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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
Unprecedented new nonadecyl parahydroperoxycinnamate isolated from Erythrina excelsa and its cytotoxic activity a
a
bc
Guy M.N. Kwamou , Louis P. Sandjo , Victor Kuete , Anaelle A.K. a
b
c
Wandja , Simplice B. Tankeo , Thomas Efferth & Augustin E. Nkengfack
a
a
Click for updates
Department of Organic Chemistry, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon b
Department of Biochemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon c
Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55128Mainz, Germany Published online: 15 Sep 2014.
To cite this article: Guy M.N. Kwamou, Louis P. Sandjo, Victor Kuete, Anaelle A.K. Wandja, Simplice B. Tankeo, Thomas Efferth & Augustin E. Nkengfack (2015) Unprecedented new nonadecyl para-hydroperoxycinnamate isolated from Erythrina excelsa and its cytotoxic activity, Natural Product Research: Formerly Natural Product Letters, 29:10, 921-925, DOI: 10.1080/14786419.2014.959519 To link to this article: http://dx.doi.org/10.1080/14786419.2014.959519
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Natural Product Research, 2015 Vol. 29, No. 10, 921–925, http://dx.doi.org/10.1080/14786419.2014.959519
Unprecedented new nonadecyl para-hydroperoxycinnamate isolated from Erythrina excelsa and its cytotoxic activity Guy M.N. Kwamoua, Louis P. Sandjoa*, Victor Kuetebc, Anaelle A.K. Wandjaa, Simplice B. Tankeob, Thomas Efferthc* and Augustin E. Nkengfacka a
Department of Organic Chemistry, University of Yaounde´ I, P.O. Box 812, Yaounde´, Cameroon; Department of Biochemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon; cDepartment of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55128 Mainz, Germany
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b
(Received 28 July 2014; final version received 26 August 2014) A new unprecedented cinnamate derivative (1) was obtained from Erythrina excelsa (Leguminosae) and identified as nonadecyl para-hydroperoxycinnamate. This compound was isolated together with three known compounds, namely lupeol (2), mixture of sitosterol and stigmasterol (3), and isoneorautenol (4). Their structures were established on the basis of NMR and mass spectroscopic data in conjunction with those reported in the literature. Compound 1 was evaluated for its capability of inhibiting cancer cell lines and growth of a panel of microbial strains. It turned out that 1 is moderately to significantly cytotoxic against six cancer cell lines and shows weak to no antimicrobial activity. Keywords: anticancer activity; drug resistance; para-hydroperoxycinnamate; Fabaceae
1. Introduction Erythrina excelsa has been up to now subject of two chemical investigations: one reported an isoflavonoid (Fomum et al. 1986) while another described a long alkyl chain ferulate (Wandji et al. 1990). In the search of new bioactive metabolites, our interest was focused on E. excelsa, which revealed in a previous reported colorimetric test the presence of terpenoids, alkaloids and phenolic metabolites (Ogutu et al. 2012). Diverse flavonoids from Erythrina species, such as 1methoxyerythrabyssin II (pterocarpan) (Rukachaisirikul et al. 2008), phaseollidin. (pterocarpan) (Iranshahi et al. 2012), wighteone (isoflavanone) (Kumar et al. 2013), alpinumisoflavone (isoflavone) and 40 -methoxylicoflavanone (flavanone), were formerly reported and some of them were described as cytotoxic metabolites (Kumar et al. 2013). Therefore, our preliminary study on E. excelsa afforded a known pterocarpan (isoneorautenol) which displayed from moderate to significant cytotoxicity against a panel of drug-resistant cancer lines (Kuete et al. 2013). The continuation of the work led to the identification of a new cinnamate congener which was biologically tested for its cytotoxicity and antimicrobial activities. We herein report the structural elucidation of the new compound and its bioactivities. 2. Results and discussion Four compounds were obtained from repeated column chromatography (CC) of the organic crude extract of E. excelsa. These secondary metabolites were identified as nonadecyl para-
*Corresponding authors. Email: [email protected], [email protected] q 2014 Taylor & Francis
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hydroperoxycinnamate (1), lupeol (2), mixture of b-sitosterol and stigmasterol (3), and isoneorautenol (4). Compound 1 was obtained as a colourless gum and its molecular formula C28H46O4 was deduced from the NMR data in conjunction with the pseudo-molecular peak at m/z 469.3301 (calcd [M þ Na]þ469.3294) found in the HR-ESI-MS. The elemental composition accounted for six double bond equivalents. The NMR spectra (see Section 3) of 1 displayed resonances similar to those of a fatty alcohol (Tanemura et al. 1994) including a CH3 group at d 0.88 (t, J ¼ 7.0 Hz)/14.3, a sequence of CH2 groups d 1.20– 1.35 (br s)/22.9 – 29.9 and a downfield signal at d 4.18 (t, J ¼ 6.8 Hz)/64.8 corresponding with an oxymethylene attached to a withdrawing electron group. This latter supposition was supported by a strong HMBC (Figure S10, see supplementary materials) correlation observed between the oxymethylene protons (d 4.18) and a carbonyl at d 167.6. Further signals observed at d 6.84 (d, J ¼ 8.7 Hz, 2H)/116.0, 7.43 (d, J ¼ 8.7 Hz, 2H)/130.1, 6.30 (d, J ¼ 16.0 Hz, 1H)/116.0, 7.62 (d, J ¼ 16.0 Hz, 1H)/144.2, 127.6 and 157.6 in the NMR spectra were assigned to a trans p-coumaroyl moiety (Nishimura et al. 2009; Ragasa & Alimboyoguen 2013). From the NMR data, only three oxygen atoms could be found; thus, the presence of a peroxide function was deduced from the mass spectrometry and located in the para position of the acrylate moiety in the aromatic system. The peroxide function was further supported by absorption bands revealed in the IR spectrum for OH (broad, 3321 cm21), for CZOOH (short, 1121 cm21) and for OZO (short 846 cm21). These absorption bands were close to those reported for tert-butyl hydroperoxide (Bernal et al. 2009). Moreover, an exchangeable proton was revealed at d 5.16 in the 1H NMR spectrum and had no HMBC contact with the aromatic carbon at d 157.6; nevertheless, it showed a weak NOESY (Figure S11, see supplementary materials) correlation with the aromatic proton at d 6.84. The foregoing data led to identify 1 as (E)-nonadecyl-3-(4hydroperoxyphenyl)acrylate (Figure 1) and the trivial name excelsaperoxide was assigned. Biosynthetically, compound 1 probably resulted from phenylalanine which was transformed into cinnamic acid by phenyl ammonia lyase. Cinnamic acid in turn was converted to cinnamyl coenzyme A. In addition acetyl coenzyme A was responsible for the elongation of the side chain to afford the adequate fatty acid reduced into fatty alcohol by a reductase enzyme. The fatty alcohol under the action of an acyl transferase was esterified by cinnamyl coenzyme A. The resulting product seems to furnish the peroxide product in the presence of oxygen under irradiation conditions (Figure S12, see supplementary materials). This assumption corroborated with the biosynthesis pathway proposed for artemisinin by Brown (2010). Structures of the known compounds were determined to be lupeol (2) (Lee et al. 2010), mixture of sitosterol and stigmasterol (3) (De-Eknamkul & Potduang 2003) and isoneorautenol (4) (Iinuma et al. 1995) based on their NMR data and by comparison with previously reported data. The results of the antibacterial assays indicated that compound 1 has a weak activity only against Enterobacter aerogenes ATCC13048 with MIC values above 100 mg/mL and no activity against other tested microorganisms (Table 1). In contrast, it showed significant cytotoxic activities with IC50 values below 10 mM against CCRF-CEM (1.02 mM), CEM/ADR5000 (1.07 mM), MDA-MB231 (3.22 mM) and U87MG (3.75 mM) (Table 1). The activity of 1 was better than that of doxorubicin towards the resistant HO
O OCH2(CH2)17CH3 O
Figure 1. Structure of compound 1.
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Table 1. Cytotoxic and antimicrobial activities of compound 1. MIC values of compound 1 and tetracycline against IC50 values of compound 1 and doxorubicin towards cancer cell lines the tested bacterial strains Compound and MIC (mg/mL) Bacterial strains E. coli
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E. aerogenes
Klebsiella pneumoniae Pseudomonas aeruginosa
1
Tetracycline
ATCC8739 – AG100 – AG102 – ATCC 13048 512 EA3 – EA27 – CM64 – ATCC 29916 –
64 128 8 32 ,4 128 ,4 ,4
KP55 KP63 PA01
16 ,4 ,4
– – –
Compound and IC50 values (mM) Cell lines
1
Doxorubicin
CCRF-CEM 1.02 ^ 0.03 0.20 ^ 0.06 CEM/ADR5000 1.07 ^ 0.02 195.12 ^ 14.30 MDA-MB231 3.22 ^ 0.19 1.10 ^ 0.28 HCT116( p53 þ /þ) 68.27 ^ 3.67 1.41 ^ 0.29 U87MG 3.75 ^ 0.40 1.06 ^ 0.15 HepG2 59.77 ^ 4.71 3.83 ^ 0.94
CEM/ADR5000 cell line. Compound 1 can, therefore, be considered as a potential cytotoxic candidate to fight malignant diseases. This conclusion is supported by significant cytotoxicity against murine leukaemia cells found for two cyclic peroxide acids isolated from a marine sponge (Toth & Schmitz 1994). Besides, several terpenoidal peroxides have already been identified from natural organisms for instance, artemisinin, one of the famous antimalarial drugs of our century and its semi-synthetic derivatives showed cytotoxicity against HeLa S3 cancer cell lines (Beekman et al. 1996). Speciosaperoxide, a peroxide-containing ursolic acid proved a weak antioxidant activities and a moderate anti-inflammatory activities (Zhang et al. 2010). 3. Experimental part 3.1. General procedure Vacuum column chromatography (VCC), CC and thin-layer chromatography (TLC) were performed over silica gel 60H (particle size 90% ,45 mm), 200–300 mesh silica gel and silica gel GF254, respectively. 1D and 2D NMR data were recorded with a Bruker DRX-400 MHz (Bru¨ker, Karlsruhe, Germany). IR spectra were carried out on a Perkin-Elmer (FT-IR system spectrum BX spectrometer, Perkin-Elmer, Rodgau, Germany) using KBr discs. HR-ESI-MS was recorded on a variant JEOL MS instrument. HPLC-ESI-MS was performed with Agilent Technology apparatus equipped with C8 and C18 column. 3.2. Plant material The bark of E. excelsa was collected in December 2010 from Mayo-Darle´, in Adamaoua, a region in the north of Cameroon. The plant was identified by a specialist of the National Herbarium where a voucher has been deposited under the registration number 61 487/HNC. 3.2.1. Extraction and isolation procedure Air-dried bark of E. excelsa (2.2 kg) was cut into small pieces, powdered and extracted with a mixture of methanol (MeOH) –dichloromethane (1:1) for 2 days. The organic solution was
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rotary evaporated to yield 209.0 g of crude extraction. The extract was fractionated using a VCC on silica gel eluted with cyclohexane (hex), the mixture hex:ethyl acetate (EA) under gradient condition and MeOH. All CC fractions were monitored with TLC profiles. Four fractions (E1 – E4) were collected including hex –EA (75:25, 26.0 g), hex – EA (50:50, 38.0 g), EA (27.0 g) and methanol (22.0 g). Repeated CC of fraction E1 on silica gel and a mixture of hex – EA as eluent under gradient conditions led to 115 sub-fractions. Lupeol 2 (10 mg) was obtained from subfractions 15– 19. Sub-fractions 28 – 40 eluted with hex – EA (9:1) yielded isoneorautenol 4 (60.0 mg). The mixture of sterol 3 (4 mg) was isolated from sub-fractions 45– 55. Compound 1 (10 mg) was obtained from repeated CC of E2. This fraction was also eluted with the mixture of hex –EA in gradient conditions yielding 70 sub-fractions and compound 1 was filtered from subfractions 6– 10. (E)-nonadecyl-3-(4-hydroperoxyphenyl)acrylate (1) Colourless gum; Rf: 0.6 (hex –EA, 85:15), IR (KBr): 3321, 2959, 1722, 1441, 1121, 1061, 925, 846 cm21; 1H NMR (400 MHz, CDCl3) 6.30 (1H, d, J ¼ 16.0 Hz, H-2), 7.62 (1H, d, J ¼ 16.0 Hz, H-3), 7.43 (1H, d, J ¼ 8.6 Hz, H-5 and H-9), 6.84 (1H, d, J ¼ 8.6 Hz, H-6 and H-8), 4.18 (2H, t, J ¼ 6.8 Hz, H-10 ), 1.69 (2H, p, J ¼ 6.8 Hz, H-20 ), 1.39 (2H, m, H-30 ), 1.21 –1.27 (26H, br s, H-40 to H-160 ), 1.29 (2H, m, H-170 ), 1.29 (2H, m, H-180 ) and 0.88 (3H, t, J ¼ 7.0 Hz, H-190 ); 13C NMR (100 MHz, CDCl3): 167.6 (C1), 116.0 (C-2), 144.2 (C-3), 127.6 (C-4), 130.1 (C-5 and C-9), 116.0 (C-6 and C-8), 157.6 (C-7), 64.8 (C-10 ), 28.9 (C-20 ), 26.1 (C-30 ), 29.5 –29.9 (C-40 to C-160 ), 22.9 (C-170 ), 32.1 (C-180 ), and 14.3 (C-190 ); HR-MS-ESI: m/z [M þ Naþ] calcd for C28H46O4: 469.3294; found: 469.3301. 4. Conclusion The phytochemical studies of E. excelsa have led to the isolation of, along with lupeol and mixture of sterols, two anti-proliferative secondary metabolites including one known flavonoid (isoneorautenol) and a new peroxide-containing compound. However, this latter turned to be the first cinnamate ester in plant kingdom bearing a peroxide function. The observed inhibition of cancer cell growth by compounds 1 and 4 suggests that they are good candidates to explore new related leads for chemotherapy. Supplementary data NMR spectra of compound 1, and the biological method have been provided in the supplementary material. Funding Victor Kuete is very grateful to the Alexander von Humboldt foundation for an 18-month fellowship to visit the department of Prof. Efferth (Johannes Gutenberg-University, Mainz, Germany) through the ‘Georg Foster Research Fellowship for Experienced Researcher’ program for funding this work.
References Beekman AC, Woerdenbag HJ, Kampinga HH, Konings AWT. 1996. Cytotoxicity of artemisinin, a dimer of dihydroartemisinin, artemisitene and eupatoriopicrin as evaluated by the MTT and clonogenic assay. Phytother Res. 10:140–144. Bernal HG, Caero LC, Finocchio E, Busca G. 2009. An FT-IR study of the adsorption and reactivity of tert-butyl hydroperoxide over oxide catalysts. Appl Catal A: Gen. 369:27– 35. Brown GD. 2010. The biosynthesis of artemisinin (Qinghaosu) and the phytochemistry of Artemisia annua L. (Qinghao). Molecules. 15:7603 –7698. De-Eknamkul W, Potduang B. 2003. Biosynthesis of beta-sitosterol and stigmasterol in Croton sublyratus proceeds via a mixed origin of isoprene units. Phytochemistry. 62:389– 398.
Downloaded by [Simon Fraser University] at 06:33 23 April 2015
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Fomum ZT, Ayafor JF, Ifeadike PN, Nkengfack AE, Wandji J. 1986. Erythrina studies. Part 31. Isolation of an isoflavone from Erythrina senegalensis and Erythrina excelsa. Planta Med. 52:341. Iinuma M, Ohyama M, Tanaka T. 1995. Flavonoids in roots of Sophora prostrate. Phytochemistry. 38:539–543. Iranshahi M, Vu H, Pham N, Zencak D, Forster P, Quinn RJ. 2012. Cytotoxic evaluation of alkaloids and isoflavonoids from the Australian tree Erythrina vespertilio. Planta Med. 78:730–736. Kuete V, Sandjo LP, Kwamou GM, Wiench B, Nkengfack AE, Efferth T. 2013. Activity of three cytotoxic isoflavonoids from Erythrina excelsa and Erythrina senegalensis (neobavaisoflavone, sigmoidin H and isoneorautenol) toward multi-factorial drug resistant cancer cells. Phytomedicine. 21:682–688. Kumar S, Pathania AS, Saxena AK, Vishwakarma RA, Ali A, Bhushan S. 2013. The anticancer potential of flavonoids isolated from the stem bark of Erythrina suberosa through induction of apoptosis and inhibition of STAT signaling pathway in human leukemia HL-60 cells. Chem Biol Interact. 205:128– 137. Lee D-Y, Jung L, Park J-H, Yoo K-H, Chung I-S, Baek N-I. 2010. Cytotoxic triterpenoids from Cornus kousa fruits. Chem Nat Compd. 46:142–145. Nishimura K, Takenaka Y, Kishi M, Tanahashi T, Yoshida H, Okuda C, Mizushina Y. 2009. Synthesis and DNA polymerase alpha and beta inhibitory activity of alkyl p-coumarates and related compounds. Chem Pharm Bull. 57:476–480. Ogutu AI, Lilechi DB, Mutai C, Bii C. 2012. Phytochemical analysis and antimicrobial activity of Phytolacca dodecandra, Cucumis aculeatus and Erythrina excelsa. Int J Biol Chem Sci. 6:692–704. Ragasa CY, Alimboyoguen AB. 2013. Long chain 4-hydroxycinnamate esters from Allamanda neriifolia hook. Am J Essent Oils Nat Prod. 1:104–107. Rukachaisirikul T, Innok P, Suksamrarn A. 2008. Erythrina alkaloids and a pterocarpan from the bark of Erythrina subumbrans. J Nat Prod. 71:156–158. Tanemura K, Suzuki T, Horagughi T. 1994. Deprotection of acetals and silyl ethers using some p-acceptors. Bull Chem Soc Jpn. 67:290–292. Toth SI, Schmitz FJ. 1994. Two new cytotoxic peroxide-containing acids from a New Guinea sponge, Callyspongia sp. J Nat Prod. 57:123–127. Wandji J, Nkengfack AE, Fomum ZT, Ubillas R, Killday KB, Tempesta MS. 1990. A new prenylated isoflavone and long chain esters from two Erythrina species. J Nat Prod. 53:1425–1429. Zhang L, Cheng Y-X, Liu A-L, Wang H-D, Wang Y-L, Du G-H. 2010. Antioxidant, anti-inflammatory and anti-influenza properties of components from Chaenomeles speciosa. Molecules. 15:8507– 8517.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Unprecedented new nonadecyl parahydroperoxycinnamate isolated from Erythrina excelsa and its cytotoxic activity a
a
bc
Guy M.N. Kwamou , Louis P. Sandjo , Victor Kuete , Anaelle A.K. a
b
c
Wandja , Simplice B. Tankeo , Thomas Efferth & Augustin E. Nkengfack
a
a
Click for updates
Department of Organic Chemistry, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon b
Department of Biochemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon c
Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55128Mainz, Germany Published online: 15 Sep 2014.
To cite this article: Guy M.N. Kwamou, Louis P. Sandjo, Victor Kuete, Anaelle A.K. Wandja, Simplice B. Tankeo, Thomas Efferth & Augustin E. Nkengfack (2015) Unprecedented new nonadecyl para-hydroperoxycinnamate isolated from Erythrina excelsa and its cytotoxic activity, Natural Product Research: Formerly Natural Product Letters, 29:10, 921-925, DOI: 10.1080/14786419.2014.959519 To link to this article: http://dx.doi.org/10.1080/14786419.2014.959519
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,
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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 & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 Vol. 29, No. 10, 921–925, http://dx.doi.org/10.1080/14786419.2014.959519
Unprecedented new nonadecyl para-hydroperoxycinnamate isolated from Erythrina excelsa and its cytotoxic activity Guy M.N. Kwamoua, Louis P. Sandjoa*, Victor Kuetebc, Anaelle A.K. Wandjaa, Simplice B. Tankeob, Thomas Efferthc* and Augustin E. Nkengfacka a
Department of Organic Chemistry, University of Yaounde´ I, P.O. Box 812, Yaounde´, Cameroon; Department of Biochemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon; cDepartment of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55128 Mainz, Germany
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b
(Received 28 July 2014; final version received 26 August 2014) A new unprecedented cinnamate derivative (1) was obtained from Erythrina excelsa (Leguminosae) and identified as nonadecyl para-hydroperoxycinnamate. This compound was isolated together with three known compounds, namely lupeol (2), mixture of sitosterol and stigmasterol (3), and isoneorautenol (4). Their structures were established on the basis of NMR and mass spectroscopic data in conjunction with those reported in the literature. Compound 1 was evaluated for its capability of inhibiting cancer cell lines and growth of a panel of microbial strains. It turned out that 1 is moderately to significantly cytotoxic against six cancer cell lines and shows weak to no antimicrobial activity. Keywords: anticancer activity; drug resistance; para-hydroperoxycinnamate; Fabaceae
1. Introduction Erythrina excelsa has been up to now subject of two chemical investigations: one reported an isoflavonoid (Fomum et al. 1986) while another described a long alkyl chain ferulate (Wandji et al. 1990). In the search of new bioactive metabolites, our interest was focused on E. excelsa, which revealed in a previous reported colorimetric test the presence of terpenoids, alkaloids and phenolic metabolites (Ogutu et al. 2012). Diverse flavonoids from Erythrina species, such as 1methoxyerythrabyssin II (pterocarpan) (Rukachaisirikul et al. 2008), phaseollidin. (pterocarpan) (Iranshahi et al. 2012), wighteone (isoflavanone) (Kumar et al. 2013), alpinumisoflavone (isoflavone) and 40 -methoxylicoflavanone (flavanone), were formerly reported and some of them were described as cytotoxic metabolites (Kumar et al. 2013). Therefore, our preliminary study on E. excelsa afforded a known pterocarpan (isoneorautenol) which displayed from moderate to significant cytotoxicity against a panel of drug-resistant cancer lines (Kuete et al. 2013). The continuation of the work led to the identification of a new cinnamate congener which was biologically tested for its cytotoxicity and antimicrobial activities. We herein report the structural elucidation of the new compound and its bioactivities. 2. Results and discussion Four compounds were obtained from repeated column chromatography (CC) of the organic crude extract of E. excelsa. These secondary metabolites were identified as nonadecyl para-
*Corresponding authors. Email: [email protected], [email protected] q 2014 Taylor & Francis
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G.M.N. Kwamou et al.
hydroperoxycinnamate (1), lupeol (2), mixture of b-sitosterol and stigmasterol (3), and isoneorautenol (4). Compound 1 was obtained as a colourless gum and its molecular formula C28H46O4 was deduced from the NMR data in conjunction with the pseudo-molecular peak at m/z 469.3301 (calcd [M þ Na]þ469.3294) found in the HR-ESI-MS. The elemental composition accounted for six double bond equivalents. The NMR spectra (see Section 3) of 1 displayed resonances similar to those of a fatty alcohol (Tanemura et al. 1994) including a CH3 group at d 0.88 (t, J ¼ 7.0 Hz)/14.3, a sequence of CH2 groups d 1.20– 1.35 (br s)/22.9 – 29.9 and a downfield signal at d 4.18 (t, J ¼ 6.8 Hz)/64.8 corresponding with an oxymethylene attached to a withdrawing electron group. This latter supposition was supported by a strong HMBC (Figure S10, see supplementary materials) correlation observed between the oxymethylene protons (d 4.18) and a carbonyl at d 167.6. Further signals observed at d 6.84 (d, J ¼ 8.7 Hz, 2H)/116.0, 7.43 (d, J ¼ 8.7 Hz, 2H)/130.1, 6.30 (d, J ¼ 16.0 Hz, 1H)/116.0, 7.62 (d, J ¼ 16.0 Hz, 1H)/144.2, 127.6 and 157.6 in the NMR spectra were assigned to a trans p-coumaroyl moiety (Nishimura et al. 2009; Ragasa & Alimboyoguen 2013). From the NMR data, only three oxygen atoms could be found; thus, the presence of a peroxide function was deduced from the mass spectrometry and located in the para position of the acrylate moiety in the aromatic system. The peroxide function was further supported by absorption bands revealed in the IR spectrum for OH (broad, 3321 cm21), for CZOOH (short, 1121 cm21) and for OZO (short 846 cm21). These absorption bands were close to those reported for tert-butyl hydroperoxide (Bernal et al. 2009). Moreover, an exchangeable proton was revealed at d 5.16 in the 1H NMR spectrum and had no HMBC contact with the aromatic carbon at d 157.6; nevertheless, it showed a weak NOESY (Figure S11, see supplementary materials) correlation with the aromatic proton at d 6.84. The foregoing data led to identify 1 as (E)-nonadecyl-3-(4hydroperoxyphenyl)acrylate (Figure 1) and the trivial name excelsaperoxide was assigned. Biosynthetically, compound 1 probably resulted from phenylalanine which was transformed into cinnamic acid by phenyl ammonia lyase. Cinnamic acid in turn was converted to cinnamyl coenzyme A. In addition acetyl coenzyme A was responsible for the elongation of the side chain to afford the adequate fatty acid reduced into fatty alcohol by a reductase enzyme. The fatty alcohol under the action of an acyl transferase was esterified by cinnamyl coenzyme A. The resulting product seems to furnish the peroxide product in the presence of oxygen under irradiation conditions (Figure S12, see supplementary materials). This assumption corroborated with the biosynthesis pathway proposed for artemisinin by Brown (2010). Structures of the known compounds were determined to be lupeol (2) (Lee et al. 2010), mixture of sitosterol and stigmasterol (3) (De-Eknamkul & Potduang 2003) and isoneorautenol (4) (Iinuma et al. 1995) based on their NMR data and by comparison with previously reported data. The results of the antibacterial assays indicated that compound 1 has a weak activity only against Enterobacter aerogenes ATCC13048 with MIC values above 100 mg/mL and no activity against other tested microorganisms (Table 1). In contrast, it showed significant cytotoxic activities with IC50 values below 10 mM against CCRF-CEM (1.02 mM), CEM/ADR5000 (1.07 mM), MDA-MB231 (3.22 mM) and U87MG (3.75 mM) (Table 1). The activity of 1 was better than that of doxorubicin towards the resistant HO
O OCH2(CH2)17CH3 O
Figure 1. Structure of compound 1.
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Table 1. Cytotoxic and antimicrobial activities of compound 1. MIC values of compound 1 and tetracycline against IC50 values of compound 1 and doxorubicin towards cancer cell lines the tested bacterial strains Compound and MIC (mg/mL) Bacterial strains E. coli
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E. aerogenes
Klebsiella pneumoniae Pseudomonas aeruginosa
1
Tetracycline
ATCC8739 – AG100 – AG102 – ATCC 13048 512 EA3 – EA27 – CM64 – ATCC 29916 –
64 128 8 32 ,4 128 ,4 ,4
KP55 KP63 PA01
16 ,4 ,4
– – –
Compound and IC50 values (mM) Cell lines
1
Doxorubicin
CCRF-CEM 1.02 ^ 0.03 0.20 ^ 0.06 CEM/ADR5000 1.07 ^ 0.02 195.12 ^ 14.30 MDA-MB231 3.22 ^ 0.19 1.10 ^ 0.28 HCT116( p53 þ /þ) 68.27 ^ 3.67 1.41 ^ 0.29 U87MG 3.75 ^ 0.40 1.06 ^ 0.15 HepG2 59.77 ^ 4.71 3.83 ^ 0.94
CEM/ADR5000 cell line. Compound 1 can, therefore, be considered as a potential cytotoxic candidate to fight malignant diseases. This conclusion is supported by significant cytotoxicity against murine leukaemia cells found for two cyclic peroxide acids isolated from a marine sponge (Toth & Schmitz 1994). Besides, several terpenoidal peroxides have already been identified from natural organisms for instance, artemisinin, one of the famous antimalarial drugs of our century and its semi-synthetic derivatives showed cytotoxicity against HeLa S3 cancer cell lines (Beekman et al. 1996). Speciosaperoxide, a peroxide-containing ursolic acid proved a weak antioxidant activities and a moderate anti-inflammatory activities (Zhang et al. 2010). 3. Experimental part 3.1. General procedure Vacuum column chromatography (VCC), CC and thin-layer chromatography (TLC) were performed over silica gel 60H (particle size 90% ,45 mm), 200–300 mesh silica gel and silica gel GF254, respectively. 1D and 2D NMR data were recorded with a Bruker DRX-400 MHz (Bru¨ker, Karlsruhe, Germany). IR spectra were carried out on a Perkin-Elmer (FT-IR system spectrum BX spectrometer, Perkin-Elmer, Rodgau, Germany) using KBr discs. HR-ESI-MS was recorded on a variant JEOL MS instrument. HPLC-ESI-MS was performed with Agilent Technology apparatus equipped with C8 and C18 column. 3.2. Plant material The bark of E. excelsa was collected in December 2010 from Mayo-Darle´, in Adamaoua, a region in the north of Cameroon. The plant was identified by a specialist of the National Herbarium where a voucher has been deposited under the registration number 61 487/HNC. 3.2.1. Extraction and isolation procedure Air-dried bark of E. excelsa (2.2 kg) was cut into small pieces, powdered and extracted with a mixture of methanol (MeOH) –dichloromethane (1:1) for 2 days. The organic solution was
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rotary evaporated to yield 209.0 g of crude extraction. The extract was fractionated using a VCC on silica gel eluted with cyclohexane (hex), the mixture hex:ethyl acetate (EA) under gradient condition and MeOH. All CC fractions were monitored with TLC profiles. Four fractions (E1 – E4) were collected including hex –EA (75:25, 26.0 g), hex – EA (50:50, 38.0 g), EA (27.0 g) and methanol (22.0 g). Repeated CC of fraction E1 on silica gel and a mixture of hex – EA as eluent under gradient conditions led to 115 sub-fractions. Lupeol 2 (10 mg) was obtained from subfractions 15– 19. Sub-fractions 28 – 40 eluted with hex – EA (9:1) yielded isoneorautenol 4 (60.0 mg). The mixture of sterol 3 (4 mg) was isolated from sub-fractions 45– 55. Compound 1 (10 mg) was obtained from repeated CC of E2. This fraction was also eluted with the mixture of hex –EA in gradient conditions yielding 70 sub-fractions and compound 1 was filtered from subfractions 6– 10. (E)-nonadecyl-3-(4-hydroperoxyphenyl)acrylate (1) Colourless gum; Rf: 0.6 (hex –EA, 85:15), IR (KBr): 3321, 2959, 1722, 1441, 1121, 1061, 925, 846 cm21; 1H NMR (400 MHz, CDCl3) 6.30 (1H, d, J ¼ 16.0 Hz, H-2), 7.62 (1H, d, J ¼ 16.0 Hz, H-3), 7.43 (1H, d, J ¼ 8.6 Hz, H-5 and H-9), 6.84 (1H, d, J ¼ 8.6 Hz, H-6 and H-8), 4.18 (2H, t, J ¼ 6.8 Hz, H-10 ), 1.69 (2H, p, J ¼ 6.8 Hz, H-20 ), 1.39 (2H, m, H-30 ), 1.21 –1.27 (26H, br s, H-40 to H-160 ), 1.29 (2H, m, H-170 ), 1.29 (2H, m, H-180 ) and 0.88 (3H, t, J ¼ 7.0 Hz, H-190 ); 13C NMR (100 MHz, CDCl3): 167.6 (C1), 116.0 (C-2), 144.2 (C-3), 127.6 (C-4), 130.1 (C-5 and C-9), 116.0 (C-6 and C-8), 157.6 (C-7), 64.8 (C-10 ), 28.9 (C-20 ), 26.1 (C-30 ), 29.5 –29.9 (C-40 to C-160 ), 22.9 (C-170 ), 32.1 (C-180 ), and 14.3 (C-190 ); HR-MS-ESI: m/z [M þ Naþ] calcd for C28H46O4: 469.3294; found: 469.3301. 4. Conclusion The phytochemical studies of E. excelsa have led to the isolation of, along with lupeol and mixture of sterols, two anti-proliferative secondary metabolites including one known flavonoid (isoneorautenol) and a new peroxide-containing compound. However, this latter turned to be the first cinnamate ester in plant kingdom bearing a peroxide function. The observed inhibition of cancer cell growth by compounds 1 and 4 suggests that they are good candidates to explore new related leads for chemotherapy. Supplementary data NMR spectra of compound 1, and the biological method have been provided in the supplementary material. Funding Victor Kuete is very grateful to the Alexander von Humboldt foundation for an 18-month fellowship to visit the department of Prof. Efferth (Johannes Gutenberg-University, Mainz, Germany) through the ‘Georg Foster Research Fellowship for Experienced Researcher’ program for funding this work.
References Beekman AC, Woerdenbag HJ, Kampinga HH, Konings AWT. 1996. Cytotoxicity of artemisinin, a dimer of dihydroartemisinin, artemisitene and eupatoriopicrin as evaluated by the MTT and clonogenic assay. Phytother Res. 10:140–144. Bernal HG, Caero LC, Finocchio E, Busca G. 2009. An FT-IR study of the adsorption and reactivity of tert-butyl hydroperoxide over oxide catalysts. Appl Catal A: Gen. 369:27– 35. Brown GD. 2010. The biosynthesis of artemisinin (Qinghaosu) and the phytochemistry of Artemisia annua L. (Qinghao). Molecules. 15:7603 –7698. De-Eknamkul W, Potduang B. 2003. Biosynthesis of beta-sitosterol and stigmasterol in Croton sublyratus proceeds via a mixed origin of isoprene units. Phytochemistry. 62:389– 398.
Downloaded by [Simon Fraser University] at 06:33 23 April 2015
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Fomum ZT, Ayafor JF, Ifeadike PN, Nkengfack AE, Wandji J. 1986. Erythrina studies. Part 31. Isolation of an isoflavone from Erythrina senegalensis and Erythrina excelsa. Planta Med. 52:341. Iinuma M, Ohyama M, Tanaka T. 1995. Flavonoids in roots of Sophora prostrate. Phytochemistry. 38:539–543. Iranshahi M, Vu H, Pham N, Zencak D, Forster P, Quinn RJ. 2012. Cytotoxic evaluation of alkaloids and isoflavonoids from the Australian tree Erythrina vespertilio. Planta Med. 78:730–736. Kuete V, Sandjo LP, Kwamou GM, Wiench B, Nkengfack AE, Efferth T. 2013. Activity of three cytotoxic isoflavonoids from Erythrina excelsa and Erythrina senegalensis (neobavaisoflavone, sigmoidin H and isoneorautenol) toward multi-factorial drug resistant cancer cells. Phytomedicine. 21:682–688. Kumar S, Pathania AS, Saxena AK, Vishwakarma RA, Ali A, Bhushan S. 2013. The anticancer potential of flavonoids isolated from the stem bark of Erythrina suberosa through induction of apoptosis and inhibition of STAT signaling pathway in human leukemia HL-60 cells. Chem Biol Interact. 205:128– 137. Lee D-Y, Jung L, Park J-H, Yoo K-H, Chung I-S, Baek N-I. 2010. Cytotoxic triterpenoids from Cornus kousa fruits. Chem Nat Compd. 46:142–145. Nishimura K, Takenaka Y, Kishi M, Tanahashi T, Yoshida H, Okuda C, Mizushina Y. 2009. Synthesis and DNA polymerase alpha and beta inhibitory activity of alkyl p-coumarates and related compounds. Chem Pharm Bull. 57:476–480. Ogutu AI, Lilechi DB, Mutai C, Bii C. 2012. Phytochemical analysis and antimicrobial activity of Phytolacca dodecandra, Cucumis aculeatus and Erythrina excelsa. Int J Biol Chem Sci. 6:692–704. Ragasa CY, Alimboyoguen AB. 2013. Long chain 4-hydroxycinnamate esters from Allamanda neriifolia hook. Am J Essent Oils Nat Prod. 1:104–107. Rukachaisirikul T, Innok P, Suksamrarn A. 2008. Erythrina alkaloids and a pterocarpan from the bark of Erythrina subumbrans. J Nat Prod. 71:156–158. Tanemura K, Suzuki T, Horagughi T. 1994. Deprotection of acetals and silyl ethers using some p-acceptors. Bull Chem Soc Jpn. 67:290–292. Toth SI, Schmitz FJ. 1994. Two new cytotoxic peroxide-containing acids from a New Guinea sponge, Callyspongia sp. J Nat Prod. 57:123–127. Wandji J, Nkengfack AE, Fomum ZT, Ubillas R, Killday KB, Tempesta MS. 1990. A new prenylated isoflavone and long chain esters from two Erythrina species. J Nat Prod. 53:1425–1429. Zhang L, Cheng Y-X, Liu A-L, Wang H-D, Wang Y-L, Du G-H. 2010. Antioxidant, anti-inflammatory and anti-influenza properties of components from Chaenomeles speciosa. Molecules. 15:8507– 8517.
