A new xanthone from the roots of Centaurium spicatum PDF

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Phytochemistry Letters 4 (2011) 126–128 Contents lists available at ScienceDirect Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol A new xanthone from the roots of Centaurium spicatum Mohamed A. El-Shanawany a, Gamal A. Mohamed b, Alaa M. Nafady b, Sabrin R.M. Ibrahim a, Moham...

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Phytochemistry Letters 4 (2011) 126–128

Contents lists available at ScienceDirect

Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol

A new xanthone from the roots of Centaurium spicatum Mohamed A. El-Shanawany a, Gamal A. Mohamed b, Alaa M. Nafady b, Sabrin R.M. Ibrahim a, Mohamed M. Radwan c,e, Samir A. Ross c,d,* a

Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut 71524, Egypt c National Center for Natural Products Research, University of Mississippi, School of Pharmacy, University 38677, USA d Department of Pharmacognosy, University of Mississippi, School of Pharmacy, University 38677, USA e Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 11 November 2010 Received in revised form 17 December 2010 Accepted 23 December 2010 Available online 5 January 2011

The chloroformic fraction of the roots of Centaurium spicatum L. afforded one new xanthone named 1,5,8trihydroxy-3,6,7-trimethoxyxanthone (1) together with six known xanthones (2–7), one of them isolated for the first time from a plant source (2). One secoiridoid glucoside (8) was also isolated. The structures of the isolated compounds were established based on 1D and 2D (1H–1H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated compounds were tested for their antimicrobial and antiprotozoal activities. Compound 6 displayed moderate antifungal activity against Candida krusei and Cryptococcus neoformans with IC50 values of 12.8 and 17.9 mg/ml respectively. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Centaurium spicatum Gentianaceae Xanthones Secoiridoid Antimicrobial Antiprotozoal

1. Introduction Centaurium spicatum L. (Gentianaceae) is an erect littlebranched annual herb, wide spread in Europe (Everard, 1969). The plant grows in specific moist habitats, mainly among cultivated crops by water sides and along ditches (Batanouny, 1986; Tackholm, 1974). A decoction of the plant is used as folkremedy for gastric and abdominal pain, hypertension, elimination of kidney and ureter stones, to relief renal colic, as healing agent for wounds, and in ointment of sciatica, and as an infusion for diabetes treatment (Batanouny, 1986; Weiss and Fintelmann, 2000; Bibi et al., 2006; Rizk and El-Ghazaly, 1995). A survey of the current literatures revealed the isolation and identification of xanthones, secoiridoids, alkaloids from the plant (Weiss and Fintelmann, 2000; El-Shanawany et al., 1989; Kazmi et al., 1989; Vander and Labadie, 1981). The important antimicrobial bioactivities of the xanthones (Dharmaratne et al., 1999; Gopalakrishnan et al., 1997; Rath et al., 1996), together with the little previous phytochemical work on C. spicatum, encouraged us to examine this species. In the present work we report the isolation and structure elucidation of

* Corresponding author at: University of Mississippi, School of Pharmacy, National Center for Natural Products Research, University, MS 38677, USA. Tel.: +1 662 915 1031; fax: +1 662 915 7989. E-mail address: [email protected] (S.A. Ross).

one new xanthone (1), together with six known xanthones (2–7), in addition to one known secoiridoid glucoside, sweroside (8). The antimicrobial, antileishmanial and antimalarial activities of them were also reported. 2. Results and discussion Compound 1 was obtained as a yellow amorphous powder. The molecular formula was assigned as C16H14O8 on the basis of HRESIMS and NMR data. The structure of 1 was derived on the basis of spectroscopic analyses. The IR spectrum showed absorption band at nmax 1628 cm 1 reminiscent of xanthone carbonyl group (Ishiguro et al., 1998; Parra et al., 1984). The 1H NMR spectrum showed the presence of three methoxyl (dH 3.86, 3.81, and 3.75) and three hydroxyl groups {dH 11.89 (1-OH), 11.72 (8-OH), and 10.75 (5OH)} (Silva and Pinto, 2005). In addition, a pair of meta-coupled aromatic protons at dH 6.35 and 6.64 assignable to H-2 and H-4, respectively were observed. The 13C and DEPT NMR spectroscopic data of 1 showed 16 signals; 12 aromatic carbons, three methoxyls, and one carbonyl at dC 183.3 indicated a double-chelated carbonyl, meaning there were two hydroxyls attached at C-1 and C-8 (Table 1) (Hua et al., 2007; Frahm and Chaudhuri, 1979). Direct correlations of protons to their respective carbons were revealed in the HMQC spectrum. The structure of 1 was confirmed by the key HMBC [OH-8/ C-8, C-8a, C-7; OH-5/C-5, C-4b, C-6; OCH3-6/C-6; OCH3-7/C-7; H-2/ C-8b, C-1, C-3, C-4; H-4/C-3, C-2, C-4a; OCH3-3/C-3] correlations

1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2010.12.008

M.A. El-Shanawany et al. / Phytochemistry Letters 4 (2011) 126–128 Table 1 1 H- and 1C-NMR data of compound 1 and 1H-NMR data of 2 in CDCl3 mult. (J Hz). 1

1 2 3 4 4a 4b 5 6 7 8 8a 8b 9 1-OH 8-OH 5-OH 3-OCH3 5-OCH3 6-OCH3 7-OCH3

2

dH

dC

dH

6.35 d (2.4) – 6.64 d (2.4) – – – – – – – – – 11.89 s 11.72 s 10.75 s 3.86 s – 3.81 s 3.75 s

162.1 98.1 167.0 93.5 157.5 145.7 128.4 131.5 149.6 157.5 100.2 101.7 183.3 – – – 56.7 – 61.6 60.7

– 6.36 d (2.2) – 6.56 d (2.2) – – 6.73 brs – 6.71 brs – – – – 11.99 s 11.40 s – 3.89 s – 3.97 s –

(Fig. 1). On the basis of the above evidences, the structure of 1 was elucidated as 1,5,8-trihydroxy-3,6,7-trimethoxyxanthone and considered as a new natural product. Compound 2 was isolated as a yellowish powder. The 1H NMR spectrum disclosed that the molecule consisted of a tetrasubstituted xanthone, containing two methoxyl groups at dH 3.97 (6OCH3) and 3.89 (3-OCH3), four meta coupled protons at dH 6.73 (brs, H-5), 6.71 (brs, H-7), 6.56 (d, J = 2.2 Hz, H-4) and 6.36 (d, J = 2.2 Hz, H-2). The downfield singlets at dH 11.99 (1-OH) and 11.40 (8-OH) indicated the presence of two hydroxyl groups chelated with the carbonyl (Table 1) (Silva and Pinto, 2005; Hua et al., 2007; Frahm and Chaudhuri, 1979). Therefore, the structure of compound 2 was elucidated as 1,8-dihydroxy-3,6-dimethoxyxanthone. Compound 2 was previously isolated from fungus Diploschistes scruposus (Thelotremataceae) (Elix et al., 1987), and this is the first report for its isolation from plant source. The known compounds [3–8] were identified by comparing their spectroscopic and physical data with those published in literature as; 1-hydroxy-3,5,8-trimethoxyxanthone (3), 1-hy[()TD$FIG] droxy-3,5,6,7,8-pentamethoxyxanthone (4), 1,5-dihydroxy-3-

OH 8

R2

8a

7 6

H3CO

O

5

OH

9

8b

4b O

4a

1

2 3

4

OCH3

R1 1 2

R1 = OH, R2 = OCH3 R2 = H R1 = H, OH

O

OH

H3CO

H

H3CO

O OH

OCH3 H

Fig. 1. HMBC correlations of compound 1.

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methoxyxanthone (5), 1,6-dihydroxy-3,5- dimethoxyxanthone (6), 1,8-dihydroxy-3,5-dimethoxyxanthone (7), and sweroside (8) (Vander and Labadie, 1985; Monache et al., 1983; Aimi et al., 1993; Yue-hua and Rui-lin, 1993). Antimicrobial and antiprotozoal assays were performed for the isolated compounds. Only compound 6 showed moderate antifungal activity against C. krusei and C. neoformans with IC50 values of 12.82 and 17.90 mg/ml, respectively without any cytotoxicity against Vero cells up to 25.0 mg/ml. None of the tested compounds showed activity against the tested bacterial, leishmanial and malarial strains. 3. Experimental 3.1. General Melting points were carried out on an Electrothermal 9100 Digital Melting Point apparatus (Electrothermal Engineering Ltd, Essex, England). The mass spectrometric analysis was performed on an Bruker Bioapex FT-MS in ESI mode. The IR was measured on a Shimadzu Infrared-400 spectrophotometer (Kyoto, Japan). 1D and 2D NMR spectra were recorded on a Varian AS 400 spectrometer. Column chromatographic separation was performed on silica gel 60 (0.04–0.063 mm), RP-18 (0.04–0.063 mm Merck), and Sephadex LH-20 (0.25–0.1 mm, Merck). Compounds were detected by UV absorption at l 255 and 366 nm followed by spraying with anisaldehyde/H2SO4 reagent and heating at 110 8C for 1–2 min. 3.2. Plant material The roots of C. spicatum L. (Gentianaceae) were collected in May 2000 from New Valley, 200 km southwest of Assiut City, Egypt. The plant was identified and authenticated by Prof. Dr. A. Fayed, Professor of Plant Taxonomy, Faculty of Science, Assiut University. A voucher specimen (CS2000) was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut, Egypt. 3.3. Extraction and isolation and characterization Air dried powdered roots (1.0 kg) of C. spicatum L. were extracted with MeOH (3 5 L) for 72 h at room temperature. The combined extracts were concentrated under reduced pressure to afford a dark brown residue (23.2 g). This residue was suspended in distilled water (100 ml) then partitioned between n-hexane (3 400 ml), chloroform (3 400 ml), and ethyl acetate (3 400 ml), successfully. Each fraction was concentrated under reduced pressure to give n-hexane fraction (2.3 g), chloroform fraction (4.5 g), ethyl acetate fraction (2.5 g) and aqueous fraction (11.3 g). Chloroform fraction (4.5 g) was chromatographed on silica gel cc (160 g  100  5 cm) using n-hexane–EtOAc gradient to afford 13 fractions (CS-1 to CS-13). Combined fractions CS-3 and CS-4 (0.76 g) were subjected to silica gel cc (100 g  2  60 cm) eluted with n-hexane/EtOAc gradient mixtures to afford compound 7 (68 mg). Fraction CS-5 (0.46 g) was subjected to silica gel cc (80 g  1.5  50 cm) using n-hexane/EtOAc gradient as an eluent to afford compound 2 (1.0 mg). Fraction CS-7 (0.42 g) was chromatographed over silica gel column (80 g  1.5  50 cm) using n-hexane/EtOAc gradient as an eluent to give two subfractions CS-7A (115 mg) and CS-7B (240 mg). Subfraction CS-7A on rechromatographing using the same adsorbent and mobile phase afforded compound 3 (26 mg), while CS-7B was subjected to RP-18 column (100 g  2  60 cm) using MeOH/H2O (90:10) to give compound 4 (42 mg). Fraction CS-8 (0.32 g) was subjected to silica gel cc using n-hexane/EtOAc gradient to afford compound 5 (61 mg). Combined fractions CS-9 and CS-10 (0.87 g) were

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subjected to Sephadex LH-20 column (150 g  5  100 cm) using MeOH/CHCl3 (90:10) as an eluent to give three subfractions CS-910A (210 mg), CS-9-10B (306 mg), and CS-9-10C (232 mg). Separately, silica gel cc of subfractions CS-9-10B and CS-9-10C using n-hexane/EtOAc gradient afforded compounds 6 (39 mg) and 1 (22 mg). Fraction CS-13 (320 mg) was subjected to silica gel column (100 g  2  60 cm) using CHCl3/MeOH gradient and further purified on RP-18 column using MeOH/H2O (70:30) as an eluent to afford pure compound 8 (120 mg). 1,5,8-Trihydroxy-3,6,7-trimethoxyxanthone (1): Yellow amorphous powder; IR (KBr): gmax = 3520, 1628, 1605, 1555, 1159, 1057 cm 1; 1H- and 13C-NMR data see Table 1; HREIMS m/ z = 333.2611 [M H] (Calc. for C16H13O8: 333.2609). 1,8-Dihydroxy-3,6-dimethoxyxanthone (2): Yellowish white amorphous powder; IR (KBr): gmax = 3425, 1652, 1616, 1580, 1169, 1084, 951 cm 1; 1H and 13C NMR data see Table 1; HREIMS: m/z = 289.1356 [M+H]+ (Calc. for C15H13O6: 289.1352). 3.4. Antimicrobial, antiprotozoal and cytotoxicity assays The antimicrobial activity of the isolated compounds was tested against Candida albicans, Candida krusei, Candida glabrata, Escherichia coli, Pseudomonas aeruginosa, Cryptococcus neoformans, Mycobacterium intracellulare, Staphylococcus aureus, Methicillin resistance Staphylococcus aureus (MRSA) and Aspergillus fumigatus. Also they were evaluated for their antimalarial and antileishmanial activities as well as the cytotoxicity against Vero cell (Baharate et al., 2007; Radwan et al., 2007; Yang et al., 2006). The details of the assays were mentioned as supplementary data. Acknowledgments The authors are thankful to Dr. Bharanti Avula for HRESIMS and to Dr. Melissa Jacobs, Dr. Shabana Khan and Dr. Babu Tekwani for antimicrobial, cytotoxicity and antiprotozoal testings. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytol.2010.12.008. References Aimi, N., Seki, H., Sakal, S., Haginiwa, J., 1993. Kinginoside, a new acyl group carrying iridoid bioside from Lonicera morrowii. Chem. Pharm. Bull. 41, 1882–1884.

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