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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
Chemical composition, antibacterial and cytotoxic activities of the essential oil from the flowers of Tunisian Convolvulus althaeoides L. a
a
a
b
M. Hassine , A. Zardi-Berguaoui , M. Znati , G. Flamini , H. Ben a
a
Jannet & M.A. Hamza a
Laboratory Heterocyclic Chemistry, Natural Products and é (LR11SE39), Team: Medicinal Chemistry and Natural Products and Reactivity, Faculty of Science of Monastir, University of Monastir, 5019 Monastir, Tunisia b
Dipartimento di Scienze Farmaceutiche sede Chimica Bioorganica e Biofarmacia, Via Bonanno 33, 56126 Pisa, Italy Published online: 05 Feb 2014.
To cite this article: M. Hassine, A. Zardi-Berguaoui, M. Znati, G. Flamini, H. Ben Jannet & M.A. Hamza (2014): Chemical composition, antibacterial and cytotoxic activities of the essential oil from the flowers of Tunisian Convolvulus althaeoides L., Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2013.879476 To link to this article: http://dx.doi.org/10.1080/14786419.2013.879476
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.
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2013.879476
Chemical composition, antibacterial and cytotoxic activities of the essential oil from the flowers of Tunisian Convolvulus althaeoides L. M. Hassinea, A. Zardi-Berguaouia, M. Znatia, G. Flaminib, H. Ben Janneta* and M.A. Hamzaa a
Laboratory Heterocyclic Chemistry, Natural Products and e´ (LR11SE39), Team: Medicinal Chemistry and Natural Products and Reactivity, Faculty of Science of Monastir, University of Monastir, 5019 Monastir, Tunisia; bDipartimento di Scienze Farmaceutiche sede Chimica Bioorganica e Biofarmacia, Via Bonanno 33, 56126 Pisa, Italy
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(Received 24 August 2013; final version received 25 December 2013) This study describes the chemical composition and evaluates the antibacterial and the cytotoxic effects of the essential oil from the flowers of Convolvulus althaeoides. Its chemical composition, determined by GC and GC – MS, is reported for the first time. A total of 24 compounds, accounting for 95.5% of the total oil, have been identified. The oil was characterised by a high proportion of sesquiterpene hydrocarbons (36.3%), followed by oxygenated sesquiterpenes (34.7%) and oxygenated monoterpenes (24.5%). The main compounds were germacrene D (12.5%), T-cadinol (11.8%) and verbenone (6.9%). The essential oil was tested for its antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and the clinical strain Acinetobacter sp. as well as facts cytotoxic activity towards the human breast cancer cells MCF-7. This oil did not exhibit significant antibacterial activity against the tested bacteria; however, it exerted a significant cytotoxic activity against the tested cell line (IC50 ¼ 8.16 mg/mL). Keywords: Convolvulus althaeoides; essential oil; composition; GC – MS; antimicrobial activity; cytotoxic activity
1. Introduction Natural products have served as important sources of drugs since ancient times. In recent years, a renewed interest in obtaining biologically active compounds from natural sources has been observed. According to different authors, approximately 3000 plant species contain essential oils among which only 300 are commercially important. Essential oils and some of their constituents are used not only in pharmaceutical products for their therapeutic activities but also in agriculture, as food preservatives and additives for human or animal use, in cosmetics and perfumes, and other industries. In many cases, they serve as plant defence mechanisms against predation by micro-organisms, insects and herbivores (Bakkali et al. 2008). The complex composition of the essential oils and a variety of chemical structures of their constituents are responsible for a wide range of biological activities many of which are of increasing interest in the field of human and animal health. The need for new anti-infective agents due to the emergence of multiple antibiotic resistances has lead to the search of new sources of potential antimicrobials (Carson & Riley 2003). Among them, the plant kingdom offers a wide range of biodiversity with a great value for the pharmaceutical industry.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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The family Convolvulaceae contains an extremely wide variety of aromatic plants mainly in temperate countries. Among this rich array of plants which yield essential oils, the genus Convolvulus includes 250 species which are spread all over the world (Alapetite 1981). In Tunisia, we count 5 genera, 16 species, 5 subspecies and 7 varieties belonging to the Convolvulaceae family. In total, 12 species for the genus Convolvulus L. are found in Tunisia (Alapetite 1981) in which the Convolvulus althaeoides L. subspecies typicus Fiori has not been studied before. It is a climbing perennial plant with solitary flowers on long peduncles. The flower is a funnel-shaped pink bloom 3– 4 cm wide. The leaves are deeply divided into narrow, finger-like lobes. This flowering plant is native to the Mediterranean Basin, distributed only in the extreme south of Tunisia (Alapetite 1981). Convolvulus species are also employed in traditional medicine in several countries, and it is beneficial in the treatment against many human diseases. In particular, roots and leaves of Convolvulus scammonia L. have an aphrodisiac effect and antimalarial (Boullard 2001). Whole organs of Convolvulus soldanella L. have an antiscorbutic, diuretic, febrifuge and purgative effect (Duke & Ayensu 1985). Le Floc’h (1983) has not reported about its possible use in popular medicine in Tunisia. To the best of our knowledge, no data have been reported on C. althaeoides, and this is the first study on the essential oil composition of this species. With an aim of continuing our contribution to the valorization of the aromatic flora from Tunisia, in view of the potential use of its natural resources, this work deals with the study, for the first time, of the essential oil from fresh flowers of C. althaeoides (Convolvulaceae), in particular its chemical composition and the evaluation of its antimicrobial and cytotoxic activities. 2. Results and discussion 2.1. Chemical composition of the essential oil The hydrodistillation of the fresh flowers of C. althaeoides furnished light yellow oil, in 7 £ 1023% (w/w) yield. The composition of the oil is reported in Table 1, together with their linear retention indices (LRIs), the percentages of compounds and the identification methods. A total of 24 constituents, representing 95.5% of the oil, were detected. Seven oxygenated monoterpenes and derivatives, nine sesquiterpene hydrocarbons and six oxygenated sesquiterpenes were identified. Obviously, this oil may be considered as sesquiterpene-rich oil. Sesquiterpene hydrocarbons (36.3%) constituted the most abundant compounds of total essential oil. Germacrene D (12, 12.5%) represented the major component. Oxygenated sesquiterpenes were the second important compounds identified in the oil (34.7%) and T-cadinol was found at a highest value (21, 11.8%) (Table 1). We noticed that oxygenated monoterpenes and derivatives represented 24.5% of the total constituent of oil with trans-verbenol (2, 6.1%), verbenone (3, 6.9%) and 2,5-dimethoxy-p-cymene (9, 6.5%) as major odorants in the oil. 2.2. Antibacterial activity The antibacterial activity of C. althaeoides oil was evaluated, using a microdilution method against Gram-positive (Staphylococcus aureus, Enterococcus faecalis and Acinetobacter sp.) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) strains. Thus, we noticed that the essential oil was not active against E. coli and S. aureus (Table 2). The relatively highest antibacterial effect was observed against P. aeruginosa and E. faecalis expressing a growth inhibition at low concentration [minimal inhibitory concentration (MIC) ¼ 0.31 ^ 0.10 mg/mL]. However, the essential oil of C. althaeoides flowers showed less antibacterial activity against the clinical isolate Acinetobacter (1.25 ^ 0.01 mg/mL).
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Table 1. Constituents of the essential oil from the flowers of C. althaeoides.
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Compound 1 trans-Pinocarveol 2 trans-Verbenol 3 Verbenone 4 trans-Carveol 5 Methyl carvacrol 6 b-Maaliene 7 a-Copaene 8 b-Caryophyllene 9 2,5-Dimethoxy-p-cymene 10 a-Humulene 11 (E)-Geranylacetone 12 Germacrene D 13 b-Selinene 14 cis-b-Guaiene 15 (E,E)-a-Farnesene 16 d-Cadinene 17 Germacrene B 18 Caryophyllene oxide 19 cis-Arteannuic alcohol 20 1-epi-Cubenol 21 T-cadinol 22 T-muurolol 23 a-Cadinol 24 Pentadecanal Oxygenated monoterpenes and derivatives Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Total Yield % (w/w)
LRIa
Composition (%)b
1140 1146 1205 1219 1245 1381 1396 1419 1425 1456 1457 1483 1487 1492 1508 1524 1558 1582 1595 1628 1641 1643 1654 1716
1.4 6.1 6.9 1.2 1.5 2.5 0.9 3.5 6.5 1.9 0.9 12.5 1.9 1.1 5.8 4.7 1.5 2.4 5.2 1.5 11.8 5.7 5.9 2.2 24.5 36.3 34.7 95.5 7.10 – 3
Identification GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS,
RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI
Note: Bold type indicates major component. a LRI, linear retention indices (HP-5 column). b %, percentage calculated by GC-FID on non-polar capillary column HP-5.
Antimicrobial activities of the essential oils are difficult to correlate with a specific compound due to their complexity and variability. Nevertheless, some researchers reported that there is a relationship between the chemical composition of the most abundant components in the essential oil and the antimicrobial activity (Farag et al. 1989; Deans & Sbodova 1990; Cox Table 2. Antibacterial activity of the essential oil from the flowers of C. althaeoides. Micro-organism
MIC (mg/mL)
MBC (mg/mL)
Imipenem
E. coli ATCC 25922 S. aureus ATCC 25923 E. faecalis ATCC 29212 P. aeruginosa ATCC 27950 Acinetobacter sp.
10.0 ^ 0.1
.10
. 10
–
0.31 ^ 0.10
0.31 ^ 0.10
0.31 ^ 0.10
0.62 ^ 0.10
–
1.25 ^ 0.01
1.25 ^ 0.01
–
0.25 0.0156 0.5
Note: MIC, minimum inhibitory concentration in (mg/mL); MBC, minimum bactericidal concentration in (mg/mL); –, not determined.
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et al. 2000). In addition, today, it is known that the synergistic or antagonistic effect of one compound in minor percentage of mixture has to be considered (Burt 2004). The essential oil evaluated in this study has a great variety of phytochemicals that could be considered as responsible for a larger or smaller part of the loss of the noted antibacterial effect. The activity of the tested oil certainly varies with its composition. Indeed, higher amount of germacrene D (12, 12.5%) was attributed to better antibacterial activity (Devendra et al. 2011). a-Humulene (10, 1.9%), b-selinene (13, 1.9%) and caryophyllene oxide (18, 2.4%), which were found in this oil, have been associated with the antibacterial activity (Shafi et al. 2005). 2.3. Cytotoxic activity The essential oil from the fresh flowers of the C. althaeoides expressed a significant cytotoxic activity against human breast cancer cells with percentage of inhibition of 8.16 mg/mL. Cytotoxic activity of the essential oil of this plant may be attributed to specific components of the oil. Some compounds in the C. althaeoides essential oil have previously been tested for cytotoxic properties. a-Humulene (10, 1.9%), caryophyllene oxide (18, 2.4%), b-caryophyllene (8, 3.5%) and germacrene D (12, 12.5%) were reported to be cytotoxic towards MCF-7, MDAMB-231, Hs 578T, PC-3 and Hep-G2 cell lines (Setzer et al. 2006). Cytotoxic activity of the essential oil of C. althaeoides may be due to a synergistic effect of these active compounds. Therefore, the anticancer activity of this medicinal plant may be used in the development of potent anticancer drug. 3. Experimental 3.1. Plant material C. althaeoides plant was collected in April 2012 in the area of Mechref, in Monastir town, on the coast of Tunisia, and identified by Professor Fethia Harzallah-Skhiri, Laboratory of Genetic, Biodiversity and Valorisation of Bioresources (LR11ES41), Higher Institute of Biotechnology of Monastir, University of Monastir, Tunisia. A voucher specimen (CA-Ap12) has been deposited in the herbarium of the above-mentioned laboratory. 3.2. Extraction of essential oil Fresh flowers of C. althaeoides (400 g) used for this investigation were weighed and then extracted by hydrodistillation with a Clevenger-type apparatus for 4 h. The essential oil was collected by decantation, dried over anhydrous sodium sulphate, concentrated, weighed and stored in sealed glass vials in a refrigerator at 4 –58C before analysis. 3.3. Chromatographic analysis GC analyses were performed using flame ionisation detector (FID), HP-5 (30 m £ 0.25 mm ID, 0.52 mm film thickness) fused silica capillary column. The carrier gas was nitrogen (1.2 mL/ min). The oven temperature programming was 1 min isothermal at 508C, then 50 to 2808C at a rate of 58C/min and held isothermal for 1 min. The injection part and detector temperatures were 250 and 2808C, respectively. Volume injected: 0.1 mL of 1% solution (diluted in hexane). GC/EI-MS analyses were performed with a Varian CP-3800 (Varian Inc., Palo Alto, CA) gas chromatograph equipped with an HP-5 capillary column (30 m £ 0.25 mm; coating thickness 0.25 mm) and a Varian Saturn (Varian Inc., Palo Alto, CA) 2000 ion-trap mass detector. Analytical conditions: injector and transfer line temperatures 220 and 2408C, respectively; oven temperature programmed from 60 to 2408C at 38C/min. The carrier gas was helium with a flow
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rate of 1 mL/min. Volume injected 0.2 mL of 1% solution (diluted in hexane); split ratio 1:30. Identification of the constituents was based on comparison of the retention times with those of authentic samples, comparing their LRI relative to the series of n-hydrocarbons, and on computer matching against commercial (NIST 98 and ADAMS) and home-made library mass spectra built from pure substances and components of known essential oils and the MS literature data (Stenhagen et al. 1974; Massada 1976; Jenning & Shibamoto 1980; Swigar & Silverstein 1981; Davies 1990; Adams 1995). Moreover, the molecular weights of all the identified substances were confirmed by GC/CI-MS, using MeOH as CI ionising gas.
3.4. Antibacterial activity
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3.4.1. Bacterial strains Both Gram-positive and Gram-negative rods were selected as micro-organisms according to their pathogenic origin. S. aureus (ATCC 25923), E. faecalis (ATCC 29212), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853) and clinical isolates of Acinetobacter sp. were used.
3.4.2. Preparation of inoculum Mueller– Hinton (M –H) broth was inoculated aseptically with the appropriate micro-organism, 24 h before testing. This was done to ensure that the bacteria was fully adapted to the broth and reached the stationary phase of growth. The inoculated bacterial strains were incubated at 378C during 18 –24 h in M –H agar; the inoculum suspension contains approximately 105 colonies forming unit of bacteria per mL.
3.4.3. Micro-well dilution assay The estimation of the MIC and minimal bactericidal concentration (MBC) was carried out by a microlitre plate dilution method (Celiktas et al. 2007; Jabrane et al. 2010). Dilutions of the essential oil were prepared to obtain concentrations ranging from 10 to 0.0775 mg/mL. Dimethyl sulfoxide (DMSO) solution was employed for sample dilution at a concentration of 10%. The MIC values were considered as the lowest essential oil fraction concentrations that prevent visible bacterial growth after 24 h of incubation at 378C and MBC as the lowest concentration that completely inhibited bacterial growth. Each experiment was repeated three times. Imipenem was employed as a positive control against Gram-positive and Gram-negative bacteria. To confirm the results of MBC, 5 mL of the experimental suspension was sub-cultured in blood agar, which was incubated at 378C for 24 h.
3.5. Cytotoxic activity Cancer is the cause of more than six million deaths each year in the world. In 2001, about 1,268,000 new cancer cases and 553,400 deaths were reported in the USA (Izevbigie 2003). In recent years, a plant-derived bioactive substance that is capable of selectively inhibiting tumour cell growth has received considerable attention in cancer chemopreventive approaches (Jang et al. 2005). Cytotoxicity of the sample was estimated on human breast cancer cells (MCF-7) as described by Natarajan et al. (2011) with modification. Cell growth was estimated by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT is a yellow water-soluble tetrazolium salt. Metabolically active cells are able to convert the dye to water-insoluble dark blue formazan by reductive cleavage of the tetrazolium ring.
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Cells were distributed in 96-well plates at 3 £ 104 cells/well in 100-mL culture medium and routinely cultured in a humidified incubator at 378C in 5% CO2 for 24 h. The essential oil at various concentrations in 1% DMSO (diluted in Phosphate-Buffered Saline (PBS)) was added and re-incubated for 24 h. Then, the medium was discarded and 30 mL of MTT dye solution (5 mg/mL in PBS) was added to every well and re-incubated for 4 h. After removing untransformed MTT reagent, 80 mL of DMSO was added to dissolve the formed formazan crystals. The amount of formazan was determined by measuring the absorbance at 540 nm using an enzyme-linked immunosorbent assay plate reader. Doxorubicin was used as a positive control. DMSO at 1% was used as control negative. The essential oil was screened three times against the tested cancer cells.
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4. Conclusion Our study is the first report on chemical composition of the fresh flowers of C. althaeoides and evaluates the antibacterial and the cytotoxic properties. The chemical constituents of the isolated essential oil were analysed by GC and GC –MS. A total of 24 compounds, accounting for 95.5% of the total oil, have been identified. The essential oil expressed relatively highest antibacterial activity against P. aeruginosa and E. faecalis. It also expressed a significant cytotoxic activity against human breast cancer cells (MCF-7). Therefore, according to these results, we suggest that the essential oil of C. althaeoides could be another potential source for new drug development. Acknowledgement The authors are grateful to Dr Fethia Harzallah Skhiri (High Institute of Biotechnology of Monastir, Tunisia) for the botanical identification.
References Adams RP. 1995. Identification of essential oil by gas chromatography/mass spectroscopy. Carol Stream, IL: Allured Publishing Co. Alapetite PG. 1981. Flore de la Tunisie [Flora of Tunisia]. Tunisia: Tunisian Scientific Publications. Bakkali F, Averbeck S, Averbeck D, Idaomar M. 2008. Biological effects of essential oils. J Food Chem Toxicol. 46:446–475. Boullard B. 2001. ‘Dictionnaire, Plantes me´dicinales du Monde’: Re´alite´s & Croyances [Dictionary, medicinal plants of the world: Realities and beliefs]. Paris: ESTEM. Burt S. 2004. Essential oils: their antibacterial properties and potential applications in foods. J Food Microbiol. 94:223–253. Carson CF, Riley TV. 2003. Non-antibiotic therapies for infectious diseases. Commun Dis Intell. 27:143–146. Celiktas OY, Kocabas EEH, Bedir E, Sukan FV, Ozek T, Baser KHC. 2007. Antimicrobial activities of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and seasonal variations. J Food Chem. 100:553–559. Cox SD, Mann CM, Markham JL, Bell HC, Gustafson JE, Warmington JR, Wyllie SG. 2000. The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil). J Appl Microbiol. 88:170–175. Davies NW. 1990. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases. J Chromatogr. 503:1– 24. Deans SG, Svoboda KP. 1990. The antimicrobial properties of marjoram (Origanum majorana L.) volatile oil. Flavour Frag J. 5:187–190. Devendra M, Shivani J, Sangeeta PS, Abhimanyu D, Ganga B. 2011. Chemical composition and antimicrobial activity of the essential oils of Senecio rufinervis DC. (Asteraceae). Indian J Nat Prod Resour. 2(1):44–47. Duke JA, Ayensu ES. 1985. ‘Medicinal plants of China’: medicinal plants of the world. Milford, CT: Near Fine. Farag RS, Daw ZY, Hewedi FM, El-Baroty GSA. 1989. Antimicrobial activity of some Egyptian spice essential oils. J Food Prot. 52:665–667. Izevbigie EB. 2003. Discovery of water-soluble anticancer agents (edotides) from a vegetable found in Benin City, Nigeria. Exp Biol Med. 228:293–298.
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Jabrane A, Ben Jannet H, Mastouri M, Mighri Z, Casanova J. 2010. Chemical composition and in vitro evaluation of antioxidant and antibacterial activities of the root oil of Ridolfia segetum (L.) Moris from Tunisia. J Nat Prod Res. 24(6):491–499. Jang HS, Kook SH, Son YO, Kim JG, Jeon YM, Jang YS, Choi YS. 2005. Flavonoids purified from Rhus verniciflua stokes actively inhibit cell growth and induce apoptosis in human osteosarcoma cells. Biochim Biophys Acta. 1726:309–316. Jenning W, Shibamoto T. 1980. Qualitative analysis of flavour and fragrance volatiles by glass capillary chromatography. New York, NY: Academic Press. Le Floc’h E. 1983. Contribution a` une e´tude ethnobotanique de la flore Tunisienne [Contribution to a ethnobotanic study of the Tunisian flora]. Tunisia: Tunisian Scientific Publications. Massada Y. 1976. Analysis of essential oils by gas chromatography and mass spectrometry. New York, NY: Wiley. Natarajan N, Thamaraiselvan R, Lingaiah H, Srinivasan P, Periyasamy BM. 2011. Effect of flavonone hesperidin on the apoptosis of human mammary carcinoma cell line MCF-7. Biomed Prev Nutr. 1:207–215. Setzer WN, Schmidt JM, Noletto JA, Vogler B. 2006. Leaf oil compositions and bioactivities of abaco bush medicines. Pharmacologyonline. 3:794–802. Shafi PM, Rosamma MK, Jamil K, Reddy PS. 2005. Antibacterial activity of the essential oil from Aristolochia indica. Fitoterapia. 73:439–441. Stenhagen E, Abrahamsson S, Mc Lafferty F. 1974. Registry of mass spectral data. New York, NY: Wiley. Swigar AA, Silverstein RM. 1981. Monoterpenes. Milwaukee, WI: Aldrich Chemical Company.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Chemical composition, antibacterial and cytotoxic activities of the essential oil from the flowers of Tunisian Convolvulus althaeoides L. a
a
a
b
M. Hassine , A. Zardi-Berguaoui , M. Znati , G. Flamini , H. Ben a
a
Jannet & M.A. Hamza a
Laboratory Heterocyclic Chemistry, Natural Products and é (LR11SE39), Team: Medicinal Chemistry and Natural Products and Reactivity, Faculty of Science of Monastir, University of Monastir, 5019 Monastir, Tunisia b
Dipartimento di Scienze Farmaceutiche sede Chimica Bioorganica e Biofarmacia, Via Bonanno 33, 56126 Pisa, Italy Published online: 05 Feb 2014.
To cite this article: M. Hassine, A. Zardi-Berguaoui, M. Znati, G. Flamini, H. Ben Jannet & M.A. Hamza (2014): Chemical composition, antibacterial and cytotoxic activities of the essential oil from the flowers of Tunisian Convolvulus althaeoides L., Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2013.879476 To link to this article: http://dx.doi.org/10.1080/14786419.2013.879476
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.
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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, 2014 http://dx.doi.org/10.1080/14786419.2013.879476
Chemical composition, antibacterial and cytotoxic activities of the essential oil from the flowers of Tunisian Convolvulus althaeoides L. M. Hassinea, A. Zardi-Berguaouia, M. Znatia, G. Flaminib, H. Ben Janneta* and M.A. Hamzaa a
Laboratory Heterocyclic Chemistry, Natural Products and e´ (LR11SE39), Team: Medicinal Chemistry and Natural Products and Reactivity, Faculty of Science of Monastir, University of Monastir, 5019 Monastir, Tunisia; bDipartimento di Scienze Farmaceutiche sede Chimica Bioorganica e Biofarmacia, Via Bonanno 33, 56126 Pisa, Italy
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(Received 24 August 2013; final version received 25 December 2013) This study describes the chemical composition and evaluates the antibacterial and the cytotoxic effects of the essential oil from the flowers of Convolvulus althaeoides. Its chemical composition, determined by GC and GC – MS, is reported for the first time. A total of 24 compounds, accounting for 95.5% of the total oil, have been identified. The oil was characterised by a high proportion of sesquiterpene hydrocarbons (36.3%), followed by oxygenated sesquiterpenes (34.7%) and oxygenated monoterpenes (24.5%). The main compounds were germacrene D (12.5%), T-cadinol (11.8%) and verbenone (6.9%). The essential oil was tested for its antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and the clinical strain Acinetobacter sp. as well as facts cytotoxic activity towards the human breast cancer cells MCF-7. This oil did not exhibit significant antibacterial activity against the tested bacteria; however, it exerted a significant cytotoxic activity against the tested cell line (IC50 ¼ 8.16 mg/mL). Keywords: Convolvulus althaeoides; essential oil; composition; GC – MS; antimicrobial activity; cytotoxic activity
1. Introduction Natural products have served as important sources of drugs since ancient times. In recent years, a renewed interest in obtaining biologically active compounds from natural sources has been observed. According to different authors, approximately 3000 plant species contain essential oils among which only 300 are commercially important. Essential oils and some of their constituents are used not only in pharmaceutical products for their therapeutic activities but also in agriculture, as food preservatives and additives for human or animal use, in cosmetics and perfumes, and other industries. In many cases, they serve as plant defence mechanisms against predation by micro-organisms, insects and herbivores (Bakkali et al. 2008). The complex composition of the essential oils and a variety of chemical structures of their constituents are responsible for a wide range of biological activities many of which are of increasing interest in the field of human and animal health. The need for new anti-infective agents due to the emergence of multiple antibiotic resistances has lead to the search of new sources of potential antimicrobials (Carson & Riley 2003). Among them, the plant kingdom offers a wide range of biodiversity with a great value for the pharmaceutical industry.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Downloaded by [University of Kent] at 03:43 01 May 2014
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The family Convolvulaceae contains an extremely wide variety of aromatic plants mainly in temperate countries. Among this rich array of plants which yield essential oils, the genus Convolvulus includes 250 species which are spread all over the world (Alapetite 1981). In Tunisia, we count 5 genera, 16 species, 5 subspecies and 7 varieties belonging to the Convolvulaceae family. In total, 12 species for the genus Convolvulus L. are found in Tunisia (Alapetite 1981) in which the Convolvulus althaeoides L. subspecies typicus Fiori has not been studied before. It is a climbing perennial plant with solitary flowers on long peduncles. The flower is a funnel-shaped pink bloom 3– 4 cm wide. The leaves are deeply divided into narrow, finger-like lobes. This flowering plant is native to the Mediterranean Basin, distributed only in the extreme south of Tunisia (Alapetite 1981). Convolvulus species are also employed in traditional medicine in several countries, and it is beneficial in the treatment against many human diseases. In particular, roots and leaves of Convolvulus scammonia L. have an aphrodisiac effect and antimalarial (Boullard 2001). Whole organs of Convolvulus soldanella L. have an antiscorbutic, diuretic, febrifuge and purgative effect (Duke & Ayensu 1985). Le Floc’h (1983) has not reported about its possible use in popular medicine in Tunisia. To the best of our knowledge, no data have been reported on C. althaeoides, and this is the first study on the essential oil composition of this species. With an aim of continuing our contribution to the valorization of the aromatic flora from Tunisia, in view of the potential use of its natural resources, this work deals with the study, for the first time, of the essential oil from fresh flowers of C. althaeoides (Convolvulaceae), in particular its chemical composition and the evaluation of its antimicrobial and cytotoxic activities. 2. Results and discussion 2.1. Chemical composition of the essential oil The hydrodistillation of the fresh flowers of C. althaeoides furnished light yellow oil, in 7 £ 1023% (w/w) yield. The composition of the oil is reported in Table 1, together with their linear retention indices (LRIs), the percentages of compounds and the identification methods. A total of 24 constituents, representing 95.5% of the oil, were detected. Seven oxygenated monoterpenes and derivatives, nine sesquiterpene hydrocarbons and six oxygenated sesquiterpenes were identified. Obviously, this oil may be considered as sesquiterpene-rich oil. Sesquiterpene hydrocarbons (36.3%) constituted the most abundant compounds of total essential oil. Germacrene D (12, 12.5%) represented the major component. Oxygenated sesquiterpenes were the second important compounds identified in the oil (34.7%) and T-cadinol was found at a highest value (21, 11.8%) (Table 1). We noticed that oxygenated monoterpenes and derivatives represented 24.5% of the total constituent of oil with trans-verbenol (2, 6.1%), verbenone (3, 6.9%) and 2,5-dimethoxy-p-cymene (9, 6.5%) as major odorants in the oil. 2.2. Antibacterial activity The antibacterial activity of C. althaeoides oil was evaluated, using a microdilution method against Gram-positive (Staphylococcus aureus, Enterococcus faecalis and Acinetobacter sp.) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) strains. Thus, we noticed that the essential oil was not active against E. coli and S. aureus (Table 2). The relatively highest antibacterial effect was observed against P. aeruginosa and E. faecalis expressing a growth inhibition at low concentration [minimal inhibitory concentration (MIC) ¼ 0.31 ^ 0.10 mg/mL]. However, the essential oil of C. althaeoides flowers showed less antibacterial activity against the clinical isolate Acinetobacter (1.25 ^ 0.01 mg/mL).
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Table 1. Constituents of the essential oil from the flowers of C. althaeoides.
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Compound 1 trans-Pinocarveol 2 trans-Verbenol 3 Verbenone 4 trans-Carveol 5 Methyl carvacrol 6 b-Maaliene 7 a-Copaene 8 b-Caryophyllene 9 2,5-Dimethoxy-p-cymene 10 a-Humulene 11 (E)-Geranylacetone 12 Germacrene D 13 b-Selinene 14 cis-b-Guaiene 15 (E,E)-a-Farnesene 16 d-Cadinene 17 Germacrene B 18 Caryophyllene oxide 19 cis-Arteannuic alcohol 20 1-epi-Cubenol 21 T-cadinol 22 T-muurolol 23 a-Cadinol 24 Pentadecanal Oxygenated monoterpenes and derivatives Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Total Yield % (w/w)
LRIa
Composition (%)b
1140 1146 1205 1219 1245 1381 1396 1419 1425 1456 1457 1483 1487 1492 1508 1524 1558 1582 1595 1628 1641 1643 1654 1716
1.4 6.1 6.9 1.2 1.5 2.5 0.9 3.5 6.5 1.9 0.9 12.5 1.9 1.1 5.8 4.7 1.5 2.4 5.2 1.5 11.8 5.7 5.9 2.2 24.5 36.3 34.7 95.5 7.10 – 3
Identification GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS, GC– MS,
RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI RI
Note: Bold type indicates major component. a LRI, linear retention indices (HP-5 column). b %, percentage calculated by GC-FID on non-polar capillary column HP-5.
Antimicrobial activities of the essential oils are difficult to correlate with a specific compound due to their complexity and variability. Nevertheless, some researchers reported that there is a relationship between the chemical composition of the most abundant components in the essential oil and the antimicrobial activity (Farag et al. 1989; Deans & Sbodova 1990; Cox Table 2. Antibacterial activity of the essential oil from the flowers of C. althaeoides. Micro-organism
MIC (mg/mL)
MBC (mg/mL)
Imipenem
E. coli ATCC 25922 S. aureus ATCC 25923 E. faecalis ATCC 29212 P. aeruginosa ATCC 27950 Acinetobacter sp.
10.0 ^ 0.1
.10
. 10
–
0.31 ^ 0.10
0.31 ^ 0.10
0.31 ^ 0.10
0.62 ^ 0.10
–
1.25 ^ 0.01
1.25 ^ 0.01
–
0.25 0.0156 0.5
Note: MIC, minimum inhibitory concentration in (mg/mL); MBC, minimum bactericidal concentration in (mg/mL); –, not determined.
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et al. 2000). In addition, today, it is known that the synergistic or antagonistic effect of one compound in minor percentage of mixture has to be considered (Burt 2004). The essential oil evaluated in this study has a great variety of phytochemicals that could be considered as responsible for a larger or smaller part of the loss of the noted antibacterial effect. The activity of the tested oil certainly varies with its composition. Indeed, higher amount of germacrene D (12, 12.5%) was attributed to better antibacterial activity (Devendra et al. 2011). a-Humulene (10, 1.9%), b-selinene (13, 1.9%) and caryophyllene oxide (18, 2.4%), which were found in this oil, have been associated with the antibacterial activity (Shafi et al. 2005). 2.3. Cytotoxic activity The essential oil from the fresh flowers of the C. althaeoides expressed a significant cytotoxic activity against human breast cancer cells with percentage of inhibition of 8.16 mg/mL. Cytotoxic activity of the essential oil of this plant may be attributed to specific components of the oil. Some compounds in the C. althaeoides essential oil have previously been tested for cytotoxic properties. a-Humulene (10, 1.9%), caryophyllene oxide (18, 2.4%), b-caryophyllene (8, 3.5%) and germacrene D (12, 12.5%) were reported to be cytotoxic towards MCF-7, MDAMB-231, Hs 578T, PC-3 and Hep-G2 cell lines (Setzer et al. 2006). Cytotoxic activity of the essential oil of C. althaeoides may be due to a synergistic effect of these active compounds. Therefore, the anticancer activity of this medicinal plant may be used in the development of potent anticancer drug. 3. Experimental 3.1. Plant material C. althaeoides plant was collected in April 2012 in the area of Mechref, in Monastir town, on the coast of Tunisia, and identified by Professor Fethia Harzallah-Skhiri, Laboratory of Genetic, Biodiversity and Valorisation of Bioresources (LR11ES41), Higher Institute of Biotechnology of Monastir, University of Monastir, Tunisia. A voucher specimen (CA-Ap12) has been deposited in the herbarium of the above-mentioned laboratory. 3.2. Extraction of essential oil Fresh flowers of C. althaeoides (400 g) used for this investigation were weighed and then extracted by hydrodistillation with a Clevenger-type apparatus for 4 h. The essential oil was collected by decantation, dried over anhydrous sodium sulphate, concentrated, weighed and stored in sealed glass vials in a refrigerator at 4 –58C before analysis. 3.3. Chromatographic analysis GC analyses were performed using flame ionisation detector (FID), HP-5 (30 m £ 0.25 mm ID, 0.52 mm film thickness) fused silica capillary column. The carrier gas was nitrogen (1.2 mL/ min). The oven temperature programming was 1 min isothermal at 508C, then 50 to 2808C at a rate of 58C/min and held isothermal for 1 min. The injection part and detector temperatures were 250 and 2808C, respectively. Volume injected: 0.1 mL of 1% solution (diluted in hexane). GC/EI-MS analyses were performed with a Varian CP-3800 (Varian Inc., Palo Alto, CA) gas chromatograph equipped with an HP-5 capillary column (30 m £ 0.25 mm; coating thickness 0.25 mm) and a Varian Saturn (Varian Inc., Palo Alto, CA) 2000 ion-trap mass detector. Analytical conditions: injector and transfer line temperatures 220 and 2408C, respectively; oven temperature programmed from 60 to 2408C at 38C/min. The carrier gas was helium with a flow
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rate of 1 mL/min. Volume injected 0.2 mL of 1% solution (diluted in hexane); split ratio 1:30. Identification of the constituents was based on comparison of the retention times with those of authentic samples, comparing their LRI relative to the series of n-hydrocarbons, and on computer matching against commercial (NIST 98 and ADAMS) and home-made library mass spectra built from pure substances and components of known essential oils and the MS literature data (Stenhagen et al. 1974; Massada 1976; Jenning & Shibamoto 1980; Swigar & Silverstein 1981; Davies 1990; Adams 1995). Moreover, the molecular weights of all the identified substances were confirmed by GC/CI-MS, using MeOH as CI ionising gas.
3.4. Antibacterial activity
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3.4.1. Bacterial strains Both Gram-positive and Gram-negative rods were selected as micro-organisms according to their pathogenic origin. S. aureus (ATCC 25923), E. faecalis (ATCC 29212), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853) and clinical isolates of Acinetobacter sp. were used.
3.4.2. Preparation of inoculum Mueller– Hinton (M –H) broth was inoculated aseptically with the appropriate micro-organism, 24 h before testing. This was done to ensure that the bacteria was fully adapted to the broth and reached the stationary phase of growth. The inoculated bacterial strains were incubated at 378C during 18 –24 h in M –H agar; the inoculum suspension contains approximately 105 colonies forming unit of bacteria per mL.
3.4.3. Micro-well dilution assay The estimation of the MIC and minimal bactericidal concentration (MBC) was carried out by a microlitre plate dilution method (Celiktas et al. 2007; Jabrane et al. 2010). Dilutions of the essential oil were prepared to obtain concentrations ranging from 10 to 0.0775 mg/mL. Dimethyl sulfoxide (DMSO) solution was employed for sample dilution at a concentration of 10%. The MIC values were considered as the lowest essential oil fraction concentrations that prevent visible bacterial growth after 24 h of incubation at 378C and MBC as the lowest concentration that completely inhibited bacterial growth. Each experiment was repeated three times. Imipenem was employed as a positive control against Gram-positive and Gram-negative bacteria. To confirm the results of MBC, 5 mL of the experimental suspension was sub-cultured in blood agar, which was incubated at 378C for 24 h.
3.5. Cytotoxic activity Cancer is the cause of more than six million deaths each year in the world. In 2001, about 1,268,000 new cancer cases and 553,400 deaths were reported in the USA (Izevbigie 2003). In recent years, a plant-derived bioactive substance that is capable of selectively inhibiting tumour cell growth has received considerable attention in cancer chemopreventive approaches (Jang et al. 2005). Cytotoxicity of the sample was estimated on human breast cancer cells (MCF-7) as described by Natarajan et al. (2011) with modification. Cell growth was estimated by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT is a yellow water-soluble tetrazolium salt. Metabolically active cells are able to convert the dye to water-insoluble dark blue formazan by reductive cleavage of the tetrazolium ring.
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Cells were distributed in 96-well plates at 3 £ 104 cells/well in 100-mL culture medium and routinely cultured in a humidified incubator at 378C in 5% CO2 for 24 h. The essential oil at various concentrations in 1% DMSO (diluted in Phosphate-Buffered Saline (PBS)) was added and re-incubated for 24 h. Then, the medium was discarded and 30 mL of MTT dye solution (5 mg/mL in PBS) was added to every well and re-incubated for 4 h. After removing untransformed MTT reagent, 80 mL of DMSO was added to dissolve the formed formazan crystals. The amount of formazan was determined by measuring the absorbance at 540 nm using an enzyme-linked immunosorbent assay plate reader. Doxorubicin was used as a positive control. DMSO at 1% was used as control negative. The essential oil was screened three times against the tested cancer cells.
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4. Conclusion Our study is the first report on chemical composition of the fresh flowers of C. althaeoides and evaluates the antibacterial and the cytotoxic properties. The chemical constituents of the isolated essential oil were analysed by GC and GC –MS. A total of 24 compounds, accounting for 95.5% of the total oil, have been identified. The essential oil expressed relatively highest antibacterial activity against P. aeruginosa and E. faecalis. It also expressed a significant cytotoxic activity against human breast cancer cells (MCF-7). Therefore, according to these results, we suggest that the essential oil of C. althaeoides could be another potential source for new drug development. Acknowledgement The authors are grateful to Dr Fethia Harzallah Skhiri (High Institute of Biotechnology of Monastir, Tunisia) for the botanical identification.
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