Fitoterapia 92 (2014) 105–110

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Four new quassinoids from the roots of Eurycoma longifolia Jack Dali Meng a,⁎, Xin Li a, Lingfei Han a, Lulu Zhang a, Weiwei An b,⁎⁎, Xian Li a a b

Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of Education, Shenyang 110016, PR China Cancer Research Institute, Harbin Medical University, No.6 BaoJian Road Nangang District, Harbin 150081, PR China

a r t i c l e

i n f o

Article history: Received 31 July 2013 Accepted in revised form 16 October 2013 Available online 26 October 2013 Keywords: Quassinoids Cytotoxic activity Single crystal X-ray diffraction

a b s t r a c t Seven compounds were isolated from the roots of Eurycoma longifolia, and characterized by comprehensive analysis of 1D and 2D NMR experiments along with single crystal X-ray diffraction. Among them, four new quassinoids were identified and three of them were diastereomers for each other. Compounds 1–7 were evaluated for cytotoxicities against HT-29, MCF-7, LOVO, BGC-823, MGC-803, HepG2, HeLa, and A549 cancer cell lines. Compounds 2 and 5 exhibited the lowest IC50 values of 24.9 μM, 11.8 μM, and 44.1 μM, 14.1 μM towards MCF-7, MGC-803 cancer cell lines, respectively, while compound 6 exhibited moderate cytotoxicity towards all the selected cancer cell lines. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Eurycoma longifolia Jack. (Simaroubaceae) is one of the most popular plants growing in the tropical rain forests of Southeast Asia. It is a tall slender shrub-tree which is known locally as ‘Tongkat Ali’ in Malaysia, ‘Pasakbumi’ in Indonesia, ‘Cay ba binh’ in Vietnam and ‘Ian-don’ in Thailand. The roots, stems, and bark of E. longifolia are all used as traditional medicines in local area, especially the roots, which are traditionally used as folk medicine for the treatment of various diseases, such as sexual insufficiency, aches, persistent fever, tertian malaria, dysentery, glandular swelling and so on [1]. In order to clarify the chemical constituents and find bio-active components from E. longifolia, a systematical phytochemical investigation on this plant was performed, which led to the discovery of seven compounds, including four new quassinoids, Δ4,5,14-hydroxyglaucarubol (1), 5-iso-eurycomadilactone (2), eurycomadilactone (3), 13-epi-eurycomadilactone (4) and three known compounds, eurycomanone (5), eurycomanol (6) and 13β,21-dihydroxyeurycomanone(7). The cytotoxic activities of

⁎ Corresponding author. Tel.: +86 24 23986475. ⁎⁎ Corresponding author. Tel.: +86 451 86665003. E-mail addresses: [email protected] (D. Meng), [email protected] (W. An). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.10.009

these compounds against several different cancer cell lines were evaluated. In this paper, the details of the isolation and structural elucidation work of the compounds, along with their potential anti-cancer activities will be discussed. 2. Results and discussion Compound 1 was obtained as a colorless cubic crystal. The molecular formula was determined as C20H28O9 based on its quasimolecular ion peak [M + Na]+ at m/z 435.1607 (calcd. 435.1626) in the HR-ESI-TOF-MS experiment. In the 1H NMR spectrum, three methyl proton signals were observed at δH 1.75 (3H, s), 1.77 (3H, d, J = 7.3 Hz), 1.97 (3H, s), and their carbon signals were δC 14.2, 19.1, 20.2, respectively, in the 13C NMR spectrum. The presence of two protons at δH 4.00 (1H, d, J = 10.0 Hz), 4.22 (1H, dd, J = 10.0, 4.0 Hz) and two carbons at δC 67.5, 84.1 suggested the existence of two methine groups. An endocyclic methylene group was conduced from two proton signals at δH 4.29 (1H, d, J = 8.8 Hz), 4.67 (1H, d, J = 8.8 Hz) with their carbon signals at δC 68.2. In the 13C NMR, it can also be found out two cyclic olefinic carbon signals at δC 127.5, 130.1, one ester carbonyl signal at δC 174.9, and one carbon signal at δC 111.0. By comparing the 1H and 13C NMR spectra data of 1 with those of △4,5-glaucarubol [2], it can be concluded that these two compounds possessed similar structure, except for one hydroxyl groups substituted at C-14. The substitution of hydroxyl could be

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further confirmed in the HMBC spectra, in which four protons, including H-9 (δH 3.19), H-12 (δH 4.14), H-15 (δH 5.48) and H-21 (δH 1.77), are all correlated to C-14 (δC 76.7) (see Fig. 2). The relative configuration of 1 was confirmed by single crystal X-ray diffraction analysis in combination with the reported [4] (see Fig. 4). Finally, the structure of 1 was confirmed as Δ4,5, 14-hydroxyglaucarubol (see Fig. 1), marked as a new compound. The molecular formulas of 2, 3, 4 were all assigned as C20H24O9 on the basis of HR-ESI-TOF-MS with a quasi-molecular ion peak [M + Na]+ at m/z 431.1312 (calcd. 431.1313, 2), m/z 431.1314(calcd. 431.1313, 3) and m/z 431.1311 (calcd. 431.1313, 4), respectively, and each of them had two more oxygen atoms than that of shinjudilactone [3]. Compound 2 was isolated as a white needle. 1H NMR spectrum showed three methyl proton signals at δH 1.33 (3H, s), 1.55 (3H, d, J = 7.5 Hz), 1.82 (3H, s), two endocyclic methylene proton signals at δH 4.30 (1H, d, J = 12.0 Hz), 5.43 (1H, d, J = 12.0 Hz), and one cyclic olefinic proton signal at δH 6.12 (1H, s). Twenty carbons were observed in 13C NMR spectrum, including three methyl carbon signals at δC 9.5, 11.4, 22.7, and two ester carbonyl carbon signals at δC 173.8, 174.2. Three sp2 hybridized carbon signals at δC 126.6, 161.6 and 197.1 indicated the existence of an α,β-unsaturated ketone moiety, which were further proved by the long-range correlations between H-1 (δH 4.48) and C-2 (δC 197.1), H-3 (δH 6.12) and C-1 (δC 83.4), C-5 (δC 43.7), C-18 (δC 22.7), respectively, in HMBC spectrum (see Fig. 2). By comparing the 1H and 13C NMR data of 2

Fig. 1. Structures of compounds 1–7.

with those reported [5], it could be concluded that 2 was a C20-quassinoid triterpenoid compound. The NOESY experiment on 2 exhibited long-range correlations between H-5 (δH 3.19) and H-1 (δH 4.48), H-6α (δH 2.38), H-9 (δH 3.44); H-15 (δH 5.40) and H-9 (δH 3.44), H-13 (δH 3.14), respectively, confirming that H-5 and H-13 were all α-orientated in 2 (see Fig. 3). The absolute configuration of 2 (see Fig. 5) was defined by the single crystal X-ray diffraction experiment as (1S, 5S, 7R, 8S, 9R, 10R, 11S, 13S, 14S, 15R)-5-iso-eurycomadilactone (see Fig. 1), designated as a new compound. Compound 3 was obtained as an amorphous white powder. Three methyl proton signals at δH 0.83, 0.76 and 1.94 were found in 1H NMR spectrum and twenty carbons were given in the 13C NMR spectrum, including three methyl carbons at δC 9.4, 15.1, 22.6, two carbonyls carbons (ester or lactone moieties) at δC 171.7 and 170.9, three α,β-unsaturated ketone carbons at δC 196.2, 160.1 and 123.3, respectively. All of these NMR data were similar to those of shinjudilactone reported in a previous paper [5], only differing in two more hydroxyl groups substitutions at C-14 and C-15. The HMBC spectrum furnished the long-range correlations between H-1 (δH 4.43) and C-2 (δC 196.2), H-3 (δH 5.84) and C-1 (δC 76.8), C-5 (δC 45.8), C-18 (δC 22.6), H-5 (δH 2.50) and C-3 (δC 123.3), C-18 (δC 22.6), C-19 (δC 15.1) (see Fig. 2), respectively, corroborating the existence of an α, β-unsaturated ketone group in ring A. Both H-7 (δH 4.19) and H-15 (δH 4.67) were correlated with C-8 (δC 47.4), C-14 (δC 76.9), C-16 (δC 170.9), indicating the presence of the lactonic ring C and two hydroxyls at C-14 and C-15. Another endocyclic lactonic ring located between C-8 and C-11 was determined from long-range correlations between H-9 (δH 3.04), H-13 (δH 2.21) and C-12 (δC 171.7) in HMBC spectrum (see Fig. 2). The NOESY experiment revealed that the stereochemistry of 3 was similar with that of 2 except for the opposite relative configuration of H-5 (δH 2.50), which were correlated with H-19 (δH 0.83), H-7 (δH 4.19), and H-6β (δH 2.03), respectively. So, it can be concluded that H-5 was β-oriented in 3, and 3 was an epimer of 2. Thus, the absolute configuration of 3 was established as (1S, 5R, 7R, 8S, 9R, 10R, 11S, 13S, 14S, 15R)-eurycomadilactone (see Fig. 3), specified as a new compound. Compound 4 was isolated as a white needle. The Co-TLC chromatography showed a little higher Rf value than that of 3. HSQC and HMBC spectra data showed that 3 and 4 shared the same planar structure. The chemical shifts of 4 were also similar to that of 3, except that C-8, C-9, C-11 and C-15 were up-field by 1.6, 1.3, 4.0, 4.0 ppm and C-12, C-13, C-14, C-16, C-21 were down-field by 2.6, 2.8, 2.2, 1.2, 3.3 ppm, respectively (see Table 1). In the NOESY experiment, the correlations from H-21 (δH 1.87) to H-9 (δH 3.72), H-15 (δH 5.34) were shown, suggesting that H-21 was α-oriented, H-13 was β-oriented obviously, which was reversed with that in 3 (see Fig. 3), elucidating that 3 was an epimer of 4. So the stereo configuration of 4 could be finally determined as (1S, 5S, 7R, 8S, 9R, 10R, 11S, 13R, 14S, 15R)-13-epi-eurycomadilactone (see Fig. 1), marked as a new compound. By comparison of data with the reported values, the structures of known compounds were identified as eurycomanone (5) [4,5], eurycomanol (6) [1,2], and 13β, 21-dihydroxyeurycomanone (7) [6]. Their structures are listed in Fig. 1. Furthermore, compounds 1–7 were tested for their cytotoxicities against Human cervical carcinoma HeLa cell, Human

D. Meng et al. / Fitoterapia 92 (2014) 105–110

Fig. 2. 1H–13C long-range correlations in the HMBC spectrum of 1–4.

Fig. 3. The NOESY correlations of compounds 2–4.

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Fig. 4. Single-crystal X-ray structure of 1.

hepatocellular liver carcinoma HepG2 cell, Human intestinal cancer HT-29 cell, Human intestinal cancer LOVO cell, Human gastric cancer BGC-823 cell, Human lung adenocarcinoma epithelial A549 cell, human breast cancer MCF-7 cell and Human gastric cancer MGC-803 cell with fluorouracil as a

positive control. Among them, 2 exhibited the lowest IC50 values of 24.9 μM and 11.8 μM towards MCF-7 and MGC-803 cell lines, respectively. 5 exhibited the IC50 values of 44.1 μM and 14.1 μM against MCF-7 and MGC-803 cell lines, respectively. Additionally, 6 exhibited moderate cytotoxicity towards all tested cancer cell

Fig. 5. Single-crystal X-ray structure of 2.

D. Meng et al. / Fitoterapia 92 (2014) 105–110

lines. But other compounds were not clearly responsible for cytotoxicity test.

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4. Experimental 4.1. Plant material

3. Conclusion Quassinoids, one of the main constituents in E. longifolia, have various biological activities, including anti-tumor, anti-malarial, anti-inflammatory, anti-diabetic, aphrodisiac effects, et.al [7]. In this study, seven quassinoids, including four new compounds 1–4, were isolated from the roots of E. longifolia. The sterical configuration of compounds 1 and 2 was confirmed by single crystal X-ray diffraction analysis. Compounds 2 and 5 exhibited potent cytotoxicity towards MCF-7 and MGC-803 cancer cell line. The cytotoxicities of 2 and 5 are close to those of picrasin B, I, and J from Quassia amara L. Leaf herbal tea [8], which exhibited the IC50 values of 8.9 μM, 14 μM, and 77 μM against MCF-7, respectively. But their activities are lower than that of simalikalactone D, which exhibited the IC50 values of 29 nM against MCF-7 [9]. Kuo [10] isolated nearly 65 compounds from the roots of E. longifolia and screened them for potential cytotoxicity, while the quassinoids displayed strong cytotoxicities toward MCF-7 and A549 cell lines. Based on this study and those data mentioned above, it could be concluded that, among different cancer cell lines, quassinoids showed best activity on MCF-7. This is an interesting and important finding, and will be very useful for the further study on E. longifolia and quassinoids for their applications as potential antitumor agents.

Table 1 1 H NMR and

The plant materials were collected from Malaysia by Ningbo Liwah Plant Extraction Technology Limited, Ningbo, China and were identified by Pro. Jincai Lu, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. A voucher specimen (NO.DGAL-1013) was deposited in School of Traditional Chinese Material Medica, Shenyang Pharmaceutical University.

4.2. Extraction and isolation The roots of the plant (25 kg) were extracted with MeOH under reflux for 2 h. After evaporation of the combined MeOH extracts in vacuo, the resultant aqueous residues were suspended in H2O and extracted with EtOAc and n-BuOH saturated with H2O, respectively. The n-BuOH layer was evaporated to dryness in vacuo (250 g) and then was chromatographed on silica gel column using a gradient CH2Cl2–MeOH system (100:1–0:100) to give nine fractions (1–9). Fraction 2 was further purified by repeated silica gel CC with CH2Cl2/MeOH (100:5) as eluant to give 2 (24.8 mg), 3 (25.3 mg) and 5 (19.6 mg) after recrystallisation with CH2Cl2–MeOH solvent. Fraction 6 was purified by repeated silica gel CC using CH2Cl2/MeOH (100:8) as eluting solvent to give 1 (20.2 mg) and 6 (20.3 mg) followed by recrystallisation. Fraction 7 was also purified by repeated silica gel CC using CH2Cl2/MeOH (100:8) as eluant and then

13

C NMR spectroscopic data of compounds 1–4.

Proton

1

2c

3d

4c

Carbon

1b

2f

3e

4g

H-1 H-2 H-3α H-3β H-5 H-6α H-6β H-7 H-9 H-12 H-13 H-15 H-18 H-19 H-20α H-20β H-21

4.00 (d, 10.0) (dd, 10.0, 4.0) 2.36 (dd, 17.0, 10.0) 2.50(dd, 17.0, 6.0)

4.48 (s)

4.43 (d, 2.4)

4.96 (s)

6.12 (s)

5.84 (s)

6.03 (s)

3.19 (dd, 15.0, 3.0) 2.38 (dt, 15.0, 3.0) 2.18(td, 15.0, 3.0) 4.63 (brt) 3.44 (s)

2.50 2.14 2.03 4.19 3.04

(overlapped) (brd, 12.0) (dd, 12.0, 6.0) (dd, 12.0, 6.0) (s)

2.80 (d, 12.0) 2.53 (brd, 12.0) 2.47(dd, 12.0, 6.0) 4.64 (dd, 12.0, 6.0) 3.72 (s)

2.21 4.67 1.94 0.83 4.09 4.60 0.76

(dd, 17.4, 7.2) (d, 6.5) (s) (s) (d, 12.0) (d, 12.0) (d, 7.2)

3.03 5.34 1.90 1.51 5.55 4.56 1.87

84.1 67.5 41.8 127.5 130.1 28.3 76.7 53.7 50.9 44.9 111.0 79.8 42.6 76.7 71.7 174.9 20.2 19.1 68.2 14.2

83.4 197.1 126.6 161.6 43.7 27.6 75.6 49.3 54.5 49.2 79.9 173.8 53.0 77.8 75.0 174.2 22.7 9.5 68.2 11.4

76.8 196.2 123.3 160.1 45.8 29.4 79.6 47.4 47.5 51.6 42.4 77.3 171.7 50.7 76.9 73.3 170.9 22.6 15.1 69.4 9.4

78.9 196.8 125.1 159.2 47.9 31.3 78.6

3.14 (d, 7.5 Hz) 5.40 (s) 1.82 (s) 1.33 (s) 4.30 (d, 12.0) 5.43 (d, 12.0) 1.55 (d, 7.5)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 19 20 21

a b c d e f g

a

3.01 2.63 5.19 3.19 4.14 2.83 5.48 1.75 1.97 4.67 4.29 1.77

(dd, 15.2, 3.5) (brd, 15.2) (overlapped) (s) (d, 4.0) (m) (s) (s) (s) (d, 8.8) (d, 8.8) (d, 7.3)

Measured at 600 MHz in Pyridine-d5. Measured at 150 MHz in Pyridine-d5. Measured at 300 MHz in Pyridine-d5. Measured at 300 MHz in DMSO-d6. Measured at 75 MHz in DMSO-d6. Measured at 75 MHz in Pyridine-d5. Measured at 150 MHz in Pyridine-d.

(dd, 15.0, 7.5) (s) (s) (s) (d, 11.0) (d, 11.0) (d, 7.5)

53.0 44.3 75.2 175.7 55.8 81.3 71.4 173.6 23.1 16.2 71.4 14.3

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chromatographed on Sephadex LH-20 CC eluted by MeOH to afford 4 (29.6 mg) and 7 (19.7 mg). 4.2.1. Compound 1 Colorless cubic crystals (MeOH); [α]25 D = −68.3 (c 0.3, MeOH); its molecular formula was deduced to be C20H28O19 from the HR-ESI-TOF-MS m/z: 435.1607[M + H]+ (calcd. for 435.1626); For 1H and 13C NMR(pyridine-d5) spectroscopic data, see Table 1. 4.2.2. Compound 2 White amorphous powder (MeOH); [α]25 D = − 16.2 (c 0.3, MeOH); its molecular formula was deduced to be C20H24O19 from the HR-ESI-TOF-MS m/z: 431.1314 [M + Na]+ at (calcd. for 431.1313); For 1H and 13C NMR(pyridine-d5) spectroscopic data, see Table 1. 4.2.3. Compound 3 White needles (MeOH); [α]25 D = − 57.6 (c 0.3, MeOH); its molecular formula was deduced to be C20H24O19 from the HR-ESI-TOF-MS m/z: 431.1311 [M + Na]+ (calcd. for 431.1313); For 1H and 13C NMR (pyridine-d5) spectroscopic data, see Table 1. 4.2.4. Compound 4 White needles (MeOH); [α]25 D = +80.1 (c 0.7, MeOH); its molecular formula was deduced to be C20H24O19 from the HR-ESI-TOF-MS m/z: 431.1312 [M + Na]+ (calcd. for 431.1313); For 1H and 13C NMR (pyridine-d5) spectroscopic data, see Table 1.

4.3. Cytotoxic assay against HT-29, MCF-7, LOVO, BGC-823, MGC-803, HepG2, HeLa, A549 cell lines HT-29, MCF-7, LOVO, BGC-823, MGC-803, HepG2, HeLa, A549 cell were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). The tumor cell lines were cultured in RPMI-1640 medium (GIBCO, Grand Island, NY, USA), minimum essential medium (Eagle) (GIBICO) containing 10% fetal bovine serum (FBS) (Yuanhengshengma Biological Reagent Institute, BeiJing, China) and 0.03% L-glutamine (GIBCO) in 5% CO2 at 37 °C. Cells in the exponential phase of growth were used in the experiments. All the cells were seeded in 96-well plate (NUNC, Roskilde, Denmark) at 5 × 104 cells/ well. After preincubation overnight, various concentrations of compounds tested (1–1000 μM) were added and cultured for 48 h. Cell growth was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method with a plate reader. IC50 values (concentration that causes 50% growth inhibition) were determined. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 81073154), Liaoning Talents Engineer Project of China (Grant No. 2010921074), and the Program for Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team in University. We appreciate the help of Prof. Jincai Lu at Shenyang Pharmaceutical University for identifying the plant material. Appendix A. Supplementary data

4.2.5. Compound 5 White needles (MeOH); [α]25 D = − 44.5 (c 0.7, MeOH); mp. 242–243 °C, its molecular formula was deduced to be C20H24O9 according to the reported literature [3,4].

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.10.009. References

4.2.6. Compound 6 White needles (MeOH); [α]25 D = + 11.6 (c 0.2, MeOH); mp. 258–260 °C, its molecular formula was deduced to be C20H26O9 according to the reported literature [3,4]. 4.2.7. Compound 7 Colorless cubic crystals (MeOH); [α]25 D = +34.8 (c 0.1, MeOH); mp. 229–230 °C, its molecular formula was deduced to be C20H26O11 according to the reported literature [3,4].

[1] Rajeev B, A.A. K. Fitoterapia 2010;81:669–79. [2] Grieco PA, Haddad J, Pineiro-Nunez MM, Huffman JC. Phytochemistry 1999;50:637–45. [3] Kubota K, Fukamiya N, Okano M, Tagahara K, Lee KH. Bull Chem Soc Jpn 1996;69:3613–7. [4] Darise M, Kohda H, Mizutani K, Tanaka O. Phytochemistry 1982;21:2091–3. [5] Darise M, Kohda H, Mizutani K, Tanaka O. Phytochemistry 1983;22:1514. [6] Tada H, Yasuda F, Otani K. Eur J Med Chem 1991;26:345–9. [7] Guo BC, Wu TSH. Nat Prod Res Dev 2005;17:88–97. [8] Emeline H, Stéphane B, Geneviève B. J Ethnopharmacol 2009;26:114–8. [9] Xu Z, Chang FR, Wang HK. J Nat Prod 2000;63:1712–5. [10] Kuo PC, Damu AG, Lee KH. Bioorg Med Chem 2004;12:537–44.

Four new quassinoids from the roots of Eurycoma longifolia Jack.

Seven compounds were isolated from the roots of Eurycoma longifolia, and characterized by comprehensive analysis of 1D and 2D NMR experiments along wi...
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