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Flavonoid constituents and cytotoxic activity of Erucaria hispanica (L.) Druce growing wild in Egypt
⁎Tel.: +20 233335455. monakhalil66@hotmail.com (M.M. Marzouk)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Thirteen flavonoid compounds were isolated for the first time from the aerial parts of Erucaria hispanica (L.) Druce growing in Egypt. Their structures were established on the basis of detailed chromatographic and spectroscopic techniques (UV, 1D and 2D NMR and ESIMS). The cytotoxic activity of the methanol extract as well as some isolated compounds against four human carcinoma cell lines; breast (MCF7), liver (HEPG2), cervix (HELA) and colon (HCT116) were evaluated.
Keywords
Brassicaceae
Erucaria hispanica (L.) Druce
Flavonoids
Biflavonoids
Cytotoxic activity
1 Introduction
Brassicaceae is one of the largest angiosperm families with approximately 338 genera and more than 3709 species distributed worldwide (Al-Shehbaz et al., 2006). In the flora of Egypt, Brassicaceae is the fourth of eleven large families, widely distributed in all phytogeographic regions (Boulos, 1995). It includes economically important ornamentals, crops, and vegetables, as well as it is a source of cooking oils and forage (Judd et al., 1999). The plants of this family are used in the treatment of several diseases because of their anticancer, antibacterial, antifungal, antirheumatic, and antidiabetic properties (Kirtikar and Basu, 1975). The genus Erucaria is represented by four species that grow in Egypt; Erucaria crassifolia, Erucaria microcarpa, Erucaria pinnata and Erucaria hispanica (Täckholm, 1974; Boulos, 1999). Four flavonoid compounds: 3-O-galactopyranoside of quercetin and isorhamnetin, lucenin-1 (luteolin 6,8-di-C-β-glucopyranoside) and robinetin (3,7,3′,4′,5′-penta hydroxy flavone) were previously isolated from the aerial parts of E. microcarpa (Hashem, 2007). The flavonoid constituents and cytotoxic activity of E. hispanica have not yet been reported.
2 Experimental
2.1 General
1D and 2D NMR experiments (1H, 13C, HMQC and HMBC) were recorded on a Jeol EX-500 spectrometer: 500 MHz (1H NMR), 125 MHz (13C NMR). UV spectrophotometer (Shimadzu model 2401 PC), EIMS: Finnigan-Mat SSQ 7000 spectrometer, ESIMS: LCQ Advantage Thermo Finnigan spectrometer. CC Polyamide 6S (Riedel-De-Haen AG, Seelze Haen AG, Seelze Hanver, Germany) using MeOH/H2O as eluent. CC Silica gel 60 (Merck, 0.063–0.2 mm) using CH2Cl2/MeOH (2:3). PC (descending) Whatman No. 1 and 3 MM papers, using solvent systems (1) H2O, (2) 15% HOAc (H2O–HOAc 85:15), (3) CAW (CHCl3–HOAc–H2O 90:45:6), (4) BAW (n-BuOH–HOAc–H2O 4:1:5, upper layer), (5) BBPW (C6H6–n-BuOH–pyridine-H2O 1:5:3:3, upper layer). Solvents 4 and 5 were used for sugar analysis, Sephadex LH-20 (Pharmazia). Authentic samples were obtained from the department of phytochemistry and plant systematics, NRC. Complete acid hydrolysis for O-glycosides (2 N HCl, 2 h, 100 °C) were carried out and followed by paper co-chromatography with authentic samples to identify the aglycones and sugar moieties. The sugar units of C-glycoside flavonoids were determined using ferric chloride degradation.
2.2 Plant material
Fresh samples of E. hispanica (L.) Druce were collected on the Alexandria–Cairo desert road near Bremely’s cave (Egypt) in April 2008. The sample was collected and identified by Prof. Dr. S.A. Kawashty and Dr. M.M. Marzouk. A voucher specimen (No. 782) has been deposited in the herbarium of the National Research Centre (CAIRC).
2.3 Extraction and isolation
Air-dried, ground, aerial parts of E. hispanica (L.) Druce (750 g) were defatted with light petroleum ether (40–60 °C) and extracted three times at room temperature with 70% methanol/water. The methanol extract was evaporated under reduced pressure and temperature affording 85 g residue, then subjected to a polyamide column (100 × 5 cm) starting with water as eluent then decreasing the polarity by increasing the concentration of methanol. A total of 48 fractions were collected, each of about 500 mL. Similar fractions were combined according to their PC properties using H2O, 15% HOAc, CAW and BAW as eluents to give six main fractions (A-F). Fraction A (20% MeOH/H2O) was applied to a Silica gel column (35 × 2.5 cm) using CH2Cl2/MeOH (2:3). Fractionation gave rise to two major fractions. They were applied to PC using CAW then purified on a Sephadex LH-20 column (30 × 1.5 cm) using methanol yielded compounds 1 (16 mg), 2 (14 mg) and 3 (24 mg). Fraction B was rechromatographed on polyamide column (35 × 2.5 cm) using 50% H2O yielded four major fractions. They were purified on a Sephadex LH-20 column using MeOH/H2O (1:1) and repeated by using methanol yielded compounds 4 (23 mg), 5 (18 mg), 6 (12 mg), 7 (22 mg) and 8 (13.5 mg). Fraction C (60% MeOH/H2O) yielded compound 9 (10 mg) by separation on PC using 15% HOAc followed by BAW. Fractions D and E (60–80% MeOH/H2O) were chromatographed on PC using BAW to yield compounds 10 (12 mg) and 11 (6 mg). Fraction F (100% MeOH) was applied to PC using 15% HOAc followed by H2O double solvent yielded compounds 12 (8 mg) and 13 (10 mg).
2.3.1 Kaempferol-3-O-β-sophoroside-7-O-β-glucopyranoside (1)
Yellow amorphous powder, mp 223–225 °C, Rf 0.22 (BAW). UV spectral data, λmax (nm): (MeOH) 265, 344 (+NaOMe), 266, 389, (+AlCl3) 275, 299, 344, 394; (+AlCl3 HCl) 275, 300, 344, 393; (+NaOAc) 265, 380; (NaOAc/H3BO3) 265, 345. 1H NMR (500 MHz, DMSO-d6, δ, ppm, J/Hz: 7.8 (2H, d, J = 9.0, H-2′,6′); 6.95 (2H, d, J = 9.0, H-3′,5′); 6.73 (1H, d, J = 2.0, H-8); 6.4 (1H, d, J = 2.0, H-6); 5.65 (1H, d, J = 7.2, H-1″); 5.07 (1H, d, J = 7.2, H-1‴), 4.46 (1H, d, J = 7.0, H-1″″); 3–4 (m, sugar protons overlapped with -OH proton signals). 13C NMR 125 MHz, DMSO-d6, ppm, 177.5 (C-4), 164 (C-7), 161.4 (C-5), 159.7 (C-4′), 158.2 (C-2), 156.8 (C-9), 133.1 (C-3), 130.6 (C-2′), 130.6 (C-6′), 121.1 (C-1′), 116.2 (C-3′), 116.2 (C-5′), 105.2 (C-10), 104.1 (C-1″″), 99.6 (C-1‴), 98.8 (C-6), 98.7 (C-1″), 94.5 (C-8), 82.5 (C-2″), 77.4 (C-5″), 77.1 (C-3″″), 76.9 (C-5″″), 76.6 (C-3″), 76.2 (C-3‴), 74.2 (C-2″″), 73.9 (C-5‴), 73.1 (C-2‴), 70.1 (C-4‴), 69.7 (C-4″″), 69.6 (C-4″), 60.9 (C-6‴), 60.8 (C-6″″), 60.5 (C-6″). Negative ESIMS; m/z 771.2 [M−H]−.
2.3.2 Kaempferol-3-O-β-sophoroside-7-O-β-2‴ feruloyl glucopyranoside (2)
Pale yellow amorphous powder, mp 187–190 °C, Rf 0.29 (BAW). UV spectral data, λmax (nm): (MeOH) 267, 346, (+NaOMe) 268, 394, (+AlCl3) 273, 302, 348, 399, (+AlCl3/HCl) 274, 300, 345, 398, (+NaOAc) 266, 350; (+NaOAc/H3BO3) 264, 314, 348. 1H NMR 500 MHz, DMSO-d6, δ, ppm, J/Hz: 7.94 (2H, d, J = 8.5, H-2′,6′); 7.36 (1H, d, J = 15.6, H-7″″′); 7.12 (1H, d, J = 1.8, H-2″″′); 6.96 (1H, dd, J = 8.1, 1.8, H-6″″′); 6.89 (2H, d, J = 8.5, H-3′,5′); 6.7 (1H, d, J = 2.0, H-8); 6.66 (1H, d, J = 8.2, H-5″″′); 6.36 (1H, d, J = 2.0, H-6); 6.28 (1H, d, J = 15.8, H-8″″′); 5.65 (1H, d, J = 7.5, H-1″); 5.17 (1H, d, J = 7.2, H-1‴), 4.54 (1H, d, J = 7.0, H-1″″); 4.12 (1H, t, H-2‴); 3.74 (3H, s, OCH3); 3–4 (m, sugar protons overlapped with -OH proton signals). 13C NMR 125 MHz, DMSO-d6, ppm, 177.8 (C-4), 167.3 (C-9″″′), 163.4 (C-7), 162.3 (C-5), 161.8 (C-4′), 158.2 (C-2), 156.8 (C-9), 150.3 (C-4″″′), 147.8 (C-3″″′), 145.6 (C-8″″′), 133 (C-3), 131 (C-2′), 131 (C-6′), 126.2 (C-1″″′), 123.5 (C-6″″′), 120.2 (C-1′), 115.9 (C-5″″′), 116.3 (C-3′), 116.3 (C-5′), 115.1 (C-7″″′), 111.4 (C-2″″′), 104.6 (C-10), 103.9 (C-1″″), 99.1 (C-1‴), 98.8 (C-1″), 98.7 (C-6), 94.5 (C-8), 82.2 (C-2″), 78.4 (C-2‴), 77.4 (C-5″), 77.2 (C-5‴), 76.9 (C-5″″), 76.8 (C-3‴), 76.6 (C-3″″), 76.3 (C-3″), 73.3 (C-2″″), 70.2 (C-4‴), 69.9 (C-4″), 69.7 (C-4″″), 60.9 (C-6‴), 60.8 (C-6″″), 60.5 (C-6″), 56.8 (OCH3). Negative ESIMS; m/z 947 [M−H]−.
2.3.3 Kaempferol-3,7-di-O-α-rhamnopyranoside (3)
Whitish yellow needles, mp 201–205 °C. Rf 0.62 (BAW). Negative ESIMS; m/z 577.2 [M−H]−.
2.3.4 Luteolin 6,8-di-C-β-glucopyranoside [Lucenin 1] (4)
Yellow amorphous powder, mp 182–184°C. Rf 0.31 (BAW). Negative ESIMS; m/z 609 [M−H]−.
2.3.5 Kaempferol-3-O-β-glucopyranoside-7-O-α-rhamnopyranoside (5)
Yellow crystals, mp 186–188 °C. Rf 0.46 (BAW). Negative ESIMS; m/z 593.3 [M−H]−.
2.3.6 Isorhamentin-3-O-β-glucopyranoside-7-O-α-rhamnopyranoside (6)
Yellow crystals, mp 202–204 °C. Rf 0.52 (BAW). Positive ESIMS; m/z 625.13 [M+H]+.
2.3.7 Luteolin 8-C-β-glucoside [Orientin] (7)
Yellow amorphous powder, mp 176–178 °C. Rf 0.31 (BAW). Negative ESIMS; m/z 447 [M−H].
2.3.8 Apigenin 8-C-β-glucopyranoside [Vitexin] (8)
Yellow crystals, mp 280–286 °C, Rf 0.39 (BAW). Negative ESIMS; m/z 431.17 [M−H]−.
2.3.9 Quercetin 7-O-β-glucopyranoside (9)
Yellow crystals, mp 277–279 °C, Rf 0.32 (BAW). Negative ESIMS; m/z 463 [M−H]−.
2.3.10 Agathisflavone 7,7″,4′,4‴-tetra methyl ether (10)
Yellow powder, mp 264–268 °C, Rf 0.79 (BAW). Negative ESIMS; m/z 595 [M−H]−.
2.3.11 Isorhamnetin (11)
Pale yellow crystals, mp 246–248 °C, Rf 0.77 (BAW). EIMS; m/z 316 [M+].
2.3.12 3,7,4′-Trihydroxyflavone (12)
Pale yellow amorphous powder, mp 203–205 °C, Rf 0.79 (BAW). EIMS; m/z 270 [M+].
2.3.13 Apigenin (5,7,4′-trihydroxy-flavone)(13)
Yellow crystals, mp 282–286 °C, Rf 0.72 (BAW). EIMS; m/z 270 [M+].
2.4 Cytotoxic activity
Potential cytotoxicity of the aqueous methanol extract of E. hispanica (L.) Druce as well as some pure compounds were tested using the method of Skehan et al. (1990). Cells were plated in a 96-multi well plate (104 cells/well) for 24 h before treatment with the extract to allow attachment of cells to the wall of the plate. Different concentrations of the extracts (0, 10, 25, 50 and 100 μg/mL) were added to the cell monolayer in triplicate. Monolayer cells were incubated with the compounds for 48 h at 37 °C and in an atmosphere of 5% CO2. After 48 h, cells were fixed, washed and stained with sulforhodamine B. Excess stain was washed with acetic acid and attached stain was recovered with Tris–EDTA buffer. Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration was plotted to obtain the survival curve of each tumor cell line as compared with Doxorubicin, the control anticancer drug.
3 Results and discussion
3.1 Identification of flavonoid compounds
The methanol extract of the aerial parts of E. hispanica was purified through chromatographic methods (Mabry et al., 1970; Markham, 1982) yielding 13 flavonoid compounds (Fig. 1). Their structures were established on the basis of detailed chemical and spectroscopic techniques (Markham, 1982; Mabry et al., 1982; Agrawal and Bansal, 1989; Markham and Geiger, 1994). Spectral data of the isolated flavonoids, were in a good accordance with those previously published ones (Nielsen et al., 1993; Ofman et al., 1995; Yuldashev and Karimov, 2001; Marzouk et al., 2009; Kim et al., 2010).
Chemical structures of flavonoid compounds (1–13) isolated from Erucaria hispanica (L.) Druce.
The structures of compounds 1 and 2 were elucidated and fully characterized by 1D and 2D NMR (HMQC and HMBC). On the other hand, compound 10 was reported for the first time in Brassicaceae.
Compound 1: Kaempferol-3-O-β-sophoroside-7-O-β-glucopyranoside, was isolated as a yellow amorphous powder. UV spectral data with diagnostic shift reagents suggested the presence of a 3,7-disubstituted flavonol glycoside with free hydroxyl groups at the 5 and 4′ positions (Mabry et al., 1970; Markham, 1982). Two intermediate spots were detected upon mild acid hydrolysis with 0.1 N HCl, before yielding the aglycone. Complete acid hydrolysis (2 N HCl, 2 h, 100 °C) yielded glucose (Co-PC) and kaempferol (3,5,7,4′-tetrahydroxyflavone) (Co-PC, UV, EIMS and 1H NMR). The negative-ion ESIMS showed a molecular ion peak [M−H]−at m/z 771.2, corresponding to a molecular formula C33H40O21. The 1H NMR spectrum showed two pairs of doublets at δ 7.8 (J = 9.0) and δ 6.95 (J = 9.0) assigned to H 2′,6′ and H 3′,5′, respectively. The two meta coupled doublets at δ 6.7 (J = 2.0) and δ 6.4 (J = 2.0) are assigned to H-8 and H-6, respectively. This downfield chemical shift confirmed that C-7 is substituted in ring A (Mabry et al., 1970; Markham and Geiger, 1994]. The 1H NMR spectrum also revealed three distinct anomeric proton resonances at δ 5.65 (J = 7.2 Hz), δ 5.07 (J = 7.2 Hz), and δ 4.46 (J = 7 Hz), attributed to H-1″, H-1‴ and H-1″″ of three β-glucopyranose units (Markham and Geiger, 1994). The 13C NMR spectrum displayed 33 carbon resonances; 15 of which were assigned to kaempferol as the aglycone moiety and 18 to three glucose moieties. The HMQC experiment showed the three anomeric protons of glucose moieties at δ 5.65, 5.07 and 4.46 were correlated with δ 98.7, 99.6 and 104.2, respectively. In the HMBC spectrum the anomeric proton of one glucopyranosyl unit (H-1″, δ 5.65) showed a correlation with C-3 (δ 134), and the second glucopyranosyl unit (H-1‴, δ 5.07) with C-7 (δ 162.4). The interglycosidic linkage at position 3 of the aglycone between two glucose moieties was found to be (1 → 2) as the signal at δ 82.5 is characteristic for C-2 in a 2-substituted glucose unit (Agrawal and Bansal, 1989; Mabry et al., 1982).
Chemical and spectral analysis of compound 2 (kaempferol-3-O-β-sophoroside-7-O-β-2‴ feruloyl glucopyranoside) showed consistence with the presence of compound 1 and ferulic acid. The negative-ion ESIMS showed a molecular ion peak [M−H]−at m/z 947. In 1H NMR spectrum, the anomeric proton of the glucose moiety at position 7 appeared at down field chemical shift (δ 5.17, d, J = 7.2 Hz) compared to that of compound 1, suggested that the 7-glucose moiety should be substituted at position 2 (Markham and Geiger, 1994), supported by the down filed shift of C-2‴ at 79.9 (Agrawal and Bansal, 1989; Mabry et al., 1982).
3.2 Cytotoxic activity
The cytotoxic activity of the methanol extract against Breast (MCF7), liver (HEPG2), cervix (HELA) and Colon (HCT116) human tumor carcinoma cell lines showed a moderate activity with IC50 values of 18, 20.8, 14.7 and 21.4 μg/mL, respectively (Fig. 2), while the pure isolated compounds; 4, 7, 9 and 10 showed an activity against cervix (HELA) cell line with IC50 values of 20.7, 15.6, 16.4 and 20.3 μg/mL, respectively (Fig. 3) and no activity evaluated against the remaining three cell lines.
Cytotoxic activity of the methanol extract of Erucaria hispanica (L.) Druce against four human carcinoma cell lines: Cervix (HELA), liver (HEPG2), breast (MCF7) and colon (HCT116).
Cytotoxic activity of compounds 4, 7, 9 and 10 against Cervix carcinoma cells line (HELA).
4 Conclusion
This is the first report of the isolation of these flavonoid compounds from E. hispanica (L.) Druce and its cytotoxic activity evaluation. It is worthy to notice that E. hispanica is the first species of the family Brassicaceae to occurrence of biflavonoid.
Acknowledgements
This work was financially supported by the Phytochemistry and Plant Systematics Department, National Research Centre, Egypt. The author is grateful to National Cancer Institute, Cairo University for measuring the cytotoxic activity.
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