COUMARINS FROM CYNANCHUM ACUTUM A. El-Demerdash, A. M. Dawidar, E. M. Keshk and M. Abdel-Mogib1 (Received November 2008; Accepted March 2009
ABSTRACT From the aerial parts of Cynanchum acutum L. several compounds have been isolated including the simple coumarins scopoletin and scoparone. The isolated compounds were identified on the basis of spectral methods (IR, UV, 1H NMR, and GC/MS). This is the first report of coumarins from the genus Cynanchum. Key words: Asclepiadaceae, Cynanchum acutum, coumarins, scopoletin, scoparone. RESUMEN De las partes aéreas de Cynanchum acutum L. se aislaron varios compuestos, entre ellos la escopoletina y escoparona. Los compuestos aislados se identificaron por métodos espectroscópicos (IR, UV, 1H NMR, y GC/MS). Este es el primer reporte de cumarinas del genero Cynanchum.
INTRODUCTION Family Asclepiadaceae comprises many medicinal parts with a wide range of therapeutic activities. The dried whole plant of Cynanchum arnottianum has been used in India and tropical America as insecticide and parasiticide (Lewis, H.W. and Lewis., 1977). C. atratum dried whole plant has been used as antifebrile and diuretic in China (Zhang et al., 1985). The dried whole plant of C. paniculatum has been used as anodyne and for the therapy of chronic tracheitis in China (Sakuma et al., 1968). C. wilfordi is used as a substitute for the tonic crude drug “Ka-Shu-Uh” in Korea (Tsukamoto et al., 1985).
The phytochemical investigation of the genus Cynanchum revealed the isolation and characterization of many classes of natural products including steroidal glycosides (Liu et al., 2007), carbohydrates (Yi-Bin et al., 2004), alkaloids (Tian-Ying et al., 2001), phenolic compounds (Lou et al., 1993), and triterpenes (Konda et al., 1990). Cynanchum acutum L. (Asclepiadaceae) is a wild perennial herb commonly distributed in Egypt and known as olliq, modeid, or libbein (Tackholm, 1974). The phytochemical investigation of this species led to the isolation of β-sitosterol, lupeol, lupyl acetate, and α-amyrin (Halim et al.,1990), sarcostine, quercetin and quercetin 3-O-β-
Chemistry Department, Faculty of Science, Mansoura University, Mansoura-35516, Mansoura, Egypt. 1 Corresponding author: E-mail
[email protected], Tel.: +2 0502242388
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A. El-Demerdash, A.M. Dawidar, E.M. Keshk
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M. Abdel-Mogib
D-galactoside (El Sayed et al.,1994), as well as four flavonoid glycosides: quercetin diO-hexoside, quercetin 3-O-rhamnosyl(1→ 2)glycoside, quercetin 3-O-galactoside, and quercetin 3-O-xyloside (Heneidak et al., 2006). In this article, we present the isolation of two simple coumarins, in addition to other compounds from C. acutum. This is the first report of coumarins from the genus Cynanchum.
Plant material
MATERIAL AND METHODS
Air dried aerial parts of C. acutum (800 g) were soaked at room temperature in methanol. After solvent vaporization, the crude extract (40 g) was dissolved in the least amount of methanol and diluted with water and successively extracted with petroleum ether (40-60 °C) and methylene chloride. The petroleum ether extract (6.7 g) was saponified using alcoholic aqueous sodium hydroxide (5%) and stirring for 2 h, then re-extracted with petroleum ether to give the non saponifiable part (2 g). Acidification by conc. HCl (5%) and re-extraction by chloroform afforded the saponifiable part (1.5 g). The non saponifiable part of petroleum ether fraction (2 g) was separated over neutral aluminum oxide (Al2O3) column chromatography, eluted with hexane/ethyl acetate solvent system with increasing polarity to afford the triterpenoidal compounds. Lupyl acetate was isolated with a mixture of hexane/EtOAc (49:1), lupeol with hexane/EtOAc (24:1), and finally a mixture of β-sitosterol and stigmasterol with hexane/EtOAc (13:1). The isolated compounds were further purified using preparative TLC using the same solvent systems mentioned above. Lupyl acetate (0.25 g, Rf = 0.72), lupeol (0.15 g, Rf = 0.3), mixture of β-sitosterol and stigmasterol (0.1 g, Rf = 0.25). The CH2Cl2 fraction (2.6 g) was separated (1 g of which) over a silica gel CC eluted with a petroleum ether/EtOAc solvent system with increasing polarity to afford scoparone (petroleum ether/EtOAc, 3:2)
General 1 H NMR spectra were recorded on a 500MHz spectrometer (Jeol) at Faculty of Science, Alexandria University. Chemical shifts are given in ppm relative to TMS as internal standard. Infrared spectra were recorded as thin film cast from CHCl3, and performed on a Mattson 5000 FT-IR spectrophotometer at Faculty of Science, Mansoura University. GC/MS analyses were performed on a Varian GC interfaced to Finnegan SSQ 7000 mass selective detector (SMD) with ICIS V2.0 data system for MS identification of the GC components. Analyses were run with a DB-5 (J&W Scientific, Folosm, CA) cross-linked fused silica capillary column (30 m long, 0.25 mm internal diameter) coated with polydimethylsiloxane (0.5 µm film thickness). The oven temperature was programmed from 50 oC for 3 min., at isothermal, then heating by 7C/ min. to 250 o C and isothermally for 10 min., at 250 oC. Injector temperature was 200 oC and the volume injected was 0.5µl. transition-line and ion source temperature was 250 oC and 150 o C respectively. The mass spectrometer had a delay of 3 min. to avoid the solvent peak and then scanned from m/z 50 to m/z 300. Ionization energy was set at 70eV (Agriculture Research Center, Dokki, Cairo, Egypt). Thin layer chromatography and preparative TLC were performed on silica gel (Kieselgel 60, GF 254) of 0.25 mm thickness.
C. acutum L. (Asclepiadaceae) was collected from New Damitta, Egypt, in May 2005 by the forth author and identified by Prof. Dr. Ibrahim Mashaly, Prof. of Plant Ecology, Botany Department, Faculty of Science, Mansoura University. Processing of the plant material
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Table 1: 1H NMR spectra data for scopoletin
Table 2: 1H NMR spectral data for scoparone.
No. of H atom δ value, ppm
No. of H atom δ value, ppm Integration, multiplicity (J, Hz)
Integration,
3 4 5 8 C-6-OMe
multiplicity (J, Hz) 6.28 7.6 6.92 6.86 3.96
1H, 1H, 1H, 1H, 3H,
d(9.6) d (9.6) s s s
3 4 5 8 C-6-OMe C-7-OMe
6.28 7.6 6.92 6.86 3.94 3.91
1H, 1H, 1H, 1H, 3H, 3H,
d (9.6) d, (9.6) s s s s
Table 3: Identified compounds from the CH2Cl2 fraction by GC/MS No. of peak
1 2 3 4
Identification of compounds2 Isovanillin 3-Oxo-alpha-ionol Syringic aldehyde (-)-Liliolide
Mol. Wt. 152.05 208.15 182.06 196.11
Rf (min.) 12.68 16.42 16.61 17.99
Area% 1.08 1.96 2.54 2.05
The identification was based on high percentage of matching with the authentic spectrum using NIST library 2
and scopoletin (petroleum ether/EtOAc, 1:1). The isolated coumarins were further purified by preparative TLC to afford scopoletin at Rf 0.21 (petroleum ether/EtOAc 6:5) (0.198 g, 19.8%) and scoparone at Rf 0.39 (petroleum ether/EtOAc, 3:2) (0.2 g, 20.0%). A sample of the CH2Cl2 fraction was analyzed by GC/MS and the identified compounds are indicated in Table 3. Scopoletin: yellow needles, m.p. 204 oC, IR, cm 1: 3396 (OH), 1713 (CO δ-lactone), 1611 ( CH=CH ), 1565 and 1514 ( aromatic benzene ring). Scoparone: colorless needles, m.p. 144 o C, IR, cm-1: 1713 (CO δ-lactone), 1611 (CH=CH ), 1565 and 1514 ( aromatic benzene ring). RESULTS AND DISCUSSION The purification of the extract of the dried aerial parts of C. acutum afforded some known natural products, including β-sitosterol, stigmasterol, lupeol, and lupyl acetate, which have been previously isolated from the same species (Halim et al., 1990) where their
structures were proven by 1H NMR data. The 1H NMR spectra of compounds scopoletin and scoparone (Tables 1 and 2) showed two doublets with coupling constant of 9.15 Hz at δ 6.27 and 7.60 ppm, which were assigned as H-3 and H-4, respectively, characteristic for coumarins. The IR spectrum of scopoletin showed absorption bands at 3396 cm-1, due to a hydroxylic group; 1713 cm-1, corresponding to carbonyl group (δ-lactone); 1611 cm-1, corresponding to CH=CH group; 1565 and 1514 cm-1, corresponding to aromatic benzene ring. The 1H NMR spectrum of scopoletin showed a methoxyl group singlet at δ 3.94 ppm and two aromatic proton singlets at δ 6.91 and 6.84 ppm which were explained by a 6,7-disubstitution, indicating either scopoletin or iso-scopoletin framework. By addition of sodium acetate solution according to Horowitz et al. (1960), a notable bathochromic shift from 340 to 390 nm with increase in the intensity of the band was observed, indicating that the isolated compound had a scopoletin structure (TzuChing et al., 2007).
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The 1H NMR spectrum of scoparone was similar to that of scopoletin, except for an additional singlet at δ 3.91 ppm indicating its identity as scoparone (Schuster et al., 1993), which was found to be in agreement with a molecular ion peak [M+] at m/z 206 in the mass spectrum. In this article we report the isolation of coumarins for the first time from the genus Cynanchum.
and
M. Abdel-Mogib
CONCLUSION The phytochemical investigation of Cynanchum acutum L. has led to isolation and identification of several known natural products including two simple coumarines scopoletin and scoparone which are reported for the first time from the genus. Acknowledgments Thanks to Prof. Dr. Ibrahim Mashaly for identification of the plant material.
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