Coumarins and xanthones from the seeds of Mammea siamensis

SHORT REPORT

Coumarins and xanthones from the seeds of Mammea siamensis

Surat Laphookhieo^I,*; Phunrawie Promnart^I; John Keith Syers^I; Akkharawit Kanjana-Opas^II; Chanita Ponglimanont^III; Chatchanok Karalai^III

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

^IIDepartment of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand

^IIIDepartment of Chemistry, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand

ABSTRACT

A new coumarin, mammea E/BB cyclo D (1), together with five known compounds, mammea E/BA cyclo D (2), suragin C (3), therapin B (4), 1,7-dihydroxyxanthone (5) and 1-hydroxy-5-methoxyxanthone (6), were isolated from the seeds of Mammea siamensis. Their structures were characterized using 1D and 2D NMR spectral data. Suragin C and therapin B showed cytotoxic activity against breast adenocarcinoma (MCF-7), human cervical cancer (HeLa), colon cancer (HT-29) and human oral cancer (KB).

Keywords: mammea E/BB cyclo D, cytotoxic activity, Mammea siamensis, guttiferrae

RESUMO

Uma cumarina inédita, mammea E/BB ciclo D (1), juntamente com cinco compostos conhecidos, mammea E/BA ciclo D (2), suragina C (3), terapina B (4), 1,7-dihidroxixantona (5) e 1-hidróxi-5-metoxyxantona (6), foram isolados de sementes de Mammea siamensis. Suas estruturas foram caracterizadas usando dados de RMN 1D e 2D. Suragina C e terapina B mostraram atividade citotóxica contra adenocarcinoma de mama (MCF-7), câncer cervical humano (HeLa), câncer de colon (HT-29) e câncer oral humano (KB).

Introduction

Mammea siamensis (Miq) T. Anders. (Guttiferae), known in Thai as "Sarapi", is a small evergreen tree distributed in Thailand, Laos, Cambodia, Vietnam and Myanmar. The flowers of this plant have been used in traditional Thai medicine as a heart tonic. Investigations of different parts of the plant have revealed the presence of several coumarins and xanthones.^1-4 We have previously reported the isolation and structure determination of phenolic compounds from the seeds of this species.⁵ In a continuation of our study on this plant, we now report herein the isolation and structure elucidation of a novel compound, mammea E/BB cyclo D (1), together with three known coumarins, mammea E/BC cyclo D (2),³ suragin C (3),⁶ therapin B (4)⁷ and two known xanthones, 1,7-dihydroxyxanthone (5)⁸ and 1-hydroxy-5-methoxyxanthone (6)⁹ from the CH₂Cl₂ extract (Figure 1). The cytotoxic activity of all isolates is also reported.

Experimental

General procedures

Melting points were determined using a Fisher-John melting point apparatus. The optical rotation [a]_D values were determined with a JASCO P-1020 polarimeter. UV spectra were measured with a UV-160A spectrophotometer (Shimadzu). The IR spectra were measured with a Perkin-Elmer FTS FT-IR spectrophotometer. The ¹H and ¹³C NMR spectra were recorded using 500 MHz Varian UNITY INOVA and 300 MHz Bruker FTNMR Ultra Shield^TM spectrometers. Chemical shifts were recorded in parts per million (d) in CDCl₃ with tetramethylsilane (TMS) as an internal reference. The EIMS was obtained from a MAT 95 XL mass spectrometer. Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60 F₂₅₄ (Merck, 230-400 Mesh ASTM) and silica gel 100 (Merck, 70-230 Mesh ASTM), respectively. Precoated plates of silica gel 60 F₂₅₄ and reversed-phase (RP-18 F_254S) were used for analytical purposes.

Plant material

The seeds of M. siamensis were collected from Mae Fah Luang University, Tasud, Muang, Chiang Rai Province, northern Thailand in August 2005. The identification was made by Professor Puangpen Sirirugsa, Department of Biology, Faculty of Science, Prince of Songkla University and a voucher specimen (No. SC09) was deposited at Prince of Songkla University Herbarium.

Extraction and Isolation

The seeds (224.5 g) of M. siamensis were extracted successively with CH₂Cl₂ (500 mL) at room temperature for 5 days. The filtered samples were combined and the solvents were evaporated under reduced pressure to provide the CH₂Cl₂ extracts (44.4 g).

The CH₂Cl₂ extract (44.4 g) was chromatographed by QCC and eluted with hexane-EtOAc mixtures to give seven fractions (F1-F7). Fraction F2 (1.92 g) was purified by RP-18 CC with acetone: H₂O (3:1) and followed by RP-18 preparative TLC with acetone: H₂O (3:1) to yield 1 (3.1 mg) and 2 (4.3 mg). Fraction F4 (3.35 g) was separated by CC with EtOAc: hexane (3:17) and followed by RP-18 preparative TLC with MeOH:H₂O (4:1) to provide five subfractions (F4a-F4e). Subfraction F4b (12.8 mg) was purified by preparative TLC with EtOAc: hexane (1:3, v/v) to give 5 (2.1 mg). Subfraction F4d (1.02 g) was purified by RP-18 CC with MeOH:H₂O (4:1) and followed by CC with EtOAc: hexane (1:3) to afford 4 (16.8 mg) and 3 (32.6 mg). Fraction F6 (167.0 mg) was separated by CC with EtOAc: hexane (2:3, v/v) to give 6 (6.3 mg).

Mammea E/BB cyclo D (1)

Yellowish viscous oil; ¹H NMR (d, CDCl₃, 300 MHz): 14.44 (7-OH), 6.74 (1H, d, J 10.0 Hz, H-4''), 6.60 (1H, dd, J 6.8, 2.8 Hz, H-1'), 6.30 (1H, s, H-3), 5.60 (1H, d, J 10.0 Hz, H-5''), 4.02 (1H, sextet, J 6.3 Hz, H-2'''), 2.17 (3H, s, H-1'-COCH₃), 1.97 (1H, m, H-2'a), 1.80 (1H, m, H-3'''a), 1.78 (1H, m, H-2'b), 1.58 (3H, s, H-7''), 1.56 (3H, s, H-8'), 1.45 (1H, m, H-3'''b), 1.26 (3H, d, J 6.3 Hz, H-5'''), 1.07 (3H, t, J 7.2 Hz, H-3') and 1.06 (3H, t, J 7.2 Hz, H-4'''); ¹³C NMR data (CDCl₃, 75 MHz): 210.8 (C-1'''), 170.3 (CH₃CO), 163.5 (C-7), 159.3 (C-2), 157.5 (C-4), 156.7 (C-8a), 155.7 (C-5), 126.8 (C-5''), 115.8 (C-4''), 106.5 (C-6), 106.4 (C-3), 103.7 (C-8), 100.9 (C-4a), 80.2 (C-6''), 73.0 (C-1'), 46.9 (C-2'''), 29.6 (C-3'''), 28.6 (C-2'), 28.4 (C-7''), 27.8 (C-8''), 21.0 (CH₃CO), 16.9 (C-5''' ), 10.6 (C-4'''), 10.0 (C-3'); EIMS m/z (rel. int.): 428 [M]⁺ (39), 413 (100), 371 (45), 353 (13), 311 (29), 283 (5); HREIMS m/z [M]⁺ 428.1813 (calc. for C₂₄H₂₈O₇, 428.1835); UV(MeOH) l_max/nm: 225, 280, 285, 300, 373; IR(CHCl₃) n_max/cm^-1: 3454, 1738, 1655, 1605; -15.0º (c 0.10, MeOH).

Cytotoxicity assay

The procedure for cytotoxic assay was performed by sulphorhodamine B (SRB) assay as described by Skehan et al.¹⁰ In this study, four cancer cell lines, MCF-7 (breast adenocarcinoma), HeLa (human cervical cancer), HT-29 (colon cancer) and KB (human oral cancer) were used. Camptothecin, the reference substance, exhibited activity toward MCF-7, HeLa, HT-29 and KB cell lines, with IC₅₀ range of 0.2-2.0 µg mL^-1 (Table 1)

Results and Discussion

Mammea E/BB cyclo D (1) was isolated as a yellowish viscous oil, with a molecular formula C₂₄H₂₈O_7, established by HREIMS analysis of its molecular ion [M]⁺ at m/z 428.1813 (Calc. for C₂₄H₂₈O₇m/z 428.1835). The UV spectrum of 1 showed absorption bands at 225, 280, 285, 300 and 373 nm suggesting the presence of conjugation in the molecule. The IR spectrum exhibited the characteristic of carbonyl (1738 and 1655 cm^-1) and hydroxyl (3454 cm^-1) functionalities. The ¹³C NMR and DEPT spectra revealed 24 carbons, including six methyls (d 10.0, 10.6, 16.9, 21.1, 27.8 and 28.4), two methylenes (d 28.6 and 29.6), five methines (d 46.9, 73.0, 106.4, 115.8 and 126.8) and eleven non-hydrogenated carbons (d 80.2, 100.9, 103.7, 106.5, 155.7, 156.7, 157.5, 159.3, 163.5, 170.3 and 210.8). The ¹H NMR spectral data showed a chelated hydroxyl proton at d 14.44 assignable to 7-OH on the basis of HMBC correlations (Figure 2). The ¹H NMR spectrum also displayed a singlet signal at d 6.30, which is a typical chemical shift for H-3 of 4-alkylcoumarin skeleton.^3,11 In addition, the ¹H NMR spectrum also showed the signals of chromene ring, 2-methyl-1-oxobutyl and 1-acetoxypropyl moieties. The ¹H NMR signals of chromene ring were appeared at d 6.74 (1H, d, J 10.0 Hz, H-4''), 5.60 (1H, d, J 10.0 Hz, H-5''), 1.58 (3H, s, H-7'') and 1.56 (3H, s, H-8''), while the 2-methyl-1-oxobutyl group showed signals at d 4.02 (1H,sextet, J 6.3 Hz, H-2'''), 1.80 (1H, m, H-3'''a), 1.45 (1H, m, H-3''b), 1.26 (3H, d, J 6.3 Hz, H-5''') and 1.06 (3H, t, J 7.2 Hz, H-4'''). Finally, the 1-acetonylpropyl moiety showed the ¹H NMR signals at d 6.60 (1H, dd, J 6.8, 2.8 Hz, H-1'), 2.17 (3H, s, H-1'-COCH₃), 1.97 (1H, m, H-2'a), 1.78 (1H, m, H-2'b), and 1.07 (3H, t, J 7.2 Hz, H-3'). The locations of the three moieties were established based on the observed key HMBC correlations (Figure 2). The 1-acetoxypropyl unit was placed at C-4 due to the oxymethine proton H-1' (d 6.60) showed ²J and ³J correlation with C-4a (d 100.9), C-4 (d 157.5) and C-3 (d 106.4) in the HMBC spectrum. In addition, the olefinic proton H-3 (d 6.30) also showed ²J and ³J correlations with C-1' (d 73.0), C-2 (d 159.3) and C-4a (d 100.9). The chromene ring was located at C-5/C-6 because the olefinic proton H-4'' (d 6.74) displayed HMBC correlations to C-5 (d 155.7), C-6 (d 106.5) and C-7 (d 163.5). Finally, the hydroxyl group was located at C-4 because the chelated hydroxyl proton showed HMBC correlations to C-6 (d 106.5), C-7 (d 163.5) and C-8 (d 103.7) and the 2-methyl-1-oxobutyl moiety had to be placed at C-8 by process of elimination. Therefore, the structure of mammea E/BB cyclo D was characterized as 1.

The reported compounds were tested for their cytotoxicity against MCF-7 (breast adenocarcinoma), HeLa (human cervical cancer), HT-29 (colon cancer) and KB human oral cancer) cell lines. The results are summarized in Table 1. Only two coumarins, 3 and 4, were found to be active in this study. Suragin C (3) showed cytotoxic activities against all four cancer cell lines better than therapin B (4) (Table 1). It should be noted that the structural difference between suragin C (3) and therapin B (4) is only at C-6 (3 possesses a geranyl group while 4 contains a prenyl group). The presence of a geranyl moiety seems to be important for enhancing the cytotoxic activity. The anticancer drug used as a standard in our cytotoxic assay is camptothecin, which has an IC₅₀ in the range of 0.2-2.0 µg mL^-1.

It is worth noting that the genus Mammea of the family Guttiferae has been known to be rich in coumarins and xanthones,^1-5,9,12-17 with more than 30 compounds having been isolated from this genus. In this study, we have observed an additional new coumarin from the seeds of M. siamensis.

Acknowledgments

We are grateful to Mae Fah Luang University for financial support (Grant No. 49101020005). We also thank Professor Puangpen Sirirugsa, Department of Biology, Faculty of Science, Prince of Songkla University for plant identification.

Received: January 9, 2007

Web Release Date: August 6, 2007

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Publication Dates

• Publication in this collection
  25 Oct 2007
• Date of issue
  2007

History

• Accepted
  06 Aug 2007
• Received
  09 Jan 2007