinese Medicine, Nanjing 210046, China Affiliated Maoming People's Hospital of Southern Medical University, Maoming 525000, China Technique Centre, Jinling Pharmaceutical Co., Ltd., Nanjing 210009, China College of Pharmacy, Nanjing University of Traditional Chinese Medicine, Nanjing 210046, China

a r t i c l e

i n f o

Article history: Received 1 April 2011 Accepted in revised form 4 May 2011 Available online 14 May 2011 Keywords: Liquidambaris Resina Hamamelidaceae Pentacyclic triterpene Cytotoxicity MDA-MB-435S Antiplatelet aggregation

a b s t r a c t Two new pentacyclic triterpenes and eight known pentacyclic triterpenes were isolated from the petroleum ether extract of Liquidambaris Resina. The structural elucidation of these compounds was determined by spectroscopic data interpretation. Their cytotoxic and antiplatelet aggregation activities were examined to find potent cytotoxic and antiplatelet aggregation compounds from natural resources. The results showed that 3-keto oleane triterpenes had strong cytotoxicity against MDA-MB-435S cancer cells and that 28-carboxyl oleane triterpenes possessed significant inhibition of platelet aggregation induced by ADP. © 2011 Elsevier B.V. All rights reserved.

1. Introduction

2. Experimental

The species of Liquidambar (Hamamelidaceae) are known for their balsamic exudations. Liquidambaris Resina is the resin of Liquidambar formosana Hance, and often used for treatment of carbuncle, herpes, swelling ulcer, hemorrhinia and toothache in Chinese medicine. Modern studies have indicated that Liquidambaris Resina mainly consisted of the chemical constituents of triterpenoids, cinnamic acid and its derivatives, and possessed the pharmacological action of antitumor, antiplatelet aggregation and anti-arrhythmia [1]. In order to further elucidate the chemical composition and bioactivities of the resin of L. formosana, we have investigated its chemical constituents and isolated ten triterpenes. This paper describes the isolation, structure elucidation, cytotoxic and antiplatelet aggregation activities of 10 triterpenes from the resin of L. formosana, including two new compounds 1–2.

2.1. General experimental procedures

⁎ Corresponding author. E-mail address: [email protected] (J.-A. Duan). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.05.003

Optical rotations were measured on a JASCO P-1020 Polarimeter. IR spectra were recorded on a Nicolet IR-100 FT-IR spectrometer with KBr disks. NMR spectra were measured on a Bruker AV-300 MHz (300 MHz for 1H NMR and 75 MHz for 13C NMR). ESI-MS and HRESI-MS spectra were tested on a Micromass Q/TOF Mass Spectrometer. Anticoagulative assay was performed on coagulation analysis instrument LG-PABER-I. The blood sample was treated on Anke TDL-40B centrifugal machine. Silica gel for column chromatography (CC, 200–300 mesh) and silica gel thin layer chromatography plates were the products of Qingdao Haiyang Chemical Co., Ltd (Qingdao, China). All solvents used were of analytical grade (Nanjing Chemical Plant, Nanjing, China). Adriamycin was purchased from Qilu Pharmaceutical Co., LTD (Ji'nan, Shangdong, China), whereas fetal bovine serum (FBS) was from Sijiqing Bioengineering Institute (Hangzhou, Zhejiang, China), and diphenyltetrazolium bromide (MTT) was from Gibco (Grand Island, New York, USA). Adenosine diphosphate (ADP) was purchased from Beijing Zhongqin Scientific

928 Table 1 1 H (300 MHz) and Position

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

N.-Y. Yang et al. / Fitoterapia 82 (2011) 927–931

13

C NMR (75 MHz) spectroscopic data of compounds 1, 2 and 10 (in CDCl3) (δ, ppm; J, Hz).

1

2

10

δH (J, Hz)

δC

δH (J, Hz)

δC

δH (J, Hz)

δC

1.91 m, 1.41 m 2.46 m, 2.49 m

39.8 34.0 218.1 47.3 54.8 19.7 33.2 40.6 50.0 36.9 21.6 25.2 35.7 40.9 28.0 28.1 46.5 83.2 48.3 72.3 24.3 28.3 26.9 21.0 16.3 15.5 13.7

1.84 m, 1.72 m 1.23 m, 1.92 m 3.45 br s

36.4 27.5 76.0 39.7 48.7 18.0 27.7 39.6 47.9 37.1 24.7 122.7 143.6 41.7 27.5 22.8 46.4 41.3 45.8 30.7 33.7 32.4 28.6 22.0 17.2 61.1 26.5 183.1 33.1 23.6

1.88 m, 1.43 m 2.38 m, 2.53 m

39.2 34.2 217.7 47.4 55.3 19.7 32.6 39.1 47.0 36.8 23.4 120.9 146.0 42.6 31.1 22.2 35.8 41.0 44.9 31.2 33.7 27.9 26.6 21.5 15.1 17.4 24.9

1.34 m 1.45 m, 1.57 m 1.45 m 1.96 m 1.57 m, 1.41 m 1.39 m, 1.57 m 1.75 m 1.05 m, 1.69 m 1.48 m 1.93 m 2.04 br d (6.0) 1.52 m, 1.88 m 1.37 m, 1.48 m 1.09 s 1.04 s 0.94 s 1.00 s 1.04 s 3.61 d (12.4), 3.40 d (12.4) 1.13 s

70.7 23.7

1.33 m 1.41 m 1.07 m, 1.79 m 1.76 m 2.23 m, 1.96 m 5.24 br t (3.5)

1.61 m, 1.97 m 2.81 m 1.62 t (13.7), 1.15 m 1.36 m, 1.21 m 1.78 m, 1.54 m 0.96 s 0.90 s 0.82 s 4.04 d (12.5), 3.94 d (12.5) 1.14 s 0.90 s 0.94 s

Instrument Co. Ltd (Beijing, China). Rabbit (3.8 kg) was supplied by Shanghai Sikelai Experimental Animal Co., Ltd (Shanghai, China).

2.2. Extraction and isolation The resin (25 g) of L. formosana, collected in November 2009 from Guangdong Province of China, was extracted with petroleum ether (60–90 °C) (2 × 300 mL) for 2 h under reflux, and the combined extracts were concentrated in vacuo. The resulting extract (23.5 g) was chromatographed on silica gel (500 g) eluting with petroleum ether (60–90 °C)–acetone, stepwise gradient (30:1 → 3:1), and four fractions (I–IV) were collected. Fraction I (5.5 g) was separated by silica gel [petroleum ether–CH2Cl2 (15:1)] to yield 10 (60 mg) and 3 (40 mg). Fraction II (9.1 g) was separated by silica gel [petroleum ether (60–90 °C)–CH2Cl2 (8:1)] to obtain 1 (2 mg), 6 (50 mg), 4 (70 mg) and 5 (20 mg). Fraction III (3.9 g) was separated by silica gel [petroleum ether–CH2Cl2 (6:1)] to obtain 7 (4 mg) and 8 (5 mg). Fraction IV (5.0 g) was separated by silica gel [petroleum ether–CH2Cl2 (4:1)] to yield 2 (15 mg) and 9 (6 mg). Liquidambarone (1): white powder, [α]D20 + 69.8 (c 0.10, CHCl3); IR (KBr) λmax 3318, 2911, 1715, 1455, 1260, 1040, 967 cm −1; 1H NMR (CDCl3, 300 MHz) see Table 1; 13C NMR (CDCl3, 75 MHz) see Table 1; ESI-MS: m/z 441 [M-H] −, 477

1.33 m 1.49 m 1.40 m, 1.50 m 1.66 m 1.92 m 5.22 t (3.5)

1.58 m, 1.22 m 1.12 m, 1.81 m 1.55 m 2.36 m 1.63 m, 1.01 m 1.08 m, 1.24 m 1.71 m, 1.37 m 1.09 s 1.05 s 1.06 s 0.92 s 1.12 s 0.87 s 0.90 s

33.6 23.9

[M + Cl] −, 443 [M + H] + and 465 [M + Na] +; HR-ESI-MS: m/z 465.3339 [M + Na] + (C29H46O3Na, calc. 465.3345). Ambradiolic acid A (2): white powder, [α]D20 + 52.6 (c 0.14, CHCl3); IR (KBr) λmax 3345, 3320, 2910, 1693, 1450, 1264, 1037, 969 cm −1; 1H NMR (CDCl3, 300 MHz) see Table 1; 13C NMR (CDCl3, 75 MHz) see Table 1; ESI-MS: m/z 471 [M-H]− and 507 [M + Cl] −; HR-ESI-MS: m/z 471.3469 [M-H] − (C30H47O4, calc. 471.3474). 2.3. Cytotoxicity bioassay Compounds were tested against Human breast carcinoma MDA-MB-435S cell lines using an established colorimetric MTT assay protocol. Adriamycin was used as a positive control. Cells were seeded in 96-well culture plates at densities of 3000–4000 cells per well. After overnight, cells were cultured with 10% FBS medium in the absence for 12 h. Then, the tested compounds at different concentrations were added into 96-well plates and cultivated for 72 h, and 20 μL of MTT (5 mg/mL) was added to each well. After 4 h, the culture medium was removed and the formazan crystal was completely dissolved with 150 μL dimethylsulfoxide to each well by vigorously shaking the plate. Finally, formazan absorbance was assessed by a Bio-Tek multi-detection microplate reader at 490 nm. The IC50 is the concentration of agent that reduced cell growth by 50% under the experimental conditions.

N.-Y. Yang et al. / Fitoterapia 82 (2011) 927–931

929

was done for the positive control drug aspirin and the blank solvent ethanol. Each analyte was tested several times, and an average value was applied.

2.4. Anticoagulative assay in vitro All samples were dissolved in absolute ethanol respectively. Two male New Zealand white rabbits (2–2.5 kg) were used for anticoagulation assay in vitro, obtained from the experimental animal center of Nanjing University of Chinese Medicine and approved by Animal Ethics Committee of Nanjing University of Chinese Medicine on April 29, 2010. Rabbit common carotid artery was cut off to take sample of blood, which was mixed with anticoagulant (3.8% sodium citrate) in the proportion of 9 to 1, then centrifuged at 1000 rpm for 10 min at room temperature to give plateletrich plasma (PRP). Centrifugation of the remaining blood at 3000 rpm for 10 min yielded platelet-poor plasma (PPP). Platelet count was adjusted to 3 × 10 5 with PPP. The samples was added to the PRP (0.200 mL), then the mixture was incubated at 37 °C with stirring for 1 min before the addition of ADP (10 μM) as inducer of platelet aggregation. Aggregation was measured by a turbidimetric method. Changes in light transmission caused by platelet clotting were measured using an aggregometer. Platelet aggregation was expressed as the percentage change with the difference of light transmittance between PRP and PPP as 100%. The same experiment

3. Results and discussion The petroleum ether extract of L. formosana resin was subjected to repeated silica gel liquid chromatography to obtain ten triterpenes 1–10. Compounds 1 and 2 were identified as new triterpenes. Other compounds were identified as ambronal (3), 3-oxo-11α,12α-epoxy-olean28,13β-olide (4), 3-oxo-12α-hydroxy-olean-28,13β-olide (5), 3-oxo-olean-11-en-28,13β-olide (6), oleanonic acid (7), amooranin (8), 3-epi-maslinic acid (9) and 28-nor-βamyrenone (10) (Fig. 1) by the analysis of their spectroscopic data and comparison with literature values [2–8]. Among them, 1 is a nor-lupane triterpene, 2–9 are oleane triterpenes, 10 is a nor-oleane triterpene, and compounds 6–10 were isolated from this plant for the first time. Compound 1 was obtained as a white powder, its molecular formula was established as C29H46O3 by a [M+ Na] + ion peak at m/z 465.3339 in its HRESIMS. IR spectrum showed the presence of hydroxyl group (3318 cm− 1) and a ketone group

30

HO

20

21

19

O

29 12

18

O 13

11 26

25 1

14 9

2

10

3

7

H

17 16

15

27

6

O

1

H 24

23

20 19 12 11

1

R1

R4

2

17

R5

13 14

9 10

R2

3

R3

8 7

5 4

6

21

18

26

16 15

4

30

29

25

O

8

5 4

O

O

22

22

R6 28

27

R5 R 1 R2 R 3 R4 2 H OH H CH 3 CH2OH 3 H O CH 3 CH3 7 H O CH3 CH3 8 H O CH 2OH CH3 9 OH OH H CH3 CH3 10 H CH3 CH3 O

R6 COOH COH COOH COOH COOH H

H 24

23

OH

O

5

O

O

O

O

O

Fig. 1. Structures of compounds 1–10.

6

930

N.-Y. Yang et al. / Fitoterapia 82 (2011) 927–931

H

O

HO H

O

H

1

H

COOH

H

O O

H

2

H H

HMBC

H

C

H

NOESY

H

Fig. 2. Key HMBC and NOESY correlations of Compounds 1–2.

presence of carboxyl group (3345, 3220, 1693 cm− 1). The 1H and 13C NMR spectral data (Table 1) of 2 were similar to those of 3-epi-oleanolic acid except that two additional hydroxymethyl proton signals [δ 4.04 (d, J = 12.5 Hz), 3.94 (d, J = 12.5 Hz)] and the corresponding carbon signal (δ 61.1) were observed in 2. The hydroxymethyl proton was established as H-26 by HMBC correlation for the proton to C-7 (δ 27.7), C-9 (δ 47.3) and C-14 (δ 41.7) (Fig. 2). 1H-1H NOESY and HSQC experiments were also run to assign all proton and carbon signals for 2. So compound 2 was identified as 3α,26-dihydroxy-olean-12-en-28-oic acid, named ambradiolic acid A. The isolated triterpenes 1–10 were tested for their cytotoxicities against human breast cancer MDA-MB-435S cells using an established colorimetric diphenyltetrazolium bromide (MTT) assay protocol, and IC50 was calculated to quantitatively compare the cytotoxic potency of the tested compounds. Adriamycin was used as a positive control. Among them, compounds 3–8 and 10 exhibited strong cytotoxicity against MDA-MB-435S cells (Table 2). Evaluation of anticoagulative activities of compounds 1–10 were performed using antiplatelet aggregation method. Antiplatelet aggregation activities of 1–10 are shown in Table 3. Analyses were carried out in triplicate and data were expressed as the inhibition rate and IC50 with aspirin as the positive control, and compounds 2 and 7–9 showed strong inhibitory activity (Table 3).

(1715 cm−1). The 1H and 13C NMR spectral data (Table 1) showed some resemblance to those of 20,29-dihydroxy-3oxolupan-30,21α-olide except that the relatively low field chemical shifts of H-29 and C-29 and no carboxyl carbon resonance signal were observed in the NMR spectra of 1 [9]. Its HMBC (Fig. 2) correlations of two oxygenated carbons [δ 70.7 (C-29) and 72.3 (C-20)] with H-30 [δ 1.13 (s)] and correlations of C-18 (δ 83.2) with H-29 (δ 3.61, 3.40) suggested that 1 was an 18,29-epoxy-20-hydroxy lupane triterpene, and the isopropanal group located at C-19 should be α-oriented. Its NOESY spectrum (Fig. 2) demonstrated that H-30, H-19 [δ 2.04 (br d, J = 6.0 Hz)] and H-13 (δ 1.75 m) are both β-oriented. The HMBC correlation of the carbonyl carbon (δ 218.1) with H-23 (δ 1.09) and H-24 (δ 1.04) revealed 3-ketone in 1. Detailed analysis of NMR and MS data also indicated 28-nor structural feature of 1 and β-orientation of H-17 (δ 1.93 m). 2D-NMR experiments allowed us to make an unambiguous and complete assignment of the 1H and 13C NMR spectra of 1. The structure of 1 was thus assigned as 20R-18,29-epoxy-28norlupan-20-ol-3-one, named liquidambarone. Compound 1 is the first example of an 18,29-epoxy-lupan type triterpene. Compound 2 was obtained as a white powder, [α]D20 +52.6 (c 0.14, CHCl3). The molecular formula was established as C30H48O4 by a [M-H]− ion peak at m/z 471.3469 (calcd. for C30H47O4, 471.3474) in its HRESIMS. IR spectrum showed the

Table 2 Cytotoxic activities of isolated compounds against MDA-MB-435S tumor cells. Compound

1

2

3

4

5

6

7

8

9

10

Adriamycin

MDA-MB-435S IC50 (μg/mL)

52.14

N 100

6.00

3.06

6.19

13.82

6.73

8.09

N 100

3.09

0.33

Table 3 Antiplatelet aggregation rate of compounds 1–10 (x± s, n = 6–8). Compound

1

2

3

4

5

6

7

8

9

10

Aspirin

Antiplatelet aggregation IC50 (μg/mL)

–

12.16

–

–

–

–

10.77

8.07

13.05

–

10.59

“–” N 50 μg⋅mL− 1

N.-Y. Yang et al. / Fitoterapia 82 (2011) 927–931

This study showed that the resin of L. formosana mainly contained oleanane and lupane triterpenoids and these triterpenoids are the important antitumor and anticoagulative constituents. From the activity data, it can be concluded that 3-keto group significantly enhance the cytotoxic activity and that 28-carboxyl group remarkably strengthens the antiplatelet aggregation activity. Oleane triterpenes with 3keto group were also reported to show remarkable cytotoxicity against other tumor cells [10,11], while 28-arboxyl triterpenes were as equivalently inhibitive as acetylsalicylic acid on ADP-induced platelet aggregation [12]. Acknowledgements This research was financially supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Construction Project for Jiangsu Key Laboratory for High Technology Research of TCM Formulae (BM2010576) and Construction Project for

931

Jiangsu Engineering Center of Innovative Drug from Bloodconditioning TCM Formulae. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]

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