Year : 2009 | Volume
: 5 | Issue : 20 | Page : 324--328
Cytotoxic and Antibacterial Constituents from the Roots of Sonchus oleraceus L. Growing in Egypt
Ehab Saad Elkhayat
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut branch, Assiut 71524, Egypt
Ehab Saad Elkhayat
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut branch, Assiut 71524
Phytochemical study of the roots of Sonchus oleraceus L. (Astraceae) Growing in Egypt, afforded loliolide 1 for the first time from the genus Sonchus in addition to 15-O- β -glucopyranosyl-11 β ,13-dihydrourospermal A 2 , ursolic acid 3 , lupeol 4 and β-sitosterol- 3-O-glucopyranoside 5 for the fi rst time from the plant. The biological evaluation of the isolated compounds showed cytotoxic activity of 1 and 2 against PC33 and L5187Y cell lines, in addition to antibacterial activity against S.aureus , B. subtilis , E. Coli and N. gohnorea . The structures of the compounds were elucidated using 1D ( 1 H and 13C), 2D (H-H COSY, H S QC and HMBC) NMR and MS spectroscopic data.
|How to cite this article:|
Elkhayat ES. Cytotoxic and Antibacterial Constituents from the Roots of Sonchus oleraceus L. Growing in Egypt.Phcog Mag 2009;5:324-328
|How to cite this URL:|
Elkhayat ES. Cytotoxic and Antibacterial Constituents from the Roots of Sonchus oleraceus L. Growing in Egypt. Phcog Mag [serial online] 2009 [cited 2021 Apr 20 ];5:324-328
Available from: http://www.phcog.com/text.asp?2009/5/20/324/58154
The genus Sonchus belongs to sub-tribe Crepidinea, tribe Lactuceae and family Astraceae  and includes more than 50 species  . This genus is represented in Egypt by five species namely: maritimus , oleraceus , asper , macrocarpus and tenerrimus . Sonchus plants are well-known with their content of sesqueterpene lactones of the eudismanolide , and guaianolide structures  . Other constituents includes ionone glycosides  , phenyl propanoids  , phenolics [flavonoids and coumarines]  , in addition to sterols and lignans  .
Sonchus oleraceus L. which is a common annual herb, with erect stem branched near a pale yellow infl orescence  and known as smooth sow-thistle  . In Upper Egypt it is commonly known as lobbain () due to its milky juice. Previous studies of S. oleraceus reported the isolation of eudesmanolide and guaianolide lacone glycosides from the plant growing in Japan  and the detection of flavones glycosides in the plant growing in Canary Island  . This paper describes the phytochemical investigation of the roots of the plant growing in Egypt as well as the biological evaluation of the isolated compounds. Where the monoterpene loliolide 1 was isolated for the first time from the genus Sonchus , in addition to 15-O- β -glucopyranosyl-11 β ,13-dihydrourospermal A 2 , ursolic acid 3 , lupeol 4 and β -sitosterol-3-O-glucopyranoside 5 which were isolated for the first time from the plant. Besides, the crude alcoholic extract, compound 1 and compound 2 showed antibacterial activites against against S. aureus , B. subtilis , E. coli and N. gohnorea . Compounds 1 and 2 showed in vitro cytotoxic activity against L5187Y cell line while compound 2 only showed cytotoxic activity against PC33 cell line.
Materials and Methods
The fresh roots of S. oleraceus L. were collected in March- April 2007 from the wild plants around the campus of Al-Azhar University, Assiut, Egypt. The plant material was kindly identifi ed by Prof. Dr. A. Fayed, Professor of Plant Taxonomy, Faculty of Science, Assiut University, Egypt. A voucher specimen was deposited in the Department of Pharmacognosy herbarium, Faculty of Pharmacy, Al-Azhar University, Assiut (Registration code W. Az-007 So).
Pre-coated silica gel 60 F 254 plates (E. Merck) were used for TLC. Vacuum liquid chromatography (VLC) was carried out using silica gel 60, 0.04-0.063 mm mesh size (Merck). The solvent systems used for TLC analyses were CHCl 3 -MeOH (97:3, system I), CHCl3 -MeOH (9:1, system II) and CHCl3 -MeOH (75:25, system III). The TLC plates were visualized by spraying with p - anisaldehyde/H2 SO4 reagent and heating at 110 o Cfor 1-2 min. HPLC was performed on semi-preparative RP-18 column (Cosmosil 5C18 ARII, 250 Χ 10 mm) with a UV detector at λ max 220 nm and fl ow rate of 2.5 ml/min. 1 H and 13 C-NMR spectra were recorded on a JEOL-JNMEX- 400 spectrometer (400 MHz for 1 H and 100 MHz for 13C, respectively). EI-MS data were obtained with a JEOL JMS-700T mass spectrometer. All solvents were distilled prior to use. NMR grade solvents (Merck) were used for NMR analysis.
Extraction and Isolation
The air-dried powdered aerial parts of S. oleraceus (1.2 kg) were extracted with 70 % MeOH (4 Χ 3 L) at room temperature; evaporation of the methanol extract under reduced pressure affords a dark brown residue (14.2 g). The residue was subjected to VLC on silica gel using CHCl3 : MeOH gradients and afforded 6 fractions. Fraction I was chromatographed on silica gel and eluted with CHCl3 : MeOH gradient and afforded compounds 1 (6.2 mg), 3 (13.7 mg) and 4 (11.4mg). Fraction II was chromatographed on silica gel and eluted with CHCl3 : MeOH gradient to afford compound 5 (18 mg). Meanwhile, fraction III was chromatographed on silica gel and eluted with CHCl3 : MeOH gradient, followed by semi-preparative RP-18 HPLC [MeOH: H 2 O (40:60)], to compound afford 2 (4.3 mg).
Loliolide 1: 1 H-NMR (CDCl3 , 400MHz): 5.69 (1H, s, H-6), 4.33 (1H, ddd, J = 3.2,3.6,3.18 Hz, H-2), 2.47 (1H, dt, J = 14.1,2.6, H-1a), 1.98 (1H, dt, J = 14.6,2.7, H-1b), 1.79 (1H, dd, J = 9.3,4 Hz, H-3a), 1.53 (1H, dd, J = 14.6,3.7 Hz, H-3b), 1.78 (3H, s, Me-12), 1.47 (3H, s, Me-11), 1.27 (3H, s, Me-10).
13 C-NMR (CDCl3 , 100MHz): 182.1 (C-5), 172 (C-7), 113.2 (C-6), 87 (C-9), 66.9 (C-2), 47.5 (C-3), 45.4 (C-1), 35.8 (C-4), 30.4 (C-10), 27.1 (C-12), 26.5 (C-11).
15-O- β -glucopyranosyl-11 β ,13-dihydrourospermal A 2: 1 H-NMR (DMSO-d6 , 400MHz): 9.6 (1H, s, H-14), 6.85 (1H, t, J = 8.5 Hz, H-1), 5.11 (1H, d, J = 10.3 Hz, H-5), 4.88 (1H, t, J = 10.3 Hz, H-6), 4.56 (1H, d, J = 11.4 Hz, H-15a), 4.28 (1H, d, J = 11.7 Hz, H-15b), 3.91 (1H, m, H-8), 2.94 (1H, m, H-9a), 2.35 (1H, d, J = 15.7 Hz, H-9b), 2.64 (1H, m, H-11), 2.51 (2H, m, H2 -2), 2.07 (2H, m, H2 -3), 1.64 (1H, m, H-7), 1.37 (3H, d, J = 6.7 Hz, Me-13). The glucose moiety 4.52 (1H, d, J = 7.4 Hz, H-1′), 3.2-3.9 (10H, m, H-2′ to H-6′).
13 C-NMR (DMSO-d6 , 100MHz): 199.1 (C-14), 181.3 (C-12), 160 (C-1), 144.8 (C-10), 136.8 (C-4), 129.7 (C-5), 76.2 (C-6), 71.3 (C-8), 67.5 (C-15), 55.9 (C-7), 41 (C-11), 33.3 (C-3), 33 (C-9), 27.7 (C-2), 16.1 (C-13). The glucose moiety: 101.8 (C-1′), 76.8 (C-3′), 76 (C-5′), 73.2 (C-2′), 69.8 (4′), 61.4 (C-6′).
Cytotoxicity Assay. The cytotoxicity was evaluated by the [ 3 H] Thymidine assay  against mouse lymphoma (L5178Y), rat brain cancer cells (PC33) and human nasopharynx carcinoma cells. All cells were mycoplasmafree and cultures were propagated under standardised Conditions  .
Antimicrobial Assay. The antibacterial and antifungal activities were evaluated using the agar plate diffusion assay  . Susceptibility discs (5.5mm) were impregnated with solution of each of the alcoholic extract and compounds 1 and 2 at concentrations of 5 and 10 μg/ml. The discs were dried and placed on agar plates inoculated with the test bacterial strains: Bacillus subtilis , Staphylococcus aureus , Escherichia coli and Neisseria gonorrheae , and the fungal strains: Candida albicans and Aspergillus flavus . Each plate was inoculated with a single organism and the test was run in duplicates. The plates were incubated at 37 oC and checked for inhibition zones afte r2 4 h for bacteria and after 48hrs for fungi.
Results and Discussion
Compound 1 . Was isolated as needle crystals, the EIMS showed molecular ion peak at m/z 197 [M+H] + with a significant fragment at m/z 178 [M- H2 O] + suggested the presence of an hydroxyl substituent. The 1 H-NMR spectrum, showed the presence of an olefenic proton at δ H 5.69 (s), three methyls at δ H 1.27 (s), 1.47 (s) and 1.78 (s). In addition to oxymethine signal at δ H 4.33 (dt, J = 3.8, 3.6 Hz). The 13 C-NMR spectrum showed the presence of 11 carbons. The carbon resonances suggested an α,β- unsaturated lactone as elicited by the carbons at δ C 182.1, 172 and 113.2, which was confi rmed by the HMBC cross peaks of the olefenic proton at δ H 5.69 (H-6) with the carbons at δ C 182.1 (C-5), 172 (C-7) and 87 (C-9). The HMBC cross peaks of Me-10 with C-4, Me-11 with C-4, H-2 with C-1 and C-3, in addition to the cross peaks of Me-12 with C-9 have assigned structure 1 . From the previous data, 1 was identifi ed as loliolide , , and this is the fi rst isolation from genus Sonchus .
Compound 2 . Was isolated as an oily residue, the EIMS showed a molecular ion peak at m/z 465 [M+Na] + . The 1 H-NMR spectrum showed signals identified as aldehydic proton at δ H 9.6 (s), two olefenic protons at δ H 6.85 (t, J = 8.5 Hz) and 5.11 (d, J = 10.3 Hz), in addition to a hydroxymethine at δ H 3.91 (m) and a tertiary methyl at δ H 1.37 (d, J = 6.7 Hz). Furthermore, the spectrum showed an anomeric proton at δ H 4.52 (d, J = 7.4 Hz). The 13 CNMR showed the presence of 21 carbons, including six carbons of a glucopyarnosyl moiety. The sequence of the aliphatic and olefenic protons was made-up using the H-H COSY experiment[Figure 2], which afforded the series from H-1 to H2 -3 and from H-5 to H2 -9, in addition to the chain from H-6 to H 3 -13 through H-7 and H-11. The HMBC experiment[Figure 2] showed the cross peaks of H-1 with C-10, H 2 -3 with C-4, H2 -9 with C-10 and H-11 with C-12 and the cross peaks of H2 -15 with C-4, have afforded a costtunolide nucleus  . The cross peaks of H-14 with C-10 and H2 -15 with C-1′ assigned structure 2. From the previous data, 2 was identifi ed as 15-O- β - glucopyranosyl-11 β ,13-dihydrourospermal A  , and this is the fi rst isolation from S. oleraceus.
Compounds ,, were identified as ursolic acid  , lupeol  and β-sitoststerol-3-O-β-glucopyranoside , respectively on comparing their physical and spectral data with literatures. These compounds were isolated for the first time from S. oleraceus .
The in vitro evaluation of the cytotoxic activity of compounds 1 and 2 using the microculture tetrazolium technique (MTT), for the isolated compounds showed that 15-O- β -glucopyranosyl-11 β ,13-dihydrourospermal A2 was active against L5178Y and PC33 cell lines (ED50 6.2 and 5.2 μg/ml, respectively) and Loliolide 1 was active only against L5178Y (ED50 4.7 μg/ml).
The antimicrobial activity of the alcoholic extract and compounds 1 and 2 , revealed antibacterial activity against; B. Subtilis , E coli , S. aureus and N. gonorrhoeae [Table 1]. The alcoholic extract (10μg/ml) showed inhibition zones of 10, 9, 9 and 11 against the tested strains, respectively. Compound 1 (10 μg/ml) was the most active as it showed inhibition zones of 16, 16, 15 and 15, while compound 2 (10 μg/ml) was less active as the inhibition zones of 12, 13, 14 and 15. None of the tested compounds or the alcoholic extract showed any activity against the fungi Candida albicans or Aspergillus flavus .
It is noteworthy to mention that this is the fi rst cytotoxic and antimicrobial evaluation of loliolide 1 and 15-O- β - glucopyranosyl-11 β ,13-dihydrourospermal A 2 , although loliolide was reported to has immunosuppressive activity against T and B-lymphocytes  .
The author thanks Mr. Mahmoud A. Hafez, Graduate School of Natural Science and Technology, Kanazawa University, Japan, for running the cytotoxicity assay.[Figure 1]
|1||Giner R. M. , Ubeda A. , Just M. J. , Serrano A. , Manez S. , Rios J. L. A Chemotaxonomic survey of Sonchus subgenus Sonchus , Biochemical Systematics and Ecology , 21 : 617 - 620 ( 1993 ).|
|2||Tomb A. S. , in the Biology and Chemistry of Compositae , ( Academic Press , London , 1977 ), p. 1067 .|
|3||Tδckholm Vivi , Student's Flora of Egypt , ( Cairo University press , Second Edition, 1974 ), p. 607 .|
|4||Berrera J. B. , Fajardo L. , Gonzales M. Terpenoids from the Sonchus . VI. Tuberiferine from Sonchus tuberifer Svent. , Tetrahedron letters , 3475 - ( 1967 ).|
|5||Mahmoud Z. , El-Masry S. , Amer M. , Ziechen J. , Grenz M. Sesqueterpene lactones from Sonchus macrocarpus , Phytochemistry , 23 : 1105 - 1107 ( 1984 ).|
|6||Helal A. M. , Nakamura N. , El-Askary H. , Hattori M. Sesqueterpene lactone glucosides from Sonchus asper , Phytochemistry , 53 : 473 - 477 ( 2000 ).|
|7||Shimizu S. , Miyase T. , Ueno A. , Usmanghani K. Sesqueterpene lactone glycosides and ionones derivative glycosides from Sonchus asper , Phytochemistry , 28 : 3399 - 3402 ( 1989 ).|
|8||Zhang Z. , Xie W. , Li P. , Shi Y. , Jia Z. Sesqueterpenoids and phenyl propane derivatives from Sonchus uliginosus , Helvetica Chemica Acta , 89 : 2927- 2932 ( 2006 ).|
|9||Mansour R. M. , Saleh N. A. , Boulos L. A Chemotaxonomic study of the phenolics of Sonchus , Phytochemistry , 22 : 489 - 492 ( 1983 ).|
|10||Mahmoud Z. , El-Masry S. , Amer M. , Zieschen J. , Bohlman F. Two eudesmanolides from Sonchus macrocarpus , Phytochemistry , 22 : 1290 - 1291 ( 1983 ).|
|11||Quereshi S. J. , Awan A. G. , Khan M. A. , Bano S. Taxonomic study of the genus Sonchus L. from Pakistan , Journal of Biological Science , 2 : 309 - 314( 2002 ).|
|12||Carnes M. D. and Carnes C. D. , The Wild Flowering Plants of Bahrain. Illustrated Guide , ( IMMEL Publishing , 1989 ), p. 225 .|
|13||Miyase T. , Fukushima S. Studies on sesqueterpene glycosides from Sonchus oleraceus L. , Chem. Pharm. Bull ., 35 : 2869 - 2874 ( 1987 ).|
|14||Carmichael J. , DeGraff W. G. , Gazdar A. F. , Minna J. D. , Mitchell J. B. Evaluation of a Tetrazolium-Based Semi-Automated Colorimetric Assay: Assessment of Radio sensitivity , Cancer Res. 47 : 943 - 946 ( 1987 ).|
|15||Kreuter M. H. , Robitzki A. , Chang S. , Steffen R. , Michaelis M. , Kljajic Z. , Bachmann M. , Schrφder H. C. , Muλller W. E. G. Production of cytotoxic agent aeroplysinin by the sponge Verongia aerophoba in vitro culture , Comp. BiochePhysiol. , 101C : 183 - 187 ( 1992 ).|
|16||Elkhayat E. , Edrada R. , Ebel R. , Wray V. , Van Soest R. , Wiryowidagdo S. , Mohammed H. M. , Muller W. E. , Proksch P. New Luffariellolide Derivatives from the Indonesian Sponge Acanthodendrilla sp. , J. Nat. Prod. , 67 : 1809 - 1817 ( 2004 ).|
|17||Conegero L. S. , Ide R. M. , Nazari A. S. , Sarragiotto M. H. Chemical constituents of Alchornea glandulosa (Eupherbiaceae) , Quim. Nova. , 26 : 825 - 827 ( 2003 ).|
|18||Borkosky S. , Valdes D. A. , Bardon A. , Diaz J. G. , Herz W. Sesqueterpene lactones and other consitiuents of Eirmocephala megaphylla and Cyrtocymura cincta , Phytochemistry , 42 : 1637 - 1639 ( 1996 ).|
|19||Ogura M. , Cordell G. A. , Farnsworth N. R. Anticancer sesqoueterpene lactones of Michelia compressa (Magnoliaceae) , Phytochemistry , 17 : 957 - 961 ( 1978 ).|
|20||Said S. , Begum S. Phytochemical Studies of Berberis vulgaris , Chemistry of Natural Compounds , 40 : 138 - 140 ( 2004 ).|
|21||Seebacher W. , Simic N. , Weis R. , Saf R. , Kunert O. Spectral assignment and complete data. Complete assignment of 1 H and 13 C-NMR resonances of oleanolic acid, 18α-oleanolic acid, ursolic acid and their 11-oxo derivatives , Mag. Reson. Chem. , 41 : 636 - 638 ( 2003 ).|
|22||Faizi S. , Ali M. , Saleem R. , Irfanullah , Bibi S. Spectral assignment and complete data. Complete assignment of 1 H and 13 C-NMR assignments of stigma-5-en-3-O- β -glucoside and its acetyl derivative , Mag. Reson. Chem. , 39 : 399 - 405 ( 2001 ).|
|23||Okada N. , Shirata K. , Niwano M. , Koshino H. , Uramoto M. Immunosuppressive activity of a monoterpene from Eucommia ulmoides , Phytochemistry , 37 : 281 - 282 ( 1994 ).|