Chemical composition and antibacterial activity of the essential oil of ononis natrix from Morocco

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Chemical Composition and Antibacterial Activity of the Essential Oil of Ononis natrix from Morocco

Abdelaziz Elamrani ^a & Mohammed Benaissa ^a

a Equipe de Chimie Agroalimentaire et Chimie des substances naturelles végétales, Faculté des Sciences Aîn Chock , Université Hassan II-Casablanca , B.P 5366 , Maarif , Casablanca , Morocco

Published online: 12 Mar 2013.

To cite this article: Abdelaziz Elamrani & Mohammed Benaissa (2010) Chemical Composition and Antibacterial

Activity of the Essential Oil of Ononis natrix from Morocco, Journal of Essential Oil Bearing Plants, 13:4, 477-488, DOI:

10.1080/0972060X.2010.10643852

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Chemical Composition and Antibacterial Activity of the Essential Oil of Ononis natrix from Morocco

Abdelaziz Elamrani*, Mohammed Benaissa

Equipe de Chimie Agroalimentaire et Chimie des substances naturelles végétales, Faculté des Sciences Aîn Chock, Université Hassan II-Casablanca - B.P 5366

Maarif, Casablanca, Morocco

Abstract: The wild samples of Ononis natrix were collected in Guelmim region (south of Morocco) during June 2006. The plant samples were distillated by Clevenger apparatus and the obtained essential oils were analyzed by GC and GC-MS. The yield obtained varies greatly with a range of 0.7 to 1.3 %. A total of 63 compounds, amounting 93.3 % of the total oils were identified. The major components of Ononis natrix essential oils were farnesyl acetone (27. 3 %), geranyl acetone (20.3 %), thymol (13.7 %) and camphor (5.5 %).The antibacterial activity of the essential oil was evaluated against several strains and was shown to be significant against Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922).

Key words: Ononis natrix, Fabaceae, Essential oil composition, Antibacterial activity.

Introduction: Ononis natrix belongs to the Fabaceae (Leguminosae) family. The perennial plant is described as a sub-shrub pushing in the dry meadows on calcareous ground. Its size varies between 20 and 30 cm. Its stem oarswoman, drawn up, thorny is covered with viscous poiles. Its Sheets trifoliate and are cogged. Grouped in broken into leaf panicles, the flowers are recognized with their beautiful yellow colour; petals being striated with crimson outside ¹. The flowers appear between May and July ².

In Morocco, O. natrix is growing wild in various regions ^3-5. It occurs particularly in southern of the country at Bou-Azzer region ⁶, Oued Tensift, the oriental part, Rif ⁷, the Anti-Atlas at Taznakht basin ⁸ and Mamora’s forest ⁹. The habitat is on calcareous ground.

O. natrix is known in Morocco by the berber vernacular names of “âfzzaz”; “âfesdad” and

“fezzaz”. In the Moroccan traditional medicine, the infusions of the roots and flowers of O.

natrix have been used for the treatment of certain disturbances of the urinary tract ¹⁰. The antibacterial and antimicrobial activities of O. natrix have been studied. Many

ISSN 0972-060X

*Corresponding author (Abdelaziz Elamrani) E- mail: < [email protected]>

Received 06 July 2009; accepted in revised form 29 January 2010

Downloaded by [Korea University] at 19:26 28 December 2014

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authors have found that the plant extract has an interesting antibacterial and antimicrobial activity ^11-13. Maruhenda et al. ^14,15evaluating the antihypertensive and the diuretic activity of O. natrix, have found that the plant has a very interesting activity. Diaz et al. ¹⁶ studied the phytochemical and antibacterial screening of some species of Spanish Fabaceae and mentioned that twenty-two of 84 plant extracts (aqueous, ethereal and ethanolic) showed some bacterial activity in tests with 3 bacterial species Bacillus subtilis, Micrococcus luteus and Escherichia coli; Ononis natrix and Vicia cracca were of interest for their wide spectrum of activity. Barrero et al.¹⁷ mentioned that the flavonoids isolated from Ononis natrix showed a greatest cytotoxic activity.

A phytochemical study was carried out on O. natrix seeds by Chebli et al.¹⁸. The authors working on O. natrix collectd in the south of Morocco found that the oil was rich on fatty acids, on glycerids and on flavonoids. In addition, the phytochemical screening indicated the presence of catechic tannins, saponins, coumarins and terpens. The GC analysis of the methyl esters of fatty acids showed that linoleic and linolenic acids are the most prominent constituents of the oil respectively 33 % and 27 %. While TLC analysis showed that the oil contains mainly triglycerides and Phospholipids. O. natrix seeds are rich in proanthocyanidins (prodelphinidin and procyanidin), and flavonics aglycones, the most important are quercetin, kaempferol, isorhamnetin, nevadensin and penta-hydroxy-5, 6, 7, 3’, 4’ methoxy-3 flavon ¹⁸. The chemical composition of the extract of O. natrix has been previously investigated.

Feliciano et al ¹⁹ have isolated from the hexane extract of the whole aerial part of flowering O. natrix, the terpenoids, sterols and three compounds: 8-hydroxy-6-methoxy-3-undecyl- 3,4-dihydroisocoumarin;5-(2-acetoxytridecyl)-3-methoxyphenol and 5-(2-hydroxytridecyl)- 3-methoxyphenol. Canedo et al.²⁰ have isolated from the hexane extract of aerial parts of O. natrix, nine 5-tridecylresorcinol derivatives. Feliciano et al.²¹ have isolated five 3-alkyl- 3,4-dihydroisocoumarins and one derivative of orsellinic acid. Barrero et al.²² have isolated from the acidic fraction of the hexane extract of O. natrix, N-Δ¹³-docosenoylanthranilic acid, (22 R)-6-(2-acetoxytridecyl)-2-hydroxy-4-methoxybenzoic acid, nine resorcinol derivatives, eugenol and the flavone nevadensin. Al Khalil et al.²³ have isolated from the chloroform extract of the aerial parts of O. natrix, the N-arachidylanthranilic acid, the gardenin B, xanthomicrol, hymenoxin, 8-hydroxy-6-methoxy-3-undecyl-3,4-dihydroisocoumarin, and medicarpin-β-D-glucoside. Wollenweber et al.²⁴mentioned that more than 20 flavonoid aglycones were identified in the Mediterranean Ononis species, O. fruticosa, O. natrix subsp. ramosissima and O. tridentata. Barrero et al.²⁵ have isolated from extracts of O. natrix subsp. ramosissima two resorcinol derivatives, 5-(2-acetoxy-8-oxotridecyl) resorcinol and 5-(2-acetoxy-7-hydroxy-8-oxotridecyl) resorcinol, β-D-glucoside, betulaprenol 6, two steroids, four chalcones, six dihydrochalcones, two flavanones, and three pterocarpans.

The synthesis and the mass spectrometric studies of the principal dihydroisocoumarins of O. natrix were studied ^26,27. A short stereoselective synthesis of (3R)-3,4-dihydro-6,8- dimethoxy-3-undecyl-1H-[2]benzopyran-1-one and derivatives isolated from O. natrix has been described by Saeed ²⁸. In addition, the author mentioned that all of the compounds synthesized were examined in vitro for antifungal activity.

Recently an anthranilic acid derivative from O. natrix of Jordanian origin was isolated by Nawasreh et al.²⁹.

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The chemical composition of the Moroccan O. natrix essential oil was reported for the first time by Khallouki et al. ³⁰. The authors mentioned that the oil of O. natrix, collected around Benihdifa, near Elhoceima in northern Morocco in June 1995, was analysed by GC and GC-MS. Twenty-six constituents were only identified, comprising of mono and sesquiterpene hydrocarbons (54.4 %), oxygenated terpenes (14.2 %) and thymol (2.9 %).

The main components of the oil were camphor (16.2 %) and β-caryophyllene (9.0 %).

The objective of this study is to fully characterize the exact composition of the essential oil extracted from Ononis natrix using GC and GC-MC techniques. The antibacterial activity of the obtained essential oil was assessed against clinically isolated and reference strains microorganism covering both Gram positive and Gram negative bacteria.

Experimental

Plant Material: The samples of Ononis natrix were collected in May 2006 from Guelmim region (at 500 Km south of Rabat). In this region, fives places with a surface of 20 m x 20 m were delimited. In each place, six individual samples of the whole plant were collected and were mixed to obtain a heterogeneous sample (about 5 Kg). Therefore, five main heterogeneous samples were obtained and were treated separately. The plant material was identified according to the flora of Morocco ³¹ and also by Prof. M. Rejdali and Prof.

A. Achhal, Agronomic Institute and Veterinary Hassan II, Rabat (Morocco). A voucher specimen is deposited in the Herbarium of the Department of Botany and Ecology at the Agronomic Institute and Veterinary Hassan II, Rabat (Morocco) and also in our laboratory at the Chemistry Department at the Science Faculty Ain chock, Casablanca (Morocco).

Isolation of essential oil: After air-drying in the shade for a week, the plant material (leaves, flowers and twigs) was subjected to hydrodistillation using a Clevenger-type apparatus for 3 hours. In parallel to each distillation we determined the moisture of the air dried leaves:

two samples (5g each) were oven dried (104°C) for 4 h. The oil yields were expressed in dry matter percentage.

The oils, which were separated from water by decantation, were dried by filtration over anhydrous sodium sulphate. The oils obtained were light yellow with a strong odour.

The physicochemical characteristics of the oils were determined according to the AFNOR

32 standards at 20°C. The specific gravity, the refractive index, the optical rotation and the solubility in alcohol, were determined using respectively, the AFNOR norms: NF T 75-111, NF T 75-112, NF T 75-113, NF T 75-101.

Oil analysis: In order to put in evidence minor compound in the essential oil, we proceeded to the fractionation on column according to a previous work on the Warionia saharae by Elamrani et al.^{33, 34}. Oil (4 ml) from a mixture of the five main heterogeneous samples, was fractionated using a column chromatography (80 cm x 2 cm) on silica gel column 60, Merck 70-270 mesh ASTM (100 g). The elution was operated as follow: hexane (150 ml), chloroforme (100 ml), isopropanol (100 ml) and methanol (150 ml). The solvents used were of the highest purity available (Merck). 50 fractions were obtained. These fractions were analyzed by Thin-Layer Chromatography (TLC), which allow us to combine the similar

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fractions. Thus 5 main fractions were obtained.

The different Ononis natrix essential oils were analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS).

Gas chromatography: The analysis was carried out using a Hewlett-Packard HP 5980 gas chromatography apparatus equipped with FID and two capillary columns DB-5 and CW20M (25 m x 0.25 mm, film thickness 0.25 μm). Analytical conditions were: injector and detector temperature 240 and 260°C respectively, oven temperature programmed from 50 to 250°C at 4°C/min. Isothermal temperature at 250°C for 30 min; carrier gas 1 ml N2/

min. Relative concentrations were calculated using peak areas as given by HP 3396A integrator, without correction for response factors.

We have identified most constituents by comparison of their GC Kovats retention indices (R.I.), determined with reference to an homologous series of C₈-C₃₀ n-alkanes and with those of authentic standards available in the authors’ laboratory.

Gas chromatography-mass spectroscopy: The analysis was done using a Hewlett- Packard HP 5980 Series II gas chromatograph equipped with HP-5 capillary column (25m x 0.3mm; film thickness 0.25 μm) and an HP 5772A mass selective detector. Analytical conditions were: injector and detector temperatures: 240 and 260°C respectively. The oven temperature is programmed from 50 to 250°C at 4°C/min, then isothermal at 250°C for 10 min; the carrier gas was 2 ml He/min; we used an ionisation mode with electronic impact at 70 eV.

The constituents were identified by the combination of retention index data and mass spectra data using NBS library and other literature data.^{35, 36}.

Bacterial strains: The six bacterial strains used in this study are: Escherichia coli ATCC 25922, Escherichia coli, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Staphylococcus aureus and Salmonella enteritidis (table 3). The bacteria, maintained on Nutrient Agar (Merck, Darmstadt, Germany) were supplied by Microbiology Laboratory of the Institute Pasteur (Casablanca, Morocco). Identity of the bacteria used in this study was confirmed by Microbial Identification System in Biotechnology Application and Research Center at Institute Pasteur.

Antibacterial activity test: The antibacterial activity of the essential oil was carried out by disc diffusion test ³⁷ using 100 ml of suspension containing10⁸CFU/ml of bacteria spread on nutrient agar (NA) medium. Sterile 6mm diameter filter paper discs were impregnated with 10 mg of essential oil and placed on to nutrient agar. Ofloxacin (5 mg/

disc), sulbactam (30 mg)+cefoperazone (75 mg) (105 mg /disc) were used as positive reference standards to determine the sensitivity of one strain in each bacterial species tested. The inoculated plates with bacteria were incubated at 27°C for 24 h.

The antimicrobial activity was evaluated by measuring the zone expressed as mm of inhibition against test organism. Five discs per plate and three plates were used, and each test was run in triplicate ³⁸.

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Microdilution assays: The minimal inhibition concentration (MIC) values were studied for different bacteria. The inocula of bacteria were prepared from 12 h broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. Ononis natrix essential oils dissolved in 0.5 % dimethylsulfoxide were first diluted to the highest concentration (500 ml /ml) to be tested, and then serial twofold dilutions were made in a concentration range from 7.80 ml /ml to 500 ml /ml in 10 ml sterile test tubes containing nutrient broth. MIC values of oils against bacterial strains were determined based on a microwell dilution method

39. The 96-well plates were prepared by dispensing into each well 95 ml of nutrient broth and 5 ml of the inoculum. A 100 ml aliquot from oils initially prepared at the concentration of 500 ml /ml was added into the first wells. Then, 100 ml from their serial dilutions was transferred into six consecutive wells. The last well containing 195 ml of nutrient broth without compound and 5 ml of the inoculums on each strip was used as negative control.

The final volume in each well was 200 ml. Maxipime (Bristol-Myers Squibb, France) at the concentration range of 500-7.8 mg /ml was prepared in nutrient broth and used as standard drug for positive control. Contents of each well were mixed on a plate shaker at 300 rpm for 20 s and then incubated at appropriate temperatures for 24 h. Microbial growth was determined by absorbance at 600 nm using the ELx800 universal microplate reader (Biotek Instrument Inc., France) and confirmed by plating 5 ml samples from clear wells on nutrient agar medium. The extract tested in this study was screened two times against each organism.

The MIC of each extract was taken as the lowest concentration that showed no growth ⁴⁰. Results and discussion: The oil yields and physicochemical properties of the Moroccan Ononis natrix can be seen in Table 1. Examination of these results shows that the oil content varies greatly with a range of 0.7 to 1.3 % (ml per 100 g of dried material).

The refractive index was 1.5211. The optical rotation was + 7.4°. Table 2 lists the retention indices and percentage composition of the compounds identified in the Moroccan Ononis natrix essential oils. A total of 63 compounds, amounting 93 % of the oils, were identified.

The major components of Ononis natrix essential oils were farnesyl acetone (27.3 %), geranyl acetone (20.3 %), thymol (13.7 %) and camphor (5.5 %).

In comparison to the alone result previously reported in the literature for the oils of Ononis natrix, we observed that our results are comparable with the other authors. Khallouki et al.³⁰identified only twenty-six constituents and reported that the main components of the oil were camphor (16.2 %), β-caryophyllene (9.0 %) and thymol (2.9 %). The same twenty- six components were also found in our oils. However, it seems that, in our essential oils samples, the percentage of thymol (13.7 %) was particularly higher than those mentioned by Khallouki et al.³⁰, but the β-caryophyllene (2.7 %) and camphor (5.5 %) contents were lower. In addition, thirty-seven constituents were found in our essential oils have not been identified by Khallouki et al.³⁰. As far as we know, these thirty-seven components have not been previously described in the Ononis natrix oil in the literature.

While working on Ononis viscosa species of Turkey origin, Erdemgil et al. ⁴¹ , have identified 43 constituents and reported that the hexahydrofarnesylacetone (12.5 %), carvacrol (10.0 %), lauric acid (8.3 %), nonanal (5.5 %), E-geranylacetone (4.8 %), and dodecanal (4.8 %) were identified as the major constituents in the essential oils. In our Ononis natrix oil, the geranyl acetone was also found and reached 20.3 %.

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The percentage composition of the different groups of compounds in our O. natrix essential oils was also studied (Table 2). We observed that the hydrocarbon fraction represented 14.3 % of the total oil composition, while the oxygenated fraction represented 79.0 %.

Table 3 and 4 summarizes the results obtained from disc diffusion method, followed by measurements of MIC. The screening for antibacterial activity indicates that the O.

natrix essential oils present comparable activity against all strains of tested bacteria (MICs ranged from 7.81 to 65.37 μL/mL) (Table 3). It is interesting to note that S. aureus (ATCC 25923) and E. coli (ATCC 25922) were more sensitive to the oils with the higher inhibition zone as 27 and 25 respectively. The MICs of essential oils against S. aureus (ATCC 25923) and E. coli (ATCC 25922) were 15.6 and 7.81 μL/mL respectively. For isolated E. coli and S. aureus, the MICs were respectively 36.44 and 39.13 μL/mL and showed a moderate sensitivity. P. aeruginosa (ATCC 27853) and S. enteritidis were less sensitive to essential oils, with MICs respectively 60.96 and 65.37 μL/mL.

The antibacterial activity of the O. natrix oils may be related to the oxygenated fraction and particularly the major components such farnesyl acetone (27.3 %), geranyl acetone (20.3 %), thymol (13.7 %) and camphor (5.5 %); the results obtained in the course of the present study are in agreement with many results obtained in the literature in which some essential oils showed that greater antimicrobial potential could be ascribed to the oxygenated terpenes 11,12,42, 43.

We conclude from this study that the Moroccan Ononis natrix essential oil inhibit E.

coli and S. aureus strains, two resistant microorganisms which make serious sanitary problems worldwide and are also characterized by higher percentage of oxygenated components.

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Table 1. Physicochemical properties of Moroccan Ononis natrix essential oils Determination at 20°C

Yield (%) 0.7 to 1.3

Specific gravity 0.924

Refractive index 1.5211

Optical rotation + 7° 40’

Solubility in alcohol 0.65 vol at 70 %

- The values of the yield given were obtained from 10 determinations: 5 samples and two distillations each

- The values of the physicochemical properties given represent the average of two determinations

Table 2. Chemical composition of Moroccan Ononis natrix essential oils

Compounds RI RI Mode of %

(DB-5) (CW 20M) identification

α-Thujene 928 1030 a,c 0.3

α-Pinene 938 1033 a,c 0.2

Camphene 952 1085 a,b,c 0.6

Sabinene 976 1370 a,c 0.1

β-Pinene 980 1113 a,c 0.3

β-Myrcene 993 1150 a,c 0.6

α-Terpinene 1018 1176 a,c 0.2

p-Cymene 1026 1270 a,c 0.5

β-Phellandrene 1032 1216 a,b,c 0.6

γ-Terpinene 1062 1250 a,b,c 0.4

Terpinolene 1089 1299 a,c 0.1

Camphor 1144 1518 a,c 5.5

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table 2. (continued).

Dodecane 1200 1200 a,c 1.5

Decanal 1205 1485 a,b 0.2

(S)-carvone 1243 1715 a,c 0.1

Thymol 1290 2210 a,b,c 13.7

2-Undecanone 1292 - b,c 1.6

Tridecane 1300 - a,c 0.8

α-Longipinene 1353 1541 a,c 1.2

Eugenol 1355 2103 a,c 0.9

Phenol, 2,3,5,6-tetramethyl 1361 - a,b,c 0.2

1-Tetradecene 1393 - a,c 0.1

Tetradecane 1400 1400 a,c 0.4

Methyl eugenol 1402 - a,c 0.3

β-Caryophyllene 1419 1617 a,c 2.7

Geranyl acetone 1453 1735 a,c 20.3

Megastigmatrienone 1454 - a,b,c 0.6

Aromadendrene 1462 1650 a,b,c 0.4

Valencene 1493 1722 a,c 0.3

2-Tridecanone 1495 - a,c 0.2

α-Farnesene 1506 1727 a,c 3.4

Tridecanal 1513 - a,b 0.5

Naphthalene, 1,2,3,4,4a,- 1528 - a,c 0.2

7-hexahydro-1,6-dimethyl-4-(1-methylethyl)

Elemicin 1554 - a,b,c 0.4

trans-Nerolidol 1564 2044 a,b,c 0.2

Caryophyllene oxide 1581 2000 a,c 0.7

13-Tetradecenal 1608 - a,c 0.1

Tetradecanal 1615 - a,c 0.2

α-Bisabolol 1673 2022 a,b,c 0.1

Tetradecanoic acid 1704 - a,c 0.1

α-Hexylcinnamaldehyde 1762 2309 a,c 0.2

Benzyl Benzoate 1765 - a,c 0.4

Pentadecanoic acid 1791 - a,c 0.1

Hexadecanal 1819 - a,c 0.2

2-Heptadecanone 1859 - a,b,c 0.3

1-Hexadecanol 1879 - a,b,c 0.4

5-Octadecene, (E) 1894 - a,c 0.1

Methyl palmitate 1926 - a,c 0.1

Farnesyl acetone 1927 - a,c 27.3

Phytol 1949 - a,b 0.4

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table 2. (continued).

Hexadecanoic acid 1975 - a,c 0.5

1-Heptadecanol 1986 - a,b,c 0.1

Octadecanal 2034 - b,c 0.2

Heptadecanoic acid 2065 - a,c 0.3

Octyl methoxy cinnamate 2088 - a,c 0.1

1-Nonadecanol 2181 - a,c 0.5

1-Docosene 2194 - a,c 0.1

Octadecanoic acid 2200 - a,c 0.2

1-Eicosanol 2210 - a,c 0.1

1-Tetracosanol 2456 - a,b,c 0.3

1-Hexacosanol 2852 - a,b,c 0.2

Hexatriacontane - - a 0.3

Tritetracontane - - a 0.1

Oxygenated fraction 79.0

Hydrocarbon fraction 14.3

Total 93.3

a = Comparison of our MS data with NBS75K library data;

b = Comparison of our MS data with literature data (Adams);

c = Comparison of our RI data with literature data ( Lasseve data base, Chicoutimi Univ., Quebec, Canada ).

- Compounds are listed in order of their elution from a DB5 column Table 3. Antibacterial activity of Moroccan Ononis natrix essential oils against different bacteria

Strains Inhibition zone Positive control standard in diameter (mm) antibiotic disc (mm)

E. coli ATCC 25922 25 28 (OFX)

E. coli 09 20 (OFX)

P. aeruginosa ATCC 27853 12 15 (SCF)

S. aureus ATCC 25923 27 12 (SCF)

S. aureus 14 30 (OFX)

S. enteritidis 08 22 (SCF)

OFX, ofloxacin (5 mg/disc);

SCF, sulbactam (30 mg) + cefoperazona (75 mg) (105 mg /disc); were used as positive reference standards antibiotic discs

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Table 4. Minimal inhibitory concentration of Moroccan Ononis natrix essential oils against different bacteria

Strains Minimal inhibitory concentration Standard drug (MIC) of essential oil (10ml) (maxipime)

E. coli ATCC 25922 7.81 7.8

E. coli 36.44 15.6

P. aeruginosa ATCC 27853 60.96 31.25

S. aureus ATCC 25923 15.60 7.8

S. aureus 39.13 12.5

S. enteritidis 65.37 21.5