Journal of Oleo Science Copyright ©2016 by Japan Oil Chemists’ Society J-STAGE Advance Publication date : March 14, 2016 doi : 10.5650/jos.ess15242 J. Oleo Sci.
Chemical Composition, Cytotoxic and Antibacterial Activities of the Essential Oil from the Tunisian Ononis angustissima L. (Fabaceae) Lotfi Ghribi1, Aymen Ben Nejma1, Malek Besbes2, Fethia Harzalla-Skhiri3, Guido Flamini4 and Hichem Ben Jannet1,＊ 1
Laboratoire de Chimie Hétérocyclique, Produit Naturels et Réactivité. Equipe : Chimie Médicinale et Produits Naturels, Département de Chimie, Faculté des Sciences de Monastir, Université de Monastir, Avenue de l’Environnement, 5019 Monastir, TUNISIE 2 Laboratoire des maladies transmissibles et des substances biologiquement actives, Faculté de Pharmacie, 5000 Monastir, TUNISIE 3 Laboratoire de génétique, biodiversité et valorisation des bio-ressources, Institut Supérieur de biotechnologie de Monastir, Université de Monastir, Rue Tahar Haddad, Monastir 5000, TUNISIE 4 Dipartimento di Farmacia, Via Bonanno 33, 56126 Pisa, ITALY
Abstract: The chemical composition, cytotoxic and antibacterial activities of the hydrodistilled essential oil of the aerial parts of Ononis angustissima from south Tunisia has been evaluated. The oil yield was 0.04% (w/w). The chemical composition, determined by GC and GC-MS is reported for the first time. Forty-five components, accounting for 93.7% of the total oil have been identified. The oil was characterized by a high proportion of oxygenated sesquiterpenes (33.2%), followed by sesquiterpene hydrocarbons (6.3%) and apocarotenoids (10.3%). The main components of the oil were α-eudesmol (22.4%), 2-tridecanone (9.3%) and acetophenone (7.4%). The essential oil was tested for its possible cytotoxic activity towards the human cervical cell line HeLa using the MTT assay and the antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and the clinical strain Acinetobacter sp. This oil exerted a cytotoxic activity with an IC50 of 0.53 ± 0.02 mg/mL and a significant antibacterial effect against P. aeruginosa and E. faecalis. Key words: Ononis angustissima, essential oil, composition, α-eudesmol, cytotoxic activity, antibacterial activity. 1 Introduction Plants provide a multitude of flavours and fragrances that have found many applications in everyday life. According to different authors, approximately 3000 plants species contain essential oils, among which only 300 are commercially important1）. Essential oils and some of their constituents are used not only in pharmaceutical products for their therapeutic activities, but also in agriculture, as food preserves and additives for human or animal use, in cosmetics and perfumes, and other industrial fields. In many cases, they serve as plant defense mechanisms against predation by microorganisms, insects and herbivores2）. The complex composition of the essential oils and the 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 fields of human and animal health. Particularly, many essential oils and
their constituents have been traditionally used for their biological activities, which are known since ancient times. Presently a series of studies has demonstrated the cytotoxic activity of several essential oils. The genus Ononis（Fabaceae）is well represented worldwide: 75 species live in Europe and Asia, others in Africa3）. In Tunisia, 9 species of this genus can be found4）. Ononis species have been used for centuries in Turkey as folk remedies for their diuretic, antiseptic and antimicrobial properties5, 6）. Members of this genus have been shown to possess antibiotic, antipyretic, anti-inflammatory, antimicrobial, antiseptic, analgesic, diuretic, cytotoxic and antidiabetic activities7）. Ononis decoctions are reported to be useful in the treatment of skin and rheumatic diseases, as well as gout7）. The aerial parts of O. arvensis have been used in traditional medicine for the treatment of infections of the
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Correspondence to: Hichem Ben Jannet, Laboratoire de Chimie Hétérocyclique, Produit Naturels et Réactivité. Equipe : Chimie Médicinale et Produits Naturels, Département de Chimie, Faculté des Sciences de Monastir, Université de Monastir, Avenue de l’Environnement, 5019 Monastir, TUNISIE E-mail:
[email protected] Accepted December 24, 2015 (received for review October 21, 2015)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs
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L. Ghribi, A. B. Nejma and M. Besbes et al.
urinary tract and for skin diseases8）. O. spinosa has been reported to have pharmacological activities due to its aperient, diuretic, antibacterial, analgesic, anti-inflammatory, antiviral, cytotoxic, and antifungal effects9−11）. O. viscosa was shown to be effective against Gram positive bacteria12）. In Jordanian folk medicine, the aerial parts of O. sicula and O. hirta have been used against skin cancer and lesions. Moreover, the ethanol extract of O. hirta aerial parts demonstrated a high cytotoxic effect towards Vero cell line（IC50 72.50 μg/mL）13）. Some species of Ononis have been extensively studied to establish the chemical composition of their essential oils. The essential oil of O. viscosa was characterized by the presence of hexahydrofarnesylacetone（12.5％）, carvacrol （10.0％） , lauric acid（8.3％） , nonanal （5.5％） ,（E） -geranylacetone （4.8％）, and dodecanal（4.8％）8）. O. natrix essential oil was characterized by high mean percentages of mono- and sesquiterpene hydrocarbons （54.4％）, oxygenand thymol （2.9％） . The main compoated terpenes （14.2％） and β-caryophyllene nents of this oil were camphor （16.2％） 14） . To the best of our knowledge, there are no avail（9％） able reports on the chemical composition of the essential oil of O. angustissima, nor concerning its cytotoxicity. Therefore, as a contribution to the phytochemistry and the biological activity of this plant growing in the westernsouth region of Tunisia, we report here the in vitro cytotoxic and antibacterial properties of the essential oil isolated from its aerial parts, in relation with its chemical composition, investigated by means of complementary techniques （GC and GC-MS） .
2 Experimental 2.1 Plant material Ononis angustissima L. was collected in the region of Mides （Touzeur, South of Tunisia） in April 2011 and identified by Professor Fethia Harzallah-Skhiri, Laboratory of Genetic, Biodiversity and Valorisation of Bioresources （LR11ES41）, Higher Institute of Biotechnology of Monastir, Tunisia, University of Monastir, Tunisia. A voucher specimen（O.A-11）has been deposited in the herbarium of the above-mentioned laboratory. 2.2 Extraction of essential oil Extraction of essential oil of aerial parts（ 300 g）was carried out by hydrodistillation with a Clevenger-type apwas collected by paratus for 4 h. The essential oil （120 mg） decantation, dried over Na2SO 4, weighed and stored in sealed glass vials at 4-5℃ until analysis. 2.3 General methods 2.3.1 Analytical GC Gas chromatograph: HP 5890-series II equipped with
flame ionization detectors （FID） , HP-5 （30 m×0.25 mm ID, 0.52 μm film thickness）fused silica capillary column, carrier gas nitrogen（1.2 mL/min）. The temperature oven was programmed from 50℃（1 min）to 280℃ at 5℃/min（1 min）. Injector and detector temperatures: 250℃ and 280℃, respectively. Volume injected: 0.1 μL of 1％ hexane solution. The identification of the components was performed by comparison of their retention times with those of pure authentic samples and by mean of their Linear Rerelative to the series of C8-C30 n-hytention Indices（L.R.I） drocarbons. 2.3.2 Analytical GC-MS GC/EI-MS analyses were performed with a Varian CP-3800 gas-chromatograph equipped with a HP-5 capillary column（30 m×0.25 mm; coating thickness 0.25 μm） and a Varian Saturn 2000 ion trap mass detector. Analytical conditions: injector and transfer line temperatures 220 and 240℃ respectively; oven temperature programmed from 60℃ to 240℃ at 3℃/min; carrier gas helium at 1 mL/min; injection of 0.2 μL （10％ hexane solution） ; split ratio 1:30. Identification of the constituents was based on comparison of the retention times with those of authentic samples, comparing their linear retention indices relative to the series of C8-C30 n-hydrocarbons, and on computer matching against commercial（NIST 98 and ADAMS）and homemade library mass spectra built up from pure substances and components of known essential oils and MS literature data15−20）. Moreover, the molecular weights of all the identified substances were confirmed by GC/CI-MS, using MeOH as CI ionizing gas. 2.4 Cytotoxic activity The Human cervical adenocarcinoma, HeLa cell line was obtained from American. Type Culture Collection（ATCC, Rockville, MD, USA）and cells were cultured in a humidified atmosphere of 5％ CO2 in air at 37℃, in RPMI1640 medium containing 10％ （v/v）fetal calf serum, 2 mM glutamine, and antibiotics（200 U of penicillin and 50 mg of streptomycin per liter）and maintained at 37℃ in a humidified 5％ CO2 atmosphere21）. Cytotoxicity was measured using the MTT test with slight modifications. Cells were seeded at 5×103 cells/well in 200 μL of growth medium and incubated at 37℃ for 24h for cell adherence. The microplates were treated with the substances and incubated for 24, 48 and 72 h. Then, 10 μL of MTT were added to each well（5 mg/mL） and the incubation was continued for further 2 h. 100 μL of DMSO were added to each well. The absorbance（A）was measured at 570 nm using a Multiskan Ascent（Ascent Software version 2.6）microplate reader. The assay was realized in triplicate as a cell viability index. The percentages of cell growth were calculated as follows:
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Chemical Composition, Cytotoxic and Antibacterial Activities of the Essential Oil from the Tunisian Ononis angustissima L. (Fabaceae)
Cell growth（％）＝ ［A （sample） /A （control） ］ ×100. Cytotoxicity is expressed as the concentration of extract . inhibiting cell growth by 50％ （IC50） 2.5 Antibacterial activity 2.5.1 Micro-organisms The test micro-organisms included the following Gramnegative bacteria: P. aeruginosa（ATCC 27853）, E. coli （ATCC 25922） and the clinical isolate of Acinetobacter sp. were used. Gram-positive bacteria: S. aureus（ ATCC 25923） , E. faecalis （ATCC 29212） . 2.5.2 Determination of MIC and MBC The estimation of the minimal inhibitory concentration （MIC）and minimal bactericidal concentration（MBC）was carried out by a microlitre plate dilution method22）. 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 was defined as the lowest concentration of the compounds to inhibit the growth of the micro-organisms. The MIC of each sample was defined as the lowest concentration which inhibited either bacterial growth, after incubation at 37℃ between 18 and 24 h. The minimal bactericidal concentration（MBC）was determined by subculture on blood agar at 37℃ between 18 and 24 h. Thymol and Gentamicin were used as antibacterial positive control. Each experiment was repeated three times.
3 Results and Discussion 3.1 Chemical composition The hydrodistillation of the fresh aerial parts of Ononis angustissima L. furnished a light yellow oil, in 0.04％（w/ w）yield. Dried aerial parts of O. viscosa L. subsp. breviflora（DC）from Turkey produced essential oil in 0.24％ yield8）, these results show that the performance depends on the species. The composition of the oil was determined by GC and GC-MS. The percentage composition, with Linear Retention Indices（L.R.I）calculated for each compound, and the identification methods are reported in Table 1. A total of 45 constituents, accounting for 93.7％ of the oil, were identified. Obviously, this oil may be considered as sesquiterpene-rich oil. Analysis of the aerial parts oil led to the identification of only one oxygenated monoterpene, twenty sesquiterpenes, two phenylpropanoids, six apocarotenoids and sixteen non-terpene aldehydes, ketones, esters and acids（Table 1） . The essential oil chiefly consisted of oxygenated sesquiterpenes（33.2％ of the total oil）. Indeed, α-eudesmol（38; 22.4％）was by far the major component of the essential oil. Tridecan-2-one（22; 9.3％）, acetophenone（2; 7.4％）and dodecanal（13; 4.7％）were also found among the major
components. Sesquiterpene hydrocarbons were mostly represented by β-caryophyllene（15; 1.7％）, γ-muurolene （18; 1.2％）, α-selinene（ 21; 1.2％）and δ-cadinene（ 24; 1.1％）. The monoterpene, which constituted 1.0％ of the identified components, consisted of 4-terpineol（4; 1.0％）. Phenylpropanoids were represented by eugenol （8; 1.1％） and methyleugenol（12; 0.9％）. Among apocarotenoids, hexahydrofarnesylacetone（45; 3.5％） ,（E） -β-damascenone （10; 3.4％）,（E）-β-damascone（14; 1.4％）,（E）-β-ionone （20; 2.9％）and dimethyl ionone（26; 1.7％）were identified. Thirteen compounds were shared with the essential oils of O.viscosa and O.angustissima 8）. They accounted for 44.6％ and 21.7％, respectively. Only dodecanal and hexahydrofarnesylacetone were in common among the major constituents of the essential oils of these two species. Dodecanal was detected in the two studies in very similar percentages（4.8％ in the essential oil of O. viscosa and 4.7％ in the case of O. angustissima）. On the other hand, hexahydrofarnesylacetone was more represented in O. viscosa essential oil （12.5％）than in that of O. angustissima （3.5％）. 3.2 Cytotoxic activity The results of the cytotoxic test obtained evaluating the essential oil extracted from the aerial parts of Ononis angustissima as well as some sequiterpene hydrocarbons （6.3％; β-caryophyllene（1.7％））, oxygenated sesquiterpenes（ 33.2％; α-eudesmol（22.4％））, apocarotenoids （10.3％;（E）-β-damascenone;（3.4％））and acetophenone （7.4％）against HeLa cell line（human cervical adenocarcinoma）, using the MTT assay, are resumed in Table 2. The essential oil was found to be able to inhibit, after 72 h, 43.01％, 48.56％ and 71.32％ of the cell growth at the concentrations of 0.25 mg/mL, 0.5 mg/mL and 1 mg/mL, respectively. The IC50 value of the essential oil after 72 h was 0.53 mg/mL. Compared to that of the essential oil from Heracleum transcaucasicum, endowed with a good activity against Hela cells（IC50＝0.594 mg/mL after 96 h of incubation） , this value can be considered satisfactory23）. In fact, as reported in Table 2, the tumoral cell is almost insensitive to the essential oil when tested at low concentrations（0.125, 0.06 and 0.03 mg/mL）. In previous studies, it has been shown that sesquiterpene-rich essential oils （e.g, that of Zanthoxylum rhoifolium leaves; IC50＝90.7 ±8.2）and pure sesquiterpenes such as ß-caryophyllene 24） , have a high cytotoxicity against HeLa （IC50＝19.8±0.6） cell line. This finding may support our results because of the high percentages in our essential oil of hydrocarbon and oxygenated sesquiterpenes, including β-caryophyllene （1.7％）. Moreover, the presence of α-eudesmol in an appreciable amount（22.4％）and γ-eudesmol（1.4％）both isomers of β-eudesmol unidentified in our essential oil and considered active against HeLa cell line25−27）could support without any proof the observed cytotoxic activity. Nonethe3
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L. Ghribi, A. B. Nejma and M. Besbes et al.
Table 1 Chemical composition of the aerial parts essential oil of O. angustissima. Compound
L.R.Ia
Composition (%)b
Identificationc
1
Benzaldehyde
963
2.6
GC-MS, RI
2
Acetophenone
1067
7.4
GC-MS, RI
3
Nonanal
1103
0.6
GC-MS, RI
4
4-Terpineol
1178
1.0
GC-MS, RI
5
Benzylacetone
1235
0.6
GC-MS, RI
6
Undecanal
1307
1.1
GC-MS, RI
7
4-Vinylguaiacol
1315
1.6
GC-MS, RI
8
Eugenol
1358
1.1
GC-MS, RI
9
2-Methylundecanal
1368
0.9
GC-MS, RI
10
(E)-β-Damascenone
1383
3.4
GC-MS, RI
11
Dodecan-2-one
1392
1.5
GC-MS, RI
12
Methyl eugenol
1403
0.9
GC-MS, RI
13
Dodecanal
1408
4.7
GC-MS, RI
14
(E)-β-Damascone
1412
1.4
GC-MS, RI
15
β-Caryophyllene
1419
1.7
GC-MS, RI
16
(E)-Geranylacetone
1455
0.9
GC-MS, RI
17
11-Methylundecan-2-one
1464
3.1
GC-MS, RI
18
g-Muurolene
1478
1.2
GC-MS, RI
19
α-Amorphene
1482
0.4
GC-MS, RI
20
(E)-β-Ionone
1487
2.9
GC-MS, RI
21
α-Selinene
1495
1.2
GC-MS, RI
22
Tridecan-2-one
1497
9.3
GC-MS, RI
23
trans-g-Cadinene
1514
0.7
GC-MS, RI
24
δ-Cadinene
1524
1.1
GC-MS, RI
25
Elemol
1551
0.5
GC-MS, RI
26
Dimethyl ionone
1565
1.7
GC-MS, RI
27
Lauric acid
1568
0.6
GC-MS, RI
28
(Z)-Hex-3-enylbenzoate
1570
1.2
GC-MS, RI
29
Presilphiperfolan-8-ol
1582
0.7
GC-MS, RI
30
Caryophyllene oxide
1583
0.8
GC-MS, RI
31
Carotol
1595
0.6
GC-MS, RI
32
Guaiol
1597
1.3
GC-MS, RI
33
1,10-di-epi-cubenol
1615
0.8
GC-MS, RI
34
1-epi-Cubenol
1629
1.0
GC-MS, RI
35
g-Eudesmol
1632
1.4
GC-MS, RI
36
T-Cadinol
1642
1.2
GC-MS, RI
37
α-Muurolol
1646
0.5
GC-MS, RI
38
α-Eudesmol
1653
22.4
GC-MS, RI
39
α-Cadinol
1655
0.7
GC-MS, RI
40
Khusinol
1676
0.5
GC-MS, RI
41
Eudesma-4(15),7-dien-1-β-ol
1686
0.8
GC-MS, RI
4
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Chemical Composition, Cytotoxic and Antibacterial Activities of the Essential Oil from the Tunisian Ononis angustissima L. (Fabaceae)
Table 1 Continued. Compound
L.R.Ia
Composition (%)b
Identificationc
42
Pentadecan-2-one
1689
0.6
GC-MS, RI
43
Methyl tetradecanoate
1725
0.4
GC-MS, RI
44
Tetradecanoic acid
1768
1.2
GC-MS, RI
45
Hexahydrofarnesylacetone
1845
3.5
GC-MS, RI
Oxygenated monoterpene
1.0
Sesquiterpene hydrocarbons
6.3
Oxygenated sesquiterpenes
33.2
Phenylpropanoids
2.0
Apocarotenoids
13.8
Others
37.4
Total
93.7
Yield % (w/w)
0.04
a
LRI: Linear Retention idices (HP-5 column). %: Percentage calculated by GC-FID on non-polar capillary column HP-5 c RI: Linear Retention index; GC-MS; Mass spectrum-gas chromatography b
Table 2 Cytotoxic activity of essential oil from the aerial parts of O. angustissima. IC50 (mg/mL)
Concentration (mg/mL)
0.03
0.06
0.125
0.25
0.5
1.0
24 h
89.09
87.53
85.88
69.68
77.05
74.69
48 h
96.69
91.57
91.37
87.12
83.42
68.93
72 h
85.64
68.53
62.93
56.99
51.44
28.68
Cell growth (%) Cisplatin
0.53 ±0.02 0.003±0.001
Results are expressed as means±SD of 3 independent observations performed in triplicate. Cisplatin was tested as a reference.
Table 3 A ntibacterial activity of the essential oil from the aerial parts of O. angustissima. Micro-organism P. aeruginosa
MIC (mg/mL)
MBC (mg/mL)
Thymol (MIC)
0.31±0.10
0.62±0.10
1
GM (MBC) 0.5
10.0 ±0.10
10.0 ±0.10
0.25
0.0039
E. faecalis
0.07±0.01
0.31±0.10
0.6
0.0078
S. aureus
＞10
−
0.2
0.01562
1.25±0.10
1.25±0.10
−
−
E. coli
Acinetobacter sp.
MIC: minimum inhibitory concentration in (mg/mL); MBC: minimum bactericidal concentration in (mg/mL); GM: gentamicin; -: not determined. less, besides the contribution of specific constituents, the synergism between various components of the essential oil could play an important role in the cytotoxic effect verified in this study against HeLa cell line. 3.3 Antibacterial activity The antibacterial activity of O. angustissima oil was evaluated, using a microdilution method against Gram-pos-
itive bacteria（Staphylococcus aureus and Enterococcus faecalis）and Gram-negative bacteria（Escherichia coli, Pseudomonas aeruginosa and Acinetobacter sp.）strains. The results showed that the essential oil was not active against S. aureus and E. coli（Table 3） . The significant antibacterial effect was observed against E. faecalis and P. aeruginosa,（MIC＝0.07±0.01, 0.31±0.10 mg/mL and MBC＝0.31±0.10, 0.62±0.10 mg/mL, respectively）. 5
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L. Ghribi, A. B. Nejma and M. Besbes et al.
However, the essential oil of O. angustissima aerial parts exhibited a moderate antibacterial effect against the clini（1.25±0.10 mg/mL） . Antibaccal isolate Acinetobacter sp. terial activity of the essential oil is difficult to correlate with a specific compound because of their variability and complexity. Nonetheless, some previous studies describe that there is a relationship between the chemical composition of the most abundant compounds in the essential oil and the antibacterial activity28−30）. Some studies reported the potent antibacterial activity of the oil of Guatteriopsis friesiana might be attributed to its high sesquiterpene content （97.13％） , and similarly for the oil of G. blepharophylla（90.56％）31）, the essential oil of leaf of Litsea kostermansii contains a high percentage of sesquiterpene （Oxygenated sesquiterpenes（66.2％）and sesquiterpene hydrocarbons（32.8％））exhibited excellent antimicrobial activities32）. The presence of high content of oxygenated sesquiterpenes （33.2％） in the aerial parts essential oil of O. angustissima could be the responsible of its activity against P. aeruginosa and E. faecalis with the value of MIC （0.31 and 0.07 mg/mL, respectively） .
4 Conclusion Our study was the first report on the chemical composition of the fresh aerial parts of Ononis angustissima and assesses the cytotoxic and the antibacterial activities. The chemical constitutes of the isolated essential oil were analysed by GC and GC-MS. A total of 45 compounds, representing 93.7％ of the total oil, have been identified. The essential oil voiced a significant cytotoxic activity against , using the HeLa cell line（human cervical adenocarcinoma） MTT assay that could be due to its richness in oxygenated sesquiterpenes. A significant antibacterial effect of the same essential oil was also observed against P. aeruginosa and E. faecalis. Our study is the first report on chemical composition and in vitro antibacterial and cytotoxic properties of the essential oil of O. angustissima. Thus this work constitutes a contribution to a better valorisation of another medicinal species from the Ononis genus growing in Tunisia.
Acknowledgements The authors thank the Tunisian Ministry of high Study and Scientific Research for financial support.
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Chemical Composition, Cytotoxic and Antibacterial Activities of the Essential Oil from the Tunisian Ononis angustissima L. (Fabaceae)
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