New coumarins from Clausena lansium twigs

ARTICLE

New coumarins from Clausena lansium twigs

Wisanu Maneerat^I; Uma Prawat^II; Nisakorn Saewan^III; Surat LaphookhieoI, ^*

^INatural Products Research Laboratory, School of Science, Mae Fah Luang University, Tasud, Muang, 57100 Chiang Rai, Thailand

^IIFaculty of Science and Technology, Phuket Rajabhat University, Muang, 83000 Phuket, Thailand

^IIISchool of Cosmetic Science, Mae Fah Luang University, Tasud, Muang, 57100 Chiang Rai, Thailand

ABSTRACT

Two new coumarins namely Clausenalansimin A (5) and B (9) together with seven known coumarins (1-4 and 6-8), were isolated from twigs of Clausena lansium. All compounds were determined by spectroscopic methods. Some of isolates had cytotoxicity against human cancer cell lines (KB, MCF7 and NCI-H187).

Keywords:Clausena lansium, coumarin, clausenalansimin A, clausenalansimin B, cytotoxicity

RESUMO

Duas novas cumarinas, Clausenalansimin A (5) e B (9), juntamente com sete cumarinas conhecidas (1-4 e 6-8), foram isoladas de galhos de Clausena lansium. Todos os compostos foram determinados por métodos espectroscópicos. Alguns dos compostos isolados apresentaram citotoxicidade contra linhagens de células humanas cancerígenas (KB, MCF7 e NCI-H187).

Introduction

Clausena lansium (Lour.) Skeels is a distant relative of citrus fruit belonging to the Rutaceae family. Several parts of this plant have been used as a folk medicine in China and Taiwan. For example, the leaves have been used for the treatment of coughs, asthma and gastro-intestinal diseases, and the seeds for acute and chronic gastro-intestinal inflammation and ulcers.¹ In addition, the fruits are used for influenza, colds and abdominal colic pains in the Philippines.² Recently, the seed extract of C. lansium was found to exhibit antifungal, antiproliferative, and HIV reverse transcriptase-inhibitory activities.³ Previous chemical investigations of this plant have revealed a number of alkaloids and coumarins.^2,4-6 As parts of our continuing study on chemical constituents and biological activity of Thai medicinal plants, we report herein the isolation and structure elucidation of two new coumarins (5 and 9) along with seven known coumarins (1-4, 6-8) from the twigs of C. lansium as well as the evaluation of cytotoxicity against KB, MCF7 and NCI-H187 cancer cell lines. In addition, the ¹H and ¹³C NMR spectral data of 7 are also reported herein for the first time.

Results and Discussion

The combination of CH₂Cl₂ and acetone extracts of twigs of C. lansium was separated by chromatographic techniques to yield two new coumarins, clausenalansimin A (5) and B (9), together with seven known compounds (1-4, 6-8). All structures were elucidated using spectroscopic data and compared with those reported in the literature.

Clausenalansimin A (5) was isolated as yellow viscous oil. Its molecular formula was established as C₂₁H₂₂O₅ by HRMS. The UV-Vis spectrum showed an absorption band of a conjugated furanocoumarin at 204-300 nm,⁷ whereas IR spectroscopic data displayed absorption bands of hydroxyl and carbonyl functionalities at 3442 and 1722 cm^-1, respectively. The ¹H NMR spectrum (Table 1) of 5 showed the common signals of furanocoumarin similar to that of 1 at δ 6.36 (1H, d, J 9.6 Hz, H-3), 7.36 (1H, d, J 2.0 Hz, H-2'), 6.81 (1H, s, H-5), 7.68 (1H, d, J 2.0 Hz, H-3') and 7.76 (1H, d, J 9.6 Hz, H-4).⁸ The main different signals were observed at 5.68 (1H, dt, J 7.2 and 1.2 Hz, H-2''), 5.11 (1H, m, H-6''), 5.04 (2H, d, J 7.2 Hz, H-1''), 4.41 (1H, m, H-5''), 2.17 (2H, m, H-4''), 1.76 (3H, s, H-9''), 1.68 (3H, s, H-8'') 1.65 (3H, s, H-10'') in the ¹H NMR spectral data and could be identified as the 5-hydroxy-3,7-dimethylocta-2,7-dienyloxy moiety. This moiety was also confirmed by COSY and HMBC correlations (Figure 2) and located at C-8 because the H-1'' (δ 5.04) showed ³J HMBC correlation with the carbon at C-8 (δ 131.5). Therefore, clausenalansimin A was deduced to be 5.

Clausenalansimin B (9) was isolated as yellow viscous oil with a molecular formula of C₁₉H₁₈O₆ on the basis of HREIMS. The ¹H NMR spectral data of 9 revealed an α,β-unsaturated lactone at δ 6.12 (1H, d, J 9.6 Hz, H-3) and 7.96 (1H, d, J 9.6 Hz, H-4) and two meta-coupling aromatic protons at δ 6.48 (1H, d, J 1.6 Hz, H-6) and 6.26 (1H, d, J 1.6 Hz, H-8).⁹ These results implied that this molecule is a 5,7-oxygenated coumarin nucleus. Moreover, the ¹H NMR spectrum also showed signals of -OCH₂-CH=C- unit at δ 4.66 (2H, m, H-1'') and 5.53 (1H, t, J 5.6 Hz, H-2'') and an α,β-unsaturated γ-lactone moiety at δ 2.35 (1H, dd, J 17.0, 7.2 Hz, H-4a''), 2.61 (1H, dd, J 17.0, 5.2 Hz, H-4b''), 5.11 (1H, m, H-5''), 7.09 (1H, br t, H-6''), 1.92 (3H, br s, H-8'').¹⁰ The side chain unit was also supported by COSY and HMBC experiments (Figure 2) and located at C-5 due to the ²J and ³J HMBC correlations of the H-4 (δ 7.96), H-6 (δ 6.48) and H-1'' (δ 4.66) with C-5 (δ 156.1). The structure of clausenalansimin B, therefore, was assigned to 9.

The remaining seven known coumarins included xanthotoxol (1),⁷ imperatorin (2),¹¹ heraclenin (3),¹² heraclenol (4),¹³ wampetin (6),¹⁴ indicolactonediol (7),¹⁵ and isoscopoletin (8)¹⁶ were determined by the 1D and 2D NMR spectral data and comparison with their reported physical and spectroscopic data. In addition, the complete assignments of ¹H and ¹³C NMR of indicolactonediol (7) are also reported herein for the first time (Table 1).

Only the stable compounds and sufficient quantity were evaluated for their cytotoxicity against three human cancer cell lines including oral cavity cancer (KB), breast cancer (MCF7) and small cell lung cancer (NCI-H187). The results of cytotoxicity of the tested compounds (1-3, 5 and 6) are summarized in Table 2. All these compounds showed weak activity with cytotoxicity against KB, MCF7 and NCI-H187 cell lines, except coumarins 3 and 6 which were found to be inactive with MCF7 cancer cell line.

Experimental

General procedures

The optical rotation [α]_D values were determined with a Bellingham & Stanley ADP440 polarimeter. UV-Vis spectra were recorded with a Perkin-Elmer UV-Vis spectrophotometer. The IR spectra were recorded with a Perkin-Elmer FTS FT-IR spectrophotometer. The NMR spectra were recorded using 400 MHz Bruker spectrometer. Chemical shifts were recorded in parts per million (δ) in CDCl₃ with tetramethylsilane (TMS) as an internal reference. The HRMS was obtained from a MicroTOF, Bruker Daltonics or MAT 95 XL mass spectrometers. Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60 H (Merck, 5-40 μm) and silica gel 100 (Merck, 63-200 μm), respectively. Precoated plates of silica gel 60 F₂₅₄ were used for analytical purposes.

Plant material

The twigs of C. lansium were collected in April 2008 from Nan Province, northern part of Thailand. Botanical identification was achieved through comparison with a voucher specimen number QBG 25077 in the herbarium collection of Queen Sirikit Garden, Mae Rim District, Chiang Mai, Thailand.

Extraction and Isolation

Air-dried twigs of C. lansium (6.73 Kg) were successively extracted with CH₂Cl₂ and acetone over a period of 3 days each at room temperature. The CH₂Cl₂ and acetone extracts were combined (34.02 g) and subjected to QCC over silica gel eluted with a gradient of hexane-acetone (100% hexane to 100% acetone) to provide seventeen fractions (A-Q). Fraction G (562.9 mg) upon standing at room temperature gave compound 2 (247.1 mg). Fraction I (1.83 g) was separated by CC using 20% EtOAc-hexane to give seven subfractions (I1-I7). Subfraction I7 (546.4 mg) was further purified by CC with 30% hexane-CH₂Cl₂ yielding compound 3 (207.8 mg). Fraction K (4.64 g) was separated by CC eluted with a gradient of CH₂Cl₂-EtOAc (5% EtOAc-CH₂Cl₂ to 100% EtOAc) to give compound 1 (5.6 mg), and nine subfractions (K1-K9). Subfraction K6 (124.2 mg) was subjected to repeated CC with 2% acetone-CH₂Cl₂ to afford compound 5 (8.7 mg). Purification of fraction M (806.5 mg) was performed by sephadex LH20 with 60% CH₂Cl₂-MeOH, yielding five subfractions (M1-M5). Subfraction M2 (199.9 mg) was further subjected to repeated CC with a gradient of CHCl₃-hexane (70% CHCl₃-hexane to 100% CHCl₃) to afford eleven subfractions (M2a-M2k). Subfraction M2b (49.9 mg) was further subjected to repeated CC with 80% CHCl₃-hexane to yield four fractions (M2b1-M2b4). Compound 6 (4.8 mg) was derived from fraction M2b2 (18.5 mg) by repeated CC using 60% CHCl₃-hexane whereas compound 7 (1.7 mg) was obtained from fraction M2b3 (21.5 mg) by repeated CC with 5% EtOAc-CHCl₃. Subfraction M2d (8.6 mg) was further purified by prep.TLC with 5% EtOAc-CHCl₃ to afford 6 (7.3 mg). Fraction N (621.1 mg) was performed by CC with 40% EtOAc-hexane, yielding eleven subfractions (N1-N11). Subfraction N8 (65.5 mg) was separated by CC using a gradient of EtOAc-CH₂Cl₂ (10% EtOAc-CH₂Cl₂ to 25% EtOAc-CH₂Cl₂) to yield compound 9 (4.0 mg) while subfraction N10 (39.4 mg) was purified by prep.TLC with 7% EtOAc-CH₂Cl₂ to give compound 4 (9.8 mg).

Clausenalansimin A (5)

Yellow viscous oil; [α]_D²⁷ +48.1° (c 0.02, CHCl₃); UV λ_max/nm (MeOH): 204, 216, 247, 299; IR (neat) ν_max/cm^-1: 3442, 2924, 2855, 1722, 1626, 1587, 1443, 1401, 1327, 1261, 1150, 1092, 1026, 870, 801, 754; ¹H NMR (CDCl₃, 400 MHz) and ¹³C NMR (CDCl₃, 100 MHz), see Table 1; HRMS (APCI, -ve) m/z: 389.1161 ([M+Cl_--]^-, calc. C₂₁H₂₂O₅Cl, 389.1156).

Clausenalansimin B (9)

Yellow viscous oil; [α]_D²⁷ +17.24° (c 0.02, CHCl₃); UV λ_max/nm (MeOH): 207 and 329; IR (neat) ν_max/cm^-1: 2921, 1726, 1609, 1454, 1364, 1237, 1154, 1115, 823, 612; ¹H NMR (CDCl₃, 400 MHz) and ¹³C NMR (CDCl₃, 100 MHz): see Table 1; HREIMS m/z: 342.1158 ([M_--]⁺, calc. C₁₉H₁₈O₆, 342.1103).

Cytotoxicity assay

The procedures for cytotoxic assay were performed by sulphorhodamine B (SRB) assay (anti-KB and MCF7) and colorimetric method (anti-NCI-H187) as described by Skehan et al.⁶ In this study, three cancer cell lines, MCF7 (breast cancer), NCI-H187 (human, small cell lung cancer) and KB (oral human epidermal carcinoma) were used. Doxorubicin was the reference substance in this study.

Supplementary Information

Supplementary data are available free of charge at

http://jbcs.sbq.org.br, as PDF file.

Acknowledgments

We would like to thank the Thailand Research Fund (Grant no. RSA5280011) and Mae Fah Luang University for financial support and to the Bioassay Research Facility of BIOTEC (Thailand) for cytotoxicity tests. WM thanks Mae Fah Luang University for a Ph.D graduate student research grant. We are indebted to Mr. Nitirat Chimnoi, Chulabhorn Research Institute, Bangkok, for recording mass spectrum and Ms. Nareerat Tongtip, Department of Chemistry, Faculty of Science, Phuket Rajabhat University for recording NMR spectral data. We also thank Assoc. Prof. Dr Chatchanok Karalai and Assoc. Prof. Chanita Ponglimanont, Department of Chemistry, Faculty of Science, Prince of Songkla University for supporting some scientific material and giving valuable suggestions.

Received: July 18, 2009

Web Release Date: December 16, 2009

SUPPLEMENTARY INFORMATION

Figure S1 - Click to enlarge

Figure S2 - Click to enlarge

Figure S3 - Click to enlarge

Figure S4 - Click to enlarge

Figure S5 - Click to enlarge

Figure S6 - Click to enlarge

Figure S7 - Click to enlarge

Figure S9 - Click to enlarge

Figure S10 - Click to enlarge

Figure S11 - Click to enlarge

Figure S12 - Click to enlarge

Figure S13 - Click to enlarge

Figure S14 - Click to enlarge

Figure S16 - Click to enlarge

Figure S17 - Click to enlarge

Figure S18 - Click to enlarge

Figure S19 - Click to enlarge

Figure S20 - Click to enlarge

Figure S21 - Click to enlarge

Figure S23 - Click to enlarge

Figure S24 - Click to enlarge

• 1. Adebajo, A. C.; Iwalewa, E. O.; Obuotor, E. M.; Ibikunle, G. F.; Omisore, N. O.; Adewunmi, C. O.; Obaparusi, O. O.; Klaes, M.; Adetogun, G. E.; Schmidt, T. J.; Verspohl, E. J.; J. Ethnopharmacol. 2009, 10, 122.
• 2. Lin, J. H.; Phytochemistry 1989, 28, 621.
• 3. Ng, T. B.; Lam, S. K.; Fong, W. P.; Biol. Chem. 2003, 384, 289.
• 4. Yang, M. H.; Chen, Y. Y.; Huang, L.; Phytochemistry 1988, 27, 445.
• 5. Kumar, V.; Vallipuram, K.; Adebajo, A. C.; Reisch, J.; Phytochemistry 1995, 40, 1563.
• 6. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, R. M.; J Natl Cancer Inst 1990, 82, 1107.
• 7. Ito, C.; Katsuno, S.; Furukawa, H.; Chem. Pharm. Bull. 1998, 46, 341.
• 8. Gellért, M.; Reisch, J.; Szendrei, K.; Novák, I.; Csedö, K.; Phytochemistry 1972, 11, 2894.
• 9. Ngadjui, B. T.; Ayafor, J. F.; Sondengam B. L.; Connolly, J. D.; Phytochemistry 1989, 28, 585.
• 10. Nakamura, K.; Takemura, Y.; Ju-ichi, M.; Ito, C.; Furukawa, H.; Heterocycles 1998, 43, 549.
• 11. Masuda, T.; Takasugi, M.; Anetai, M.; Phytochemistry 1998, 47, 13.
• 12. Adityachaudhury, N.; Ghosh, D.; Choudhuri, A.; Phytochemistry 1974, 13, 235.
• 13. Razdan, T. K.; Kachroo, V.; Harkar, S.; Koul, S.; Phytochemistry 1982, 21, 923.
• 14. Khan, N. U.; Navqi, S. W. I.; Ishratullah, K.; Phytochemistry 1983, 22, 2624.
• 15. Rakash, D.; Raj, K.; Kapil, R. S.; Popli, S. P.; Phytochemistry 1978, 17, 1194.
• 16. Al-Barwani, F. M.; Eltayeb, E. A.; Biochem. Syst. Ecol. 2004, 32, 1097.

Publication Dates

• Publication in this collection
  21 May 2010
• Date of issue
  2010

History

• Received
  18 July 2009
• Accepted
  16 Dec 2009