Cleistanthane diterpenes from the seed of ...

Fitoterapia 91 (2013) 148–153

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Cleistanthane diterpenes from the seed of Caesalpinia sappan and their antiausterity activity against PANC-1 human pancreatic cancer cell line Hai Xuan Nguyen a, Mai Thanh Thi Nguyen a, Thy Anh Nguyen a, Nhi Y Thi Nguyen a, Dao Anh Thi Phan a, Phuoc Ho Thi a, Trong Huu Phan Nguyen a, Phu Hoang Dang a, Nhan Trung Nguyen a, Jun-ya Ueda b, Suresh Awale b,⁎ a b

Faculty of Chemistry, University of Science, Vietnam National University-Ho Chi Minh City, Viet Nam Frontier Research Core for Life Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan

a r t i c l e

i n f o

Article history: Received 19 July 2013 Accepted in revised form 24 August 2013 Available online 31 August 2013 Keywords: Caesalpinia sappan Cleistanthane diterpene Antiausterity strategy Anticancer drug discovery PANC-1 Nutrient starvation

a b s t r a c t Three new cleistanthane diterpenes named tomocinon (1), tomocinol A (2), and tomocinol B (3), were isolated from the EtOAc extract of the seed of Caesalpinia sappan. Their structures were determined by extensive NMR spectroscopic analysis. The absolute stereochemistry of tomocinon (1) has been established by CD spectroscopic analysis. Cleistanthane diterpenes (1–3) represents the novel class of antiausterity agents having preferential cytotoxicity against PANC-1 human pancreatic cancer cell line under nutrient deprived condition with PC50 value of 34.7 μM, 42.4 μM and 39.4 μM, respectively. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Caesalpinia sappan Linn (Caesalpiniaceae) is a shrubby tree distributed in China, India, Thailand, Indonesia, and Viet Nam. In Viet Nam, C. sappan is known as “To Moc”, and the decoction of its heartwood has been used in traditional medicine for the treatment of rheumatism and inflammatory diseases and as an emmenagogue and homeostatic agent [1]. The heartwood of C. sappan contains various structural types of phenolic components, such as brazilins, dibenzoxocins, homoisoflavonoids, and chalcone [2], while, the seed of this plant, as a member of the genus Caesalpinia, is a rich source of cassane-type diterpenes [3]. As a part of our continued research on the screening of medicinal plants of different origins [4–9], we found that the EtOAc extract of the seed of Vietnamese C. sappan showed preferential cytoxicity against PANC-1 human pancreatic cancer cells in nutrient deprived ⁎ Corresponding author. Tel./fax: +81 076 434 7640. E-mail address: [email protected] (S. Awale). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ﬁtote.2013.08.018

condition with PC50 value (a concentration at which 50% cells were preferentially killed in nutrient deprived medium without causing toxicity in nutrient rich medium) of 43.6 μg/ mL. Therefore phytochemical investigation was carried out that led the isolation of three new biogenetically exclusive cleistanthane-type diterpenes, tomocinon (1), tomocinol A (2), and tomocinol B (3) (Chart 1). In this paper, we report the isolation and structural elucidation of these compounds by spectroscopic techniques and the antiausterity activity of the isolates against the PANC-1 human pancreatic cancer cell line. 2. Experimental 2.1. General Optical rotations were measured on a JASCO DIP-140 digital polarimeter. CD measurements were carried out on a JASCO J-805 spectropolarimeter. IR spectra were measured with a Shimadzu IR-408 spectrophotometer in CHCl3 solution.

H.X. Nguyen et al. / Fitoterapia 91 (2013) 148–153

a

b

O

20

11

12 13

OH

O

14

9

O

16

O

7

19

OH

14 9

5

3

7

18

15

1 16

5

3

13

20

O

14

16

5

15

1

17

12

13

20

15

9

19

c 17

12

1

3

OH

17

149

7

18

19

18

Chart 1. Structure of compounds isolated from Caesalpinia sappan.

Analytical Chemistry of the University of Science, VNU-HCM, Viet Nam.

NMR spectra were taken on a Bruker Advance III 500 MHz spectrometer with tetramethylsilane (TMS) as an internal standard, and chemical shifts are expressed in δ values. HRFAB-MS and HR-ESI-MS measurements were carried out on a JEOL JMS-AX505HAD mass spectrometer and Agilent 6310 Ion Trap mass spectrometer, respectively. Analytical and preparative TLC was carried out on precoated Merck Kieselgel 60F254 or RP-18F254 plates (0.25 or 0.5 mm thickness).

2.3. Extraction and isolation Air-dried seed of C. sappan (2.8 kg) were extracted with MeOH (10 L, reflux, 3 h × 3) to yield MeOH extract (300 g). The MeOH extract was suspended in H2O and partitioned successively with CH2Cl2 and EtOAc to yield CH2Cl2 (150 g), EtOAc (15 g), and H2O (110 g) fractions. The EtOAc extract was subjected to a series of silica gel column (10 cm × 120 cm) chromatography eluted with MeOH/CHCl3 (0–100%) to give 5 fractions: Fr.1–Fr.5. Fr.2 (1.6 g) was chromatographed on silica gel with MeOH/CHCl3 (0–30%) to give 3 subfractions Fr.2.1– Fr.2.3; Fr.2.2 was subjected to silica gel with EtOAc/hexane (2:8) and followed by normal-phase preparative TLC with

2.2. Plant material The seed of C. sappan was collected at An Giang Province, Viet Nam, in October 2009 and was identified by Ms. Hoang Viet, Faculty of Biology, University of Science, Vietnam National University-Ho Chi Minh City (VNU-HCM). A voucher sample AN-2213 has been deposited at the Department of

Table 1 1 H NMR (500 MHz) and Position

13

C NMR (125 MHz) data for compounds 1–3 in CDCl3 (J values in parentheses).

1

2 δC

δH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 16-OCOCH3 16-OCOCH3

0.87 1.59 1.45 1.12 1.40

m m m m m

0.88 1.55 1.69 1.23 2.03 1.69 1.13

m m m m m m m

1.84 t (14.2) 2.20 dd (14.2; 1.9)

1.06 1.56 3.82 4.36 1.22 0.85 0.80 0.78 2.05

m m (12.0; 8.6) (12.0; 6.0) s s s s s

38.1 18.6 42.0 33.2 54.6 22.0 35.2 36.8 56.9 37.3 36.4 211.2 33.4 38.7 33.2 63.8 14.3 33.4 21.5 14.1 20.9 171.0

3

δH

δC

0.75 1.59 1.48 1.08 1.32

m m m m m

0.81 1.23 1.56 1.08 1.87 1.32 0.54

m m m m m m m

0.56 m 1.68 m 3.72 dd (10.2; 6.0) 0.41 0.98 3.80 4.22 1.15 0.78 0.73 0.67 2.00

dd (5.0; 1.0) m dd (11.7; 8.6) dd (11.7; 6.7) s s s s s

38.6 18.8 42.2 33.2 55.1 22.2 36.0 36.4 53.4 36.7 29.1 75.6 27.6 34.3 24.4 65.5 19.4 33.5 21.6 14.4 21.1 171.3

δH

δC

0.82 1.60 1.43 1.11 1.33

m m m m m

0.85 1.26 1.64 1.16 1.91 1.34 0.57

m m m m m m m

0.62 m 1.72 m 3.82 dd (10.4; 5.9) 0.40 0.99 3.38 3.87 1.24 0.83 0.78 0.73

dd (5.2; 1.2) dt (9.0; 5.9) dd (11.3; 9.0) dd (11.3; 5.9) s s s s

38.8 18.9 42.4 33.4 55.3 22.4 36.2 36.6 53.6 36.8 29.3 76.0 27.6 33.9 28.6 63.3 19.3 33.6 21.7 14.6

150


acetone/hexane (15:85) to give 1 (15 mg). Fr.4 (2.1 g) was separated with EtOAc/hexane (2:8) and then MeOH/CHCl3 (2:8) to yield 3 subfractions Fr.4.1–Fr.4.3; Fr.4.3 was recrystallized from MeOH/CHCl3 (3:7) to afford 2 (150 mg) and 3 (3.0 mg). Tomocinon (1): Colorless amorphous solid; [α]25D +84 (c 0.2, CHCl3); CD (c 2.8 × 10−4 M, EtOH) [θ]282 +8058, [θ]202 −2337; IR νmax (CHCl3) 1690, 1735 cm−1; 1H and 13 C NMR, see Table 1; HR-FAB-MS m/z: 369.2366 [M + Na]+ (Calcd for C22H34O3Na: 369.2406). Tomocinol A (2): Colorless amorphous solid; [α]25D +25 (c 1.0, CHCl3); IR νmax (CHCl3) 3370, 1735 cm−1; 1H and 13C NMR, see Table 1; HR-ESI-MS m/z: 371.2552 [M + Na]+ (Calcd for C22H36O3Na: 371.2562). Tomocinol B (3): Colorless amorphous solid; [α]25D +31 (c 1.1, CHCl3); IR νmax (CHCl3) 3370, 1735 cm−1; 1H and 13C NMR, see Table 1; HR-ESI-MS m/z: 329.2459 [M + Na]+ (Calcd for C20H34O2Na: 329.2457). 2.4. Preferential cytotoxic activity against PANC-1 cells in nutrient deprived medium The preferential cytotoxicity of the isolated compounds was determined by a previously described procedure with a slight modification [4]. Briefly, human pancreatic cancer PANC-1 cells were seeded in 96-well plates (1.5 × 104/well) and incubated in fresh Dulbecco's modified Eagle's medium (DMEM, Wako Pure Chemical, Osaka, Japan) at 37 °C under humidified 5% CO2 and 95% air for 24 h. After the cells were washed with Dulbecco's phosphate-buffered saline (D-PBS, Nissui Pharmaceutical, Tokyo, Japan), the medium was changed to serially diluted test samples in either DMEM or nutrient deprived medium (NDM, prepared according to previously described protocol [13]) with control and blank in each well. After 24 h incubation, 100 μL of DMEM containing 10% WST-8 cell counting kit solution was added to the each wells directly. After 3 h incubation, the absorbance at 450 nm was measured (PerkinElmer EnSpire Multilabel Reader). Cell viability was calculated from the mean values of data from three wells by using the following equation: Cell viability ð% Þ h

i ¼ Absðtest sampleÞ AbsðblankÞ =AbsðControlÞ AbsðblankÞ 100%:

a

b

2.5. Morphological assessments of PANC-1 cells PANC-1 cells were seeded in 60 mm dishes (1 × 106 cells) and incubated DMEM at 37 °C under 5% CO2 for 24 h. The cells were then washed twice with PBS, and treated with 1 at indicated concentration in NDM and incubated for 24 h. The cells were then treated with ethidium bromide/acridine orange (EB/AO) and morphology was observed using an inverted Nikon Eclipse TS 100 microscope (40× objective) with phasecontrast and fluorescent mode. Microscopic images were taken with a Nikon DS-L-2 camera directly attached to the microscope. Live cells are stained with AO to give green fluorescence while dead cells are stained with EB to give red fluoresce.

3. Results and discussion Tomocinon (1) was obtained as a colorless amorphous solid and showed the quasimolecular ion at m/z 369.23658 [M + Na]+, corresponding to the molecular formula C22H34O3Na in HR-FAB-MS. The IR spectrum of 1 showed absorption of ketone carbonyl (1690 cm−1) and ester carbonyl (1735 cm−1) groups. The 1H NMR spectrum of 1 (Table 1) displayed four methyls (δH = 0.78, 0.80, 0.85, 1.22), an acetyl methyl (δH = 2.05), an oxymethylene (δH = 3.82, dd, J =12.0, 8.6 Hz; δH = 4.36, dd, J = 12.0, 6.0 Hz), five aliphatic methines, and six aliphatic methylenes. The 13C NMR (Table 1) and DEPT spectra of 1 exhibited 22 carbons including a ketone carbonyl carbon (δC = 211.2), an ester carbonyl carbon (δC =171.0), together with an oxymethyle carbon (δC = 63.8). Analysis of the COSY spectrum led to the partial structures depicted by the bold lines in Fig. 1a, which were connected on the basis of the long-range correlations observed in the HMBC spectrum. The presence of a cyclopropane ring was indicated by HMBC correlations from H-15 to C-8, C-12, C-13, C-14, and C-17 and also from H2-16 to C-13, C-14, and C-15. These correlations, together with a correlation from H3-17 to carbonyl carbon C-12, established the cleistanthane skeleton of 1. The location of acetoxymethylene moiety was deduced to be at C-15 on the basis of HMBC correlations of the protons of acetyl methyl and H-16 with the ester carbonyl carbon (δC = 171.0), of H2-16 with C-13, C-14, and C-15. The relative stereochemistry of 1 was determined by NOESY spectral analysis. Tomocinon (1) showed NOESY correlations from H-11β to H-8 and H3-20, and from H-8 to H-15,

c

Fig. 1. Connectivities (bold lines) deduced by the COSY and HSQC spectra and significant HMBC correlations (solid arrows) of 1–3.


suggesting that these groups are β-axially oriented. Correlations were also apparent from H-14 to H-9, H2-16, and H3-17, but not to H-15 suggesting that these groups are α-oriented. These data supported a cis cyclopropane ring with an α-acetoxymethylene substituent (Fig. 2a). Finally, the absolute stereochemistry of 1 was determined by CD spectral analysis which gave a positive cotton effect at 282 nm (Fig. 3), which in accord to octant rule suggests the absolute configuration of 1 to be 5S, 8R, 9S, 10R, 13R, 14R, 15R. The HR-ESI-MS of tomocinol A (2) showed the quasimolecular ion at m/z 371.2552 [M + Na]+, consistent with the molecular formula C22H36O3. The IR spectrum of 2 showed absorption of carbonyl (1735 cm−1) and hydroxyl (3370 cm−1) groups. The 1H and 13C NMR spectral data of 2 (Table 1) were similar to those of tomocinon (1), but it lacked the signal due to a ketone carbonyl carbon and displayed one more oximethine group (δH = 3.72, dd, J = 10.2 & 6.0 Hz). From the HMBC experiments, the additional oximethine proton H-12 showed long-range correlations to C-9, C-11, C-14, C-15, and C-17 (Fig. 1b). These data suggested that a hydroxy group was attached at C-12 instead of the ketone group in 1. The orientation of hydroxy group at C-12 was determined to be β-equatorial from the NOESY correlations between H-12 and H-9, and large J value (10.2 Hz) between H-12 and H-11β (Fig. 2b). The relative stereochemistry

151

Fig. 3. The circular dichroism spectrum of 1 showing positive cotton effect at 282 nm and determination of absolute configuration in accordance with the application of octant rule.

a

b

c

Fig. 2. Key NOESY correlations observed for compounds 1–3.

of 2 was determined from the NOESY correlations, which were similar to those of 1. Tomocinol (2) could have formed from 1 through the reduction of the carbonyl group at C-12. Based on the biogenetic consideration and NOESY data, the absolute stereochemistry of tomocinol A is concluded as 5S, 8R, 9S, 10R, 12R, 13R, 14R, 15R. The molecular formula of tomocinol B (3) was determined as C22H36O3 by HR-ESI-MS. The 1H and 13C NMR spectra of 3 (Table 1) were similar to those of 2, but they were characterized by the disappearance of signals due to an acetyl group in 2. The location of deacetylation was determined to be at C-16 based on the HMBC spectral analysis (Fig. 1c). The relative as well as absolute stereochemistry of 3 was determined concluded same as 2 based on NOESY spectral analysis (Fig. 2c) and biogenetic consideration. Therefore, the structure of tomocinol B was concluded as 3. Human pancreatic cancer is known to be the most fatal form of cancer worldwide with the worst 5-year survival rate of less than 5%, known for all forms of cancer [10]. It is the 5th leading cause of cancer related mortality in the industrialized countries such as Japan, USA and UK [11]. The conventional chemotherapeutic drugs such as paclitaxel, doxorubicin, cisplatin showed resistance to this malignancy and no effective drugs are available at present [12]. When compared to normal cells, human pancreatic tumors cells such as PANC-1 cells show the remarkable tolerance to nutrient starvation that enables these cells to survive in the hypovascular (austere) tumor microenvironment [13]. Therefore, the search for the agents that eliminates this tolerance to nutrient starvation is a novel approach in anti-cancer drug discovery [4,14]. Working on this hypothesis, we had identified a number of potent anticancer agents such as arctigenin [4], angelmarin [5], keyaassamins A–I

152


Fig. 4. Preferential cytotoxicity of 1–3 against PANC-1 human pancreatic cancer cell line in nutrient deprived medium (NDM).

[8,9], and panduratins [7,15], from the medicinal plants used in Japanese Kampo medicine and Southeast Asian countries. In the present study, cleistanthane diterpenes, 1–3 were also found to show preferential cytotoxicity against PANC-1 human pancreatic cell line in the nutrient starvation condition (NDM) in a concentration dependent manner (Fig. 4) without causing apparent toxicity in normal nutrient-rich condition (DMEM). Among them, tomocinon (1) showed stronger activity (PC50, 34.7 μM), followed by tomocinol B (3) (PC50, 39.4 μM) and tomocinol A (2) (PC50, 42.4 μM), respectively. In contrary to this, Gemcitabine and 5-FU which are clinically used anticancer drugs [16] for the treatment of pancreatic cancer were inactive at the maximum dose (PC50, N200 μM), when tested for 24 h. Arctigenin, a positive control showed the most potent preferential cytotoxicity with PC50 value of 0.8 μM [4]. Among these isolates, tomocinon (1) was further studied for its effect on the PANC-1 cells morphology in NDM. The microscopic images were analyzed under with phase-contrast and fluorescence mode using ethidium bromide/acridine orange (EB/AO) reagent. AO is cell permeable dye and gives green fluorescence in live cells. EB is permeable to dead cells only and gives red fluorescence. As shown in Fig. 5, tomocinon (1) at a concentration of 25 μM did not induce PANC-1 cells death, as indicted by

predominantly AO (green fluorescence) stained cells with intact morphology. However, tomocinon (1) at a concentration of 50 μM caused dramatic alteration of PANC-1 cells morphology and gave exclusive red fluorescence indicating the total cell death. Therefore, cleistanthane diterpenes represents a novel class of antiausterity agents, which could be lead compounds for the drug development against pancreatic cancer.

Conﬂict of interest The authors declare no conflict of interest.

Acknowledgments This work was supported by grant B2011-18-04TD from the Vietnam National University-Ho Chi Minh City (VNU-HCM) to MTTN, a grant from the Toyama Support Center for Young Principal Investigators in Advanced Life Sciences, Japan, and a Grant in Aid for Scientific Research (No. 24510314) from the Japan Society for the Promotion of Science (JSPS) to SA.

Fig. 5. Effect of tomocinon (1) on the morphology of PANC-1 cells.


Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.08.018.

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