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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Cytotoxity of two new coumarin derivatives isolated from Launaea mucronata ab
Eman R. El-Sharkawy
c
& Khaled Mahmoud
a
Department of Eco-physiology, Desert Research Center, 15753 Cairo, Egypt b
Chemical Department, Faculty of Science, Northern Borders University, Arar, Kingdom of Saudi Arabia c
Click for updates
Pharmacognosy Department, National Research Centre, ElBehooth St., 12622 Dokki, Cairo, Egypt Published online: 09 Mar 2015.
To cite this article: Eman R. El-Sharkawy & Khaled Mahmoud (2015): Cytotoxity of two new coumarin derivatives isolated from Launaea mucronata, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1017492 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1017492
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1017492
Cytotoxity of two new coumarin derivatives isolated from Launaea mucronata Eman R. El-Sharkawyab* and Khaled Mahmoudc
Downloaded by [University of Nebraska, Lincoln] at 19:00 08 April 2015
a
Department of Eco-physiology, Desert Research Center, 15753 Cairo, Egypt; bChemical Department, Faculty of Science, Northern Borders University, Arar, Kingdom of Saudi Arabia; cPharmacognosy Department, National Research Centre, El-Behooth St., 12622 Dokki, Cairo, Egypt (Received 23 December 2014; final version received 5 February 2015)
The chloroform fraction of methanol (MeOH) extract of the aerial parts of Launaea mucronata was in vitro investigated for cytotoxicity against HCT116, HepG2 and MCF7 cell lines, and resulted with IC50 ¼ 20.0, 18.6 and 14.30 mg/mL, respectively. The chloroform fraction of the MeOH extract was subjected to further fractionation, which led to the isolation of two new coumarin compounds (6-isobutyl coumarin and 6isobutyl-7-methyl- coumarin). The structures of the new compounds were elucidated by high field 1D and 2D NMR and ESI-MS spectroscopies. Low polar fractions revealed the isolation of other known triterpene compounds which were identified according to its spectral data and comparison with the literature. New coumarin compounds show high cytotoxicity against MCF-7, HCT116 and HepG2 cell lines. Keywords: Launaea mucronata; coumarin; triterpene; anticancer
1. Introduction The genus Launaea belong to family asteraceae, are annual to perennial herbs, glabrous, rarely spinescent, leaves often rosulate, dentate or pinnately lobed; teeth and lobes with white, often callous tips, about 40 species, 10 species in Saudi Arabia (Alfarhan et al. 1998). Several species of this genus are used in folk medicine for bitter stomachic and skin diseases, and are reported to have antitumour, insecticide and cytotoxic activities (Rashid et al. 2000). The antimicrobial activities of coumarin constituents (Ashraf & Nabil 2006) and the neuropharmacological properties (Auzi et al. 2007) have been investigated as well. Phytochemical studies of some genera of the Lactuceae tribe (Ahmed et al. 2006) revealed to be rich in secondary metabolites, specifically sesquiterpene lactones exhibiting the eudesmane, germacrane and guaiane.
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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E.R. El-Sharkawy and K. Mahmoud
Previous work on a methanol extract of Launaea mucronata revealed that the main constituents are flavonoids (rutin, quercetin, isorhamnetin and coumarins) (El-Fiky 1981; Mansour et al. 1983) and terpenoids (Zidorn et al. 2007). The physiological properties of natural and synthetic coumarins have been reviewed by various workers. Coumarin is a simple molecule and many of its derivatives have been known for more than a century; its vital role in plant and animal biology has not been fully exploited. Coumarins have multiple biological activities including disease prevention, growth modulation and anti-oxidant and anti-tumour effects (Lacy & OKennedy 2004). So we decided to isolate and study the biological activity of coumarin compounds to complete the previous study on this plant.
Downloaded by [University of Nebraska, Lincoln] at 19:00 08 April 2015
2. Result and discussion The chloroform fraction of L. mucronata (Forsk.) Muschler. was subjected to chromatographic separation yielding five pure compound, two of them (6-iso butyl-coumarin and 7-methyl-6isobutyl-coumarin) are new compounds which were isolated for the first time, and another three triterpene known compounds, 3 b-acetoxy- oleanen, germenicol and lupenyl acetate. The structures of the isolated compounds were elucidated on the basis of spectroscopic data (1H, 13C NMR and HSQC, HMBC, DEPT135, 1H – 1H COSY). In addition, the ethyl acetate fraction subjected to GC –MS revealed the presence of 20 important compounds (as shown in Table S1). 2.1. Cytotoxic activity The cytotoxic activity of the chloroform fraction of the methanol extract of L. mucronata was in vitro assessed against HCT116, HepG2 and MCF7. The percentages of inhibition related to the reference drug (doxorubicin) are given in Table (S2). The crude extract at concentration 50 and 25 mg/mL showed very high cytotoxicity against HepG2 with ratio of 85.91%, while giving good cytotoxic activity of 62.60% against MCF7 cell line at concentration of 25 mg/mL, and high cytotoxicity of 79.5% against HCT116 at 50 mg/mL, while at low concentration the extract shows normal activity against all cell lines when compared with doxorubicin. Chloroform fraction is rich in triterpene compounds, which have been reported to act as selective catalytic inhibitors of human DNA topoisomerases (Wada et al. 2001), and play important roles in replication, transcription, recombination and chromosome segregation at mitosis (Beretta et al. 2008). Betulin elicits anticancer effects in tumour primary cultures and cell lines in vitro, whereas it exhibited antiproliferative effect, altered tumour cell morphology, decreased their motility and induced apoptotic cell death (Rzeski et al. 2009). Coumarin and their derivatives are important and useful compounds with diverse pharmacological properties. In this study, we have evaluated the in vitro cytotoxic activity of two new coumarin derivatives, (6-iso-butyl coumarin and 6-iso butyl-7-methyl-coumarin). The in vitro cytotoxic activity of compound F1 was evaluated at different concentrations against A549, HCT116, HepG2 and MCF7 cell line, and the percentages of inhibition related to the reference drug (doxorubicin) are presented, the LC50 values for all tested compounds in the above cell lines are shown in Table S3. The new compound exhibiting cytotoxic activity (LC50 , 100 mg/mL) against all tested cancer cell lines was considered as active compound. The new compound F1 shows high cytotoxic activity of 100% for MCF7 at 100 ppm, and very good cytotoxic activity against HCT116 of 975% and HepG2 of 90.4%, and against lung cancer cell line (A549) it shows 90.1% inhibition, while for another compound F2, the amount of compound is not enough to study in vitro cytotoxic activity on all cell lines like F1 but was only evaluated at different concentrations against A549, HCT116, cell line, which shows high cytotoxic activity against HCT116, but very week activity against A549 cell line as the results tabulated in Table S3. Twenty phytoconstituents were identified from ethyl acetate fractions of
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the plant by gas chromatogram and mass spectrometry (GC – MS) analysis (Adams 1995). Compounds were identified by comparison of mass spectra with those in the Wiley and NIST Libraries. The genus Launaea-derived bioactive compounds which used as a source of antibiotic, antioxidant, anti-inflammatory anticancer properties in pharmaceutical industries were used for drug formulation. Andrographolide one of the major constituents of the fraction of chloroform extract (3.47%) is implicated towards its pharmacological activity. There are many studied for the cellular processes and targets modulated by andrographolide treatment in human cancer and immune cells. The reported previous study shows that andrographolide treatment inhibited the in vitro proliferation of different tumour cell lines, representing various types of cancers. These results suggest that andrographolide is an interesting pharmacophore with anticancer and immunomodulatory activities and hence has the potential for being developed as a cancer therapeutic agent. (Rajagopal et al. 2003) 3. Exepermintal 3.1. Plant material Flowering aerial parts of L. mucronata were collected from a wild population growing in the Arar/Rfhaa road, in north region, Saudi Arabia in April 2013; the identity of the plants has been kindly verified by Prof. Dr A. Kamal, Faculty of Science, Northern Border University. Voucher specimens were deposited in the herbarium of faculty of science, Northern Border University, girl department, (S.C.L.m36). 3.2. Extraction and purification The air-dried powder of the arial part (leaf and flower) (1 kg) was extracted by percolation in 70% methanol and filtered off; the marc lifted was extracted by the same way (this process was repeated four times). The combined methanol extracts were concentrated under reduced pressure at a temperature not exceeding 408C till dryness. The dried extract was dissolved in small amount of water and extracted successively using chloroform, ethyl acetate and n-butanol by a separating funnel; this study concerns with the chloroform fraction, while n-butanol fraction is subject of another study. A total of 13 g of chloroform extract were passed through a silica gel column, eluted with a hexane – EtOAc gradient solvent system starting with 100% hexane with increasing amounts of EtOAc to 100%; the column was finally washed with 5% and 10% MeOH in EtOAc, triterpene and coumarin compounds were present in the eluates containing 30% EtOAc to 100% EtOAc, after separation and purification by prep. TLC on silica gel, subjected to analytical GLC and GC –MS for direct comparison with library, revealed the identification of terpenoid compounds, lupeol-3-acetoxy, 12-ursanol, olean-12-en-3-yl-acetate, germinicol, olean-12-ene-28-al and olean-12-en-28-al, cyclic 1,2-ethanediyl mercaptal. Wherever possible elution of the column with hexane – EtOAc (7:3 to 1:1, v/v) followed by prep TLC (Hexane –EtOAc, 3:2) yielded F1 (3.2 mg) and F2 (1.2 mg), pure compound. F1 and F2 were identified by spectroscopic methods (UV, H NMR, C13 NMR, two dimension and mass spectroscopy, detection and fragmentation patterns of these compounds by electrospray ionisation-mass spectrometry (ESI-MS) have been recently reported (Chen et al. 2007)). 3.3. A new compound F1: (Figure 1) UV spectral data of compound F1 in MeOH showed two absorption bands at lmax 271 and 356 NM for bands II and I, respectively, indicating a coumarin nature.
4
E.R. El-Sharkawy and K. Mahmoud O
O
O
O
, F1: 6-isobutyl –coumarin
F2: 6-isobutyl –7-methyl-coumarin
Figure 1. Structure of new compounds F1 and F2.
H NMR (chloroform-500 MHz): a shows signal at 7.61(1H-d,H-3) nand 7.41(1H, d, H-4), 6.7(1H, s, H-8),6.1(1H,d, H-5), 1.64 (d, J ¼ 6.6 Hz, 3H) 1.69 (d, J ¼ 6.6 Hz, 3H) ppm, 4.3(1H, m, C-9). 13C NMR (100 MHz, CDCl3): a 159.7 (CvO), 147.8 (C), 145.7 (CH), 139.2 (CH), 130.3 (CH), 125.3 (C), 119.7 (C), 116.8, (CH), 114.4 (CH2), 25.1 (CH3) 116.0 (CH) ppm. The isolated compound F1: White crystal, molecular weight of 202, it from the molecular ion (m/z) and molecular formula C13H14O2. The fragmentation of the compound shows base beak at 174 m/z with a reduction of CO molecule, 145 m/z with a reduction in C4H9 (ISO butyl, m/z 57). Supporting the proposed structure of compound F1 measuring the two-dimensional HMBC, HSQC spectral data which show the correlation of carbon atom in the lactone ring with hydrogen atoms, 1H binding to C-3 (7.6 p.m.) and C-4 (7.4 ppm), then in the aromatic ring was found that 1H binding to C-5 (6.1 p.m.) and C-8 (6.7 ppm) at Figure 1, and HMBC show correlation of proton H-9 with C-8 and C-7, C-7 and C-9, also, the signal of H-11(d 4.42) showed a correlation with C-5, C-6 (d) and C-7 (d) confirmed its substitution with the isobutyl group to C 6.
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3.4. Identification of new compound F2 The isolated compound F2: White needle crystal, molecular weight of 216, from the molecular ion (m/z), molecular formula, C14H16O2. The fragmentation of compound F2, show molecular ion of 145 m/z for coumarin nucleus, fragment with molecular ion of 173 m/z, with reduction of CH3, then CO. H1NMR of compound F2: s: shows signal at 7.51(1H-d, H-3) nand 7.21(1H, d, H-4), 6.2 (1H, s, H-8), 6.1(1H,s, H-5),1.4(6H, 2CH3) 4.3(1H,m, C-9), 1.69 (d, J ¼ 6.6 Hz, 3H) ppm. 13 C NMR (100 MHz, CDCl3): a: 164. 7 (CvO), 147.8 (C), 145.7 (CH), 139.2 (CH), 130.3 (CH), 125.3 (C), 119.7 (C), 116.8 (CH), 114.4 (CH2), 25.1 (CH3) 116.0 (CH) ppm. HMBC showed correlation of proton H-9 with C-8 and C-7, C-7 and C-9, also the signal of H-11(d 4.42) showed a correlation with C-5, C-6 and C-7, confirming its substitution with the isobutyl group to C 6. 3.5. Cytotoxic effect on human cell line (HePG2 – MCF 7 – HCT116 – A549) Cell viability was assessed by the mitochondrial-dependent reduction of yellow MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to purple formazan, (Mosmann, 1983). 4. Conclusion This study underscores that the crude extract of L. mucronata had various cytotoxic activity against HCT116, HepG2 and MCF-7. The new compound coumarin-6-isobutyl showed high significant cytotoxic activity against different cell lines. Whereas the other new compound, coumarin-6-isobutyl-7-methyl, had high cytotoxity against HCT116. Coumarin and triterpenes are common components of the human diet and appear to be of interest in cancer prevention or
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therapy. Searching on many chemotherapeutic agents against tumour cells without sparing normal cells remain a major obstacle and development of multi-drug resistance further limits chemotherapy in cancer, the promising results will stimulate the development of coumarin compound for cancer chemoprevention and chemotherapy. Supplementary material Supplementary material relating to this article is available online, alongside Figure S1 and Tables S1 –S3.
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Acknowledgements The authors are thankful to the Bioassay-Cell Culture Laboratory, National Research Centre, El-Tahrir St., Dokki, Cairo 12622, Egypt for conducting the analysis and determining the results.
References Auzi ARA, Hawisa NT, Sherif FM, Sarker SD. 2007. Neuropharmacological properties of Launaea resedifolia. Braz J Phamacogn. 17:160–165. Adams RP. 1995. Identification of essential oil components by gas chromatography/mass spectrometry. Carol Stream, IL: Allured Publishing Corporation. Ahmed Z, Ali D, Malik A. 2006. Structure determination of ursene-type triterpenes by NMR techniques. Magn Reson Chem. 44:717–719. doi:10.1002/mrc.1803. Alfarhan AH, Chaudhary SA, Thomas J. 1998. Notes on the flora of Saudi Arabia (Third edition). J King Saud Univ. 10:31–40. Ashraf AE, Nabil AA. 2006. Antibacterial coumarins isolated from Launaea resedifolia. Chem Plant Raw Mater. 1:65–68. Beretta GL, Perego P, Zunino F. 2008. Targeting topoisomerase I: molecular mechanisms and cellular determinants of response to topoisomerase I inhibitors. Expert Opin Ther Targets. 12:1243– 1256. Chen Y, Fan G, Zhang Q, Wu H, Wu Y. 2007. Fingerprint analysis of the fruits of Cnidium monnieri extract by highperformance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry. J Pharm Biomed Anal. 43:926– 936. doi:10.1016/j.jpba.2006.09.015. El-Fiky. 1981. Chemical constituent from Launaea resedifolia. Egypt J Pharm Sci. 29:507–513. Lacy A, O’Kennedy R. 2004. Studies on coumarins and coumarin-related compounds to determine their therapeutic role in the treatment of cancer. Curr Pharm Des. 10:3797–3811. doi:10.2174/1381612043382693. Mansour RMA, Ahmed AA, Saleh NAM. 1983. Flavone glycosides of some Launaea species. Phytochemistry. 22:2630–2631. doi:10.1016/0031-9422(83)80189-7. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 65:55–63. doi:10.1016/0022-1759(83)90303-4. Rajagopal S, Kumar RA, Deevi DS, Satyanarayana C, Rajagopalan R. 2003. Andrographolide, a potential cancer therapeutic agent isolated from Andrographis paniculata. J Exp Ther Oncol. 3:147–158. doi:10.1046/j.13594117.2003.01090.x. Rashid S, Ashraf M, Bibi S, Anjum R. 2000. Insecticidal and cytotoxic activities of Launaea Nudicaulis (Roxb.) and Launaea Resedifolia (Linn.). Pak. J Biol Sci. 3:808–809. Rzeski W, Stepulak A, Szyman´ski M, Juszczak M, Grabarska A, Sifringer M, Kaczor J, Kandefer-Szerszen´ M. 2009. Betulin elicits anti-cancer effects in tumour primary cultures and cell lines in vitro. Basic Clin Pharmacol Toxicol. 105:425–432. doi:10.1111/j.1742-7843.2009.00471.x. Thao NT, Hung TM, Lee MK, Kim JC, Min BS, Bae K. 2010. Triterpenoids from Camellia japonica and their cytotoxic activity. Chem Pharm Bull. 58:121–124. ISSN: 0009-2363. doi:10.1248/cpb.58.121. Wada S, Iida A, Tanaka R. 2001. Screening of triterpenoids isolated from phyllanthus flexuosus for DNA topoisomerase inhibitory activity. J Nat Prod. 64:1545– 1547. doi:10.1021/np010176u. Zidorn C, Ellmerer EP, Heller W, Johrer K, Frommberger M, Greil R, Guggenberger M, Ongania K, Stuppner H. 2007. A new sesquiterpene lactone sulfate from Reichardia gaditana. Z Naturforsch. 62B:132–134.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Cytotoxity of two new coumarin derivatives isolated from Launaea mucronata ab
Eman R. El-Sharkawy
c
& Khaled Mahmoud
a
Department of Eco-physiology, Desert Research Center, 15753 Cairo, Egypt b
Chemical Department, Faculty of Science, Northern Borders University, Arar, Kingdom of Saudi Arabia c
Click for updates
Pharmacognosy Department, National Research Centre, ElBehooth St., 12622 Dokki, Cairo, Egypt Published online: 09 Mar 2015.
To cite this article: Eman R. El-Sharkawy & Khaled Mahmoud (2015): Cytotoxity of two new coumarin derivatives isolated from Launaea mucronata, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1017492 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1017492
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [University of Nebraska, Lincoln] at 19:00 08 April 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1017492
Cytotoxity of two new coumarin derivatives isolated from Launaea mucronata Eman R. El-Sharkawyab* and Khaled Mahmoudc
Downloaded by [University of Nebraska, Lincoln] at 19:00 08 April 2015
a
Department of Eco-physiology, Desert Research Center, 15753 Cairo, Egypt; bChemical Department, Faculty of Science, Northern Borders University, Arar, Kingdom of Saudi Arabia; cPharmacognosy Department, National Research Centre, El-Behooth St., 12622 Dokki, Cairo, Egypt (Received 23 December 2014; final version received 5 February 2015)
The chloroform fraction of methanol (MeOH) extract of the aerial parts of Launaea mucronata was in vitro investigated for cytotoxicity against HCT116, HepG2 and MCF7 cell lines, and resulted with IC50 ¼ 20.0, 18.6 and 14.30 mg/mL, respectively. The chloroform fraction of the MeOH extract was subjected to further fractionation, which led to the isolation of two new coumarin compounds (6-isobutyl coumarin and 6isobutyl-7-methyl- coumarin). The structures of the new compounds were elucidated by high field 1D and 2D NMR and ESI-MS spectroscopies. Low polar fractions revealed the isolation of other known triterpene compounds which were identified according to its spectral data and comparison with the literature. New coumarin compounds show high cytotoxicity against MCF-7, HCT116 and HepG2 cell lines. Keywords: Launaea mucronata; coumarin; triterpene; anticancer
1. Introduction The genus Launaea belong to family asteraceae, are annual to perennial herbs, glabrous, rarely spinescent, leaves often rosulate, dentate or pinnately lobed; teeth and lobes with white, often callous tips, about 40 species, 10 species in Saudi Arabia (Alfarhan et al. 1998). Several species of this genus are used in folk medicine for bitter stomachic and skin diseases, and are reported to have antitumour, insecticide and cytotoxic activities (Rashid et al. 2000). The antimicrobial activities of coumarin constituents (Ashraf & Nabil 2006) and the neuropharmacological properties (Auzi et al. 2007) have been investigated as well. Phytochemical studies of some genera of the Lactuceae tribe (Ahmed et al. 2006) revealed to be rich in secondary metabolites, specifically sesquiterpene lactones exhibiting the eudesmane, germacrane and guaiane.
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
2
E.R. El-Sharkawy and K. Mahmoud
Previous work on a methanol extract of Launaea mucronata revealed that the main constituents are flavonoids (rutin, quercetin, isorhamnetin and coumarins) (El-Fiky 1981; Mansour et al. 1983) and terpenoids (Zidorn et al. 2007). The physiological properties of natural and synthetic coumarins have been reviewed by various workers. Coumarin is a simple molecule and many of its derivatives have been known for more than a century; its vital role in plant and animal biology has not been fully exploited. Coumarins have multiple biological activities including disease prevention, growth modulation and anti-oxidant and anti-tumour effects (Lacy & OKennedy 2004). So we decided to isolate and study the biological activity of coumarin compounds to complete the previous study on this plant.
Downloaded by [University of Nebraska, Lincoln] at 19:00 08 April 2015
2. Result and discussion The chloroform fraction of L. mucronata (Forsk.) Muschler. was subjected to chromatographic separation yielding five pure compound, two of them (6-iso butyl-coumarin and 7-methyl-6isobutyl-coumarin) are new compounds which were isolated for the first time, and another three triterpene known compounds, 3 b-acetoxy- oleanen, germenicol and lupenyl acetate. The structures of the isolated compounds were elucidated on the basis of spectroscopic data (1H, 13C NMR and HSQC, HMBC, DEPT135, 1H – 1H COSY). In addition, the ethyl acetate fraction subjected to GC –MS revealed the presence of 20 important compounds (as shown in Table S1). 2.1. Cytotoxic activity The cytotoxic activity of the chloroform fraction of the methanol extract of L. mucronata was in vitro assessed against HCT116, HepG2 and MCF7. The percentages of inhibition related to the reference drug (doxorubicin) are given in Table (S2). The crude extract at concentration 50 and 25 mg/mL showed very high cytotoxicity against HepG2 with ratio of 85.91%, while giving good cytotoxic activity of 62.60% against MCF7 cell line at concentration of 25 mg/mL, and high cytotoxicity of 79.5% against HCT116 at 50 mg/mL, while at low concentration the extract shows normal activity against all cell lines when compared with doxorubicin. Chloroform fraction is rich in triterpene compounds, which have been reported to act as selective catalytic inhibitors of human DNA topoisomerases (Wada et al. 2001), and play important roles in replication, transcription, recombination and chromosome segregation at mitosis (Beretta et al. 2008). Betulin elicits anticancer effects in tumour primary cultures and cell lines in vitro, whereas it exhibited antiproliferative effect, altered tumour cell morphology, decreased their motility and induced apoptotic cell death (Rzeski et al. 2009). Coumarin and their derivatives are important and useful compounds with diverse pharmacological properties. In this study, we have evaluated the in vitro cytotoxic activity of two new coumarin derivatives, (6-iso-butyl coumarin and 6-iso butyl-7-methyl-coumarin). The in vitro cytotoxic activity of compound F1 was evaluated at different concentrations against A549, HCT116, HepG2 and MCF7 cell line, and the percentages of inhibition related to the reference drug (doxorubicin) are presented, the LC50 values for all tested compounds in the above cell lines are shown in Table S3. The new compound exhibiting cytotoxic activity (LC50 , 100 mg/mL) against all tested cancer cell lines was considered as active compound. The new compound F1 shows high cytotoxic activity of 100% for MCF7 at 100 ppm, and very good cytotoxic activity against HCT116 of 975% and HepG2 of 90.4%, and against lung cancer cell line (A549) it shows 90.1% inhibition, while for another compound F2, the amount of compound is not enough to study in vitro cytotoxic activity on all cell lines like F1 but was only evaluated at different concentrations against A549, HCT116, cell line, which shows high cytotoxic activity against HCT116, but very week activity against A549 cell line as the results tabulated in Table S3. Twenty phytoconstituents were identified from ethyl acetate fractions of
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the plant by gas chromatogram and mass spectrometry (GC – MS) analysis (Adams 1995). Compounds were identified by comparison of mass spectra with those in the Wiley and NIST Libraries. The genus Launaea-derived bioactive compounds which used as a source of antibiotic, antioxidant, anti-inflammatory anticancer properties in pharmaceutical industries were used for drug formulation. Andrographolide one of the major constituents of the fraction of chloroform extract (3.47%) is implicated towards its pharmacological activity. There are many studied for the cellular processes and targets modulated by andrographolide treatment in human cancer and immune cells. The reported previous study shows that andrographolide treatment inhibited the in vitro proliferation of different tumour cell lines, representing various types of cancers. These results suggest that andrographolide is an interesting pharmacophore with anticancer and immunomodulatory activities and hence has the potential for being developed as a cancer therapeutic agent. (Rajagopal et al. 2003) 3. Exepermintal 3.1. Plant material Flowering aerial parts of L. mucronata were collected from a wild population growing in the Arar/Rfhaa road, in north region, Saudi Arabia in April 2013; the identity of the plants has been kindly verified by Prof. Dr A. Kamal, Faculty of Science, Northern Border University. Voucher specimens were deposited in the herbarium of faculty of science, Northern Border University, girl department, (S.C.L.m36). 3.2. Extraction and purification The air-dried powder of the arial part (leaf and flower) (1 kg) was extracted by percolation in 70% methanol and filtered off; the marc lifted was extracted by the same way (this process was repeated four times). The combined methanol extracts were concentrated under reduced pressure at a temperature not exceeding 408C till dryness. The dried extract was dissolved in small amount of water and extracted successively using chloroform, ethyl acetate and n-butanol by a separating funnel; this study concerns with the chloroform fraction, while n-butanol fraction is subject of another study. A total of 13 g of chloroform extract were passed through a silica gel column, eluted with a hexane – EtOAc gradient solvent system starting with 100% hexane with increasing amounts of EtOAc to 100%; the column was finally washed with 5% and 10% MeOH in EtOAc, triterpene and coumarin compounds were present in the eluates containing 30% EtOAc to 100% EtOAc, after separation and purification by prep. TLC on silica gel, subjected to analytical GLC and GC –MS for direct comparison with library, revealed the identification of terpenoid compounds, lupeol-3-acetoxy, 12-ursanol, olean-12-en-3-yl-acetate, germinicol, olean-12-ene-28-al and olean-12-en-28-al, cyclic 1,2-ethanediyl mercaptal. Wherever possible elution of the column with hexane – EtOAc (7:3 to 1:1, v/v) followed by prep TLC (Hexane –EtOAc, 3:2) yielded F1 (3.2 mg) and F2 (1.2 mg), pure compound. F1 and F2 were identified by spectroscopic methods (UV, H NMR, C13 NMR, two dimension and mass spectroscopy, detection and fragmentation patterns of these compounds by electrospray ionisation-mass spectrometry (ESI-MS) have been recently reported (Chen et al. 2007)). 3.3. A new compound F1: (Figure 1) UV spectral data of compound F1 in MeOH showed two absorption bands at lmax 271 and 356 NM for bands II and I, respectively, indicating a coumarin nature.
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E.R. El-Sharkawy and K. Mahmoud O
O
O
O
, F1: 6-isobutyl –coumarin
F2: 6-isobutyl –7-methyl-coumarin
Figure 1. Structure of new compounds F1 and F2.
H NMR (chloroform-500 MHz): a shows signal at 7.61(1H-d,H-3) nand 7.41(1H, d, H-4), 6.7(1H, s, H-8),6.1(1H,d, H-5), 1.64 (d, J ¼ 6.6 Hz, 3H) 1.69 (d, J ¼ 6.6 Hz, 3H) ppm, 4.3(1H, m, C-9). 13C NMR (100 MHz, CDCl3): a 159.7 (CvO), 147.8 (C), 145.7 (CH), 139.2 (CH), 130.3 (CH), 125.3 (C), 119.7 (C), 116.8, (CH), 114.4 (CH2), 25.1 (CH3) 116.0 (CH) ppm. The isolated compound F1: White crystal, molecular weight of 202, it from the molecular ion (m/z) and molecular formula C13H14O2. The fragmentation of the compound shows base beak at 174 m/z with a reduction of CO molecule, 145 m/z with a reduction in C4H9 (ISO butyl, m/z 57). Supporting the proposed structure of compound F1 measuring the two-dimensional HMBC, HSQC spectral data which show the correlation of carbon atom in the lactone ring with hydrogen atoms, 1H binding to C-3 (7.6 p.m.) and C-4 (7.4 ppm), then in the aromatic ring was found that 1H binding to C-5 (6.1 p.m.) and C-8 (6.7 ppm) at Figure 1, and HMBC show correlation of proton H-9 with C-8 and C-7, C-7 and C-9, also, the signal of H-11(d 4.42) showed a correlation with C-5, C-6 (d) and C-7 (d) confirmed its substitution with the isobutyl group to C 6.
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3.4. Identification of new compound F2 The isolated compound F2: White needle crystal, molecular weight of 216, from the molecular ion (m/z), molecular formula, C14H16O2. The fragmentation of compound F2, show molecular ion of 145 m/z for coumarin nucleus, fragment with molecular ion of 173 m/z, with reduction of CH3, then CO. H1NMR of compound F2: s: shows signal at 7.51(1H-d, H-3) nand 7.21(1H, d, H-4), 6.2 (1H, s, H-8), 6.1(1H,s, H-5),1.4(6H, 2CH3) 4.3(1H,m, C-9), 1.69 (d, J ¼ 6.6 Hz, 3H) ppm. 13 C NMR (100 MHz, CDCl3): a: 164. 7 (CvO), 147.8 (C), 145.7 (CH), 139.2 (CH), 130.3 (CH), 125.3 (C), 119.7 (C), 116.8 (CH), 114.4 (CH2), 25.1 (CH3) 116.0 (CH) ppm. HMBC showed correlation of proton H-9 with C-8 and C-7, C-7 and C-9, also the signal of H-11(d 4.42) showed a correlation with C-5, C-6 and C-7, confirming its substitution with the isobutyl group to C 6. 3.5. Cytotoxic effect on human cell line (HePG2 – MCF 7 – HCT116 – A549) Cell viability was assessed by the mitochondrial-dependent reduction of yellow MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to purple formazan, (Mosmann, 1983). 4. Conclusion This study underscores that the crude extract of L. mucronata had various cytotoxic activity against HCT116, HepG2 and MCF-7. The new compound coumarin-6-isobutyl showed high significant cytotoxic activity against different cell lines. Whereas the other new compound, coumarin-6-isobutyl-7-methyl, had high cytotoxity against HCT116. Coumarin and triterpenes are common components of the human diet and appear to be of interest in cancer prevention or
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therapy. Searching on many chemotherapeutic agents against tumour cells without sparing normal cells remain a major obstacle and development of multi-drug resistance further limits chemotherapy in cancer, the promising results will stimulate the development of coumarin compound for cancer chemoprevention and chemotherapy. Supplementary material Supplementary material relating to this article is available online, alongside Figure S1 and Tables S1 –S3.
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Acknowledgements The authors are thankful to the Bioassay-Cell Culture Laboratory, National Research Centre, El-Tahrir St., Dokki, Cairo 12622, Egypt for conducting the analysis and determining the results.
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