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Effect of gamma irradiation on chemical composition, antimicrobial and antioxidant activities of Thymus vulgaris and Mentha pulegium essential oils
Author’s Accepted Manuscript Effect Of Gamma Irradiation On Chemical Composition,Antimicrobial And Antioxidant Activities Of Thymus Vulgarisand Mentha Pulegium Essential Oils Said Zantar, Rachid Haouzi, Mohammed Chabbi, Amin Laglaoui, Mohammed Mouhib, Boujnah Mohammed, Mohammed Bakkali, Mounir Hassani Zerrouk
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S0969-806X(15)00187-5 http://dx.doi.org/10.1016/j.radphyschem.2015.05.019 RPC6816
To appear in: Radiation Physics and Chemistry Received date: 25 February 2015 Revised date: 27 April 2015 Accepted date: 9 May 2015 Cite this article as: Said Zantar, Rachid Haouzi, Mohammed Chabbi, Amin Laglaoui, Mohammed Mouhib, Boujnah Mohammed, Mohammed Bakkali and Mounir Hassani Zerrouk, Effect Of Gamma Irradiation On Chemical Composition,Antimicrobial And Antioxidant Activities Of Thymus Vulgarisand Mentha Pulegium Essential Oils, Radiation Physics and Chemistry, http://dx.doi.org/10.1016/j.radphyschem.2015.05.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
EFFECT OF GAMMA IRRADIATION ON CHEMICAL COMPOSITION, ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES OF THYMUS VULGARIS AND MENTHA PULEGIUM ESSENTIAL OILS AUTHORS: Said Zantara*, Rachid Haouzib, Mohammed Chabbib, Amin Laglaouid, Mohammed Mouhiba, Boujnah Mohammeda , Mohammed Bakkalid, Mounir Hassani Zerrouke a Irradiation
plant of Tangier, National Institute of Agronomic Research (INRA), Tangier, Morocco. b Departement of Chemistry, Faculty of Science and Technology, Tangier, Morocco. d Departement of Biology, Faculty of Science and Technology, Tangier, Morocco. e Departement of Biology, Polydisciplinary Faculty of Larache, Morocco.
*Corresponding author: Said Zantar Address: Centre Régional de la Recherche Agronomique de Tanger.78 Avenue Sidi. Mohamed Ben Abdellah, Tanger 90010 Morocco. Phone number: +212 539938033 Fax Number: +212 539394523 E-mail: [email protected] Abstract: The effects of gamma irradiation doses (10, 20 and 30 KGy) on chemical composition, antimicrobial and antioxidant activities of T. vulgaris and M. pulegium essential oils (EOs) have been studied. The chromatographic analysis showed that the studied EOs were constituted mainly by carvacrol for T. vulgaris and pulegone for M. pulegium. Gamma irradiation on the studied doses, affects quantitatively and not qualitatively some components of the investigated oils. This effect was dose dependent. While the antioxidant activity remains stable at any dose applied for the plants studied, the antimicrobial activity increased in the irradiated samples for gram negative bacteria and did not change for gram + bacteria. This study supports that gamma irradiation employed at sterilising doses did not compromise the biological activities of medicinal and aromatic plants. Keywords: essential oils, gamma radiation, chemical composition, antimicrobial activity, antioxidant activity
1. INTRODUCTION The importance of aromatic and medicinal plants (AMPs) and their extracts have been demonstrated all over the world for years. In fact, they are used for their nutraceutical, therapeutic, and cosmetic benefits. For this reason, they are subject to a high demand. In a globalized context and free trade, hygienic quality is primordial for a large commercialization of these products. Like foods, AMPs are susceptible to be
contaminated during their collection, treatment, transport, and storage by a several number of pathogen microorganisms and chemical contaminants like pesticides, heavy metals and dioxins (Machhour et al., 2008; Legnani et al., 2001; Idu et al., 2008). According to the World Health Organization, the numbers of intoxications are due to an incorrect utilization of medicinal plants which are in continuous increase (WHO, 2003). Logically, the situation is more alarming in developing countries. The Anti-poison and pharmacology Centre of Morocco, published that in 2012, 12 persons died due to an improper use of medicinal plants. One of the more important intoxication causes is the plant hygienic states that contain habitually a high number of microorganisms (Abu-dounia, 2008). The problem is more dramatic in the sense that the medicinal plants are in many cases used craw without any pretreatment. (Beuchat, 1996). In such a situation, the conformity of the international standards in terms of hygiene and safety is crucial for a wide commercialization of these plants. In addition to the good manufacturing practices, establishment of techniques for a good decontamination of AMPs is of a big importance. Two techniques of plants decontamination are actually used. The thermal processes like steam and ohmic heating and non-thermal processes like fumigation by ethylene oxide and high hydrostatic pressure (Machhour et al. 2008). When the thermal processes are destructive and affect the quality of plants, fumigation uses chemical products that are in most cases dangerous for public health (Machhour et al., 2008; Loaharanu et al., 1990). Another non-thermal technique decontamination that has proved its efficacy for microbial inactivation is irradiation (Raso and Barbosa-Canobvas, 2003). This technique, which is widely used for food preservation, has been proposed for plants decontamination by several authors (Pezzutti et al., 2005; Kume et al., 2009). Although irradiation efficacy for microbial inactivation has been widely demonstrated (Lacroix and Ouattara, 2000 ; Hong et al., 2008), the effects on chemical composition and biological activities of medicinal plants raised the interest of several authors in last past years (Khattak et al., 2008; Khattak, 2012; Pérez et al., 2011). From these works it can be concluded that biological activities of medicinal plants are affected by gamma irradiation which can be modified depending on several factors like plants species, irradiation doses, time of exposition and the solvent used.
Despite that the fact that the effect of gamma irradiation on the biological activities of aqueous and organic extracts has been already studied, surprisingly, the effect on essential oils has not been well studied while they represent one of the most important fraction in medicinal plants. In food and pharmaceutical sector, essential oils are used for their antimicrobial and antioxidant activities (Burt, 2004). Preservation of these activities is very important to design and validate the decontamination process of a plant. T. vulgaris and M. pulegium are two spontaneous AMPs belonging to the Lamiaceae family that grow in several regions of Morocco. They have an undeniable commercial interest, too. However, to the best of our knowledge, there are no studies on scientific literature that evaluate the effect of gamma irradiation on chemical composition, and biological activities of their essential oils. The main objective of this work is to evaluate the effect of gamma irradiation at very high doses on the chemical composition, antioxidant and antimicrobial activity of T. vulgaris and M. pulegium essential oils.
2. MATERIALS AND METHODS 2.1. Plant material The studied plants (T. vulgaris and M. Pulegium) were collected during the period of March 2011 to June 2011. T. vulgaris was harvested in the region of Beni Idder in the Northeast of Morocco and M. pulegium from Bougedour in the North. The plants were dried from 24 to 48 hours at a temperature of 40°C under ventilation. Leaves and flowers were used for the extraction of EOs. 2.2. Irradiation
For gamma irradiation, the dried leaves and flowers (100 g) were packaged in plastic bags and then exposed to gamma irradiation for 10, 20 and 30 kGy at ambient temperature (20°C) in the Boukhalef Ionization Plant (Tangier, Morocco), using a cobalt 60 radio-isotopic source . A dosimetry work has been done using samples in same packaging and position to have dose rate and dose uniformity. For this we used Gamma Chrome dosimeter batch YR3. The read out of result has been done using a spectrophotometer under 530 nm Wavelength after 2 hours for stabilization of dosimeters. Five dosimeters were used in bags of product to determine dose rate and
doses Uniformity. We have got a minimum dose rate of 1.17 Gy/min and dose uniformity of 1.06. Using this result we irradiated specified samples to 10, 20 and 30 kGy according the time of irradiation issue to dosimetry result and corrected with daily decay factor of cobalt60. Dosimeters Amber batch 3042K for 10 kGy and red Perspex batch 4034CV for 20 and 30 kGy were used for control in order to be in the best result of calibration curve of dosimeters and have maximum precision to be within 3% given in calibration curve. After irradiation, essential oils were immediately extracted and stored at -18°C until their use. 2.3. Essential oils extraction EOs extraction was carried out by steam distillation for the two plants. 100 g of dried leaves and flowers of T. vulgaris (50 g for M. pulegium) were introduced into flask with distilled water; the mixture was boiled for 3 hours. 2.4.
Essential oils analysis
Gas chromatography/mass spectrometry (GC/MS) analysis: The GC/MS analyses were carried out using a gas chromatograph coupled with Mass Spectrophotometer (MS) (GC-MS Trace GC ultra - ITQ900, Thermo Scientific, USA) operating in electron-impact (70 eV, m/z 40-450) mode. The capillary column used was 1MS (30 m x 0.25 mm x 0.25 µm film thickness). Analytical conditions were: injector and transfer line temperatures 250 and 300°C, respectively, oven temperature programmed from 50 to 200°C at 10°C/min and from 200 to 290°C at 35°C/min, carrier gas helium, at 1mL/min, injection of 1 µl (10% hexane solution), the split ratio was 1:20. The identification of the EOs compounds was performed by comparing their mass spectra with data bank (NIST MS search V.2.0) and homemade library mass spectra built up from pure substances and components of known essential oils and MS literature data (Mclafferty and Stauffer, 1989; Adams, 2001), and by coinjection with an authentic sample. Also the constituents of essential oils were identified based on their Kovats Index, calculated in relation to the retention time of a series of alkanes (C4- C28) as reference products, in comparison with those of the chemical compounds gathered by Adams Table (Adams, 2001).
2.5.
Antimicrobial activity
Antimicrobial activity was determined by paper disc agar plates. Microbial strains used were provided by the Spanish Collection of Type Cultures (CECT). Two Gram negative bacteria Escherichia coli (SCTC 471), Salmonella Senftenberg 775W (ATCC 43845) and two Gram-positive Listeria monocytogenes (SCTC 4031), Staphylococcus aureus (SCTC 976) were used. A volume of 10 mL of Muller Hinton Agar medium (Liofilchem, Roseto degli Abruzzi, Italy) (MHA) was poured into Petri plate. 100 μL of culture bacteria are plated at approximately final concentration of 106 bacteria/mL. After 15 min, a paper disc Whatman No. 1 of 6 mm (Whatman International L7d Maidstone, England), impregnated with 10μl of the EO was put to the surface of the agar. The plates were incubated in an incubator at 37°C for 24h and the inhibition diameter was measured. 2.6.
Antioxydant activity
DPPH method: 1 mL of a solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (sigma Aldrich INC .P.O box14508 St Louis, MO 63178 USA) radical at 1 mmol/L in ethanol was transferred into tubes containing 3 mL of the diluted EO in ethanol. After agitation, the tubes were placed in the dark in a temperature room for 30 min. For each concentration, the tests were repeated three times. The negative control was composed by 1 mL of ethanol DPPH solution and 3 mL of ethanol. The positive control was represented by a standard solution of an antioxidant butylated hydroxytoluene (BHT) (Fluka, Sigma-Aldrich Chemie GmbH, CH-9471 Buchs, Spain). Reading is performed by measuring the absorbance at 517 nm by a spectrophotometer. Results expression The antioxidant activity "AA%" is given by the following formula: AA% = [(Ac –Ap) /Ac] x 100 Ac: absorbance of the control reaction (containing all reagents except the tested essential oil).
Ap: absorbance with essential oil. 2.7.
Statistical analysis
Analyses of variance were performed by Statgraphics Plus 4.0 statistical software. All experiments were performed in triplicate. The results were presented as the mean with its standard deviation (n = 3) in each case. Differences were considered significant at P <0.05. 3. RESULTS AND DISCUSSION Although gamma irradiation has proved its efficacy for microbial inactivation in plants and species, the study of side effects is actually the goal of many works. In this paper, we have decided to investigate the effect of high irradiation doses (until 30 kGy) on the chemical composition, antimicrobial and antioxidant activities of essential oils from two AMPs widely used in food and pharmaceutical sectors (T. vulgaris and M. pulegium). Most studies that have evaluated the effect of gamma irradiation on medicinal plants and species were conducted on aqueous or organic extracts when essential oils are one of the most important fractions in plants. 3.1. Chemical composition The study of the effect of gamma irradiation on chemical composition of T. vulgaris and M. pulegium has been carried out, firstly, because the biological activities of plants extract are essentially due to their wealth in chemical compounds and, secondly, to understand the results obtained in antimicrobial and antioxidant posteriorly. The effect of gamma irradiation at 10, 20 and 30 kGy doses on chemical composition of T. vulgaris and M. pulegium essential oils have been analyzed by GC/MS chromatography. Briefly, chromatographic analysis has highlighted five main components for T. vulgaris that represent more than 1% levels. Carvacrol was the major component with a content ranging from 78.33 to 81.49% accompanied with other components at relatively low levels: -Terpinene (0.7 to 0.8%), p-cymene (3.9 to 6.7%), δ-terpinene (2.8 to 4.0 %), β-humulene (2.0 to 2.5%). With respect to M. pulegium EO, four major compounds were found. Pulegone was the major constituent with a content of 80 %, accompanied by small amounts of limonene (1.3 to 1.5 %),
menthone (1.3 to 2.3 %) and periperitone (6,5 to 8.16 %). Tables 1 and 2 show that gamma irradiation process affects quantitatively but not qualitatively the chemical composition of T. vulgaris and M. pulegium EOs. The effect was dose dependent. With respect to T. vulgaris (table 1), gamma irradiation with 10 kGy, caused the increase in concentration of one component (p-cymene) that raise from 3.89 to 4.31 %, the content of other compounds was similar to the control. Table 1. Chemical composition of Thymus vulgaris essential oils extracted from unirradiated and irradiated plant at 10, 20 and 30 kGy. Retention
Kovats
time (min)
Index
α-thujene
5.16
α-pinene
Compounds
0 kGy
10 kGy
20 kGy
30 kGy
930
0.82b±0.09
0.82b±0.08
0.78b±0.06
0.55a±0.06
5.27
939
0.32±0.04
0.35±0.08
0.38±0.04
0.3±0.02
δ-terpinene
6.5
1021
0.75a±0.05
0.76a±0.02
0.86b±0.06
0.79ab±0.06
p-cymene
6.64
1030
3.89a±0.04
4.31b±0.13
-terpinene
7.14
1064
2.77a±0.07
2.51a±0.13
2.54a±0.08
2.49b±0.06
carvacrol
10.67
1308
81.29a±0.25
82.47ab±1.38
82.86ab±1.99
84b±0.32
O-acetylthymol
11.61
1380
0.28a±0.02
0.33a±0.02
0.34ab±0.02
0.41b±0.08
β-humulene
12.38
1440
2.07a±0.05
2.04a±0.04
3.07b±0.08
3.35c±0.13
92.19%
93.59%
95.66%
TOTAL a,b, means
4.83c±0.15
4.1ab±0.45
95.99%
on the same line followed by the same letters are not significantly different (P>0.05).
Table 2. Chemical composition of Mentha pulegium essential oils extracted from unirradiated and irradiated plant at 10, 20 and 30 kGy.
a,b,c means
on the same line followed by the same letters are not significantly different (P>0.05).
Compounds α-pinene β-pinene
Retention Kovats time index (min) 5.28 939 5.95
985
0 kGy
10 kGy
20 kGy
30 kGy
0.37a±0.02
0.42b±0.01
0.35a±0.02
0.36a±0.01
0.20±0.01
0.28±0.03
0.25±0.01
0.20±0.01
3-octanol
6.13
997
0.23 ±0.01
0.33 ±0.01
0.29 ±0.02
0.32c±0.01
limonene
6.70
1035
1.59c±0.01
1.33a±0.06
1.42b±0.04
1.30a±0.04
p-mentha 3,8 diene
7.33
1076
0.10a±0.01
0.23c±0.02
0.14b±0.01
0.16b±0.01
menthone
8.52
1157
1.37a±0.04
2.36c±0.13
1.93b±0.08
1.90b±0.07
isomenthol
8.96
1187
0.43a±0.03
0.66b±0.02
0.69c±0.01
0.70c±0.03
pulegone
9.88
1252
77.16±0.25
79.51±1.05
78.98±0.53
78.03±0.40
piperitone oxide
10.85
1277
1.82c±0.04
0.84b±0.02
0.52a±0.01
0.60a±0.01
piperitenone
11.31
1375
6.54a±0.04
7.97b±0.07
8.06c±0.14
8.16c±0.20
β-humulene
12.38
1440
0.25b±0.01
0.37c±0.02
0.12a±0.02
0.14a±0.02
Germacrene D
12.88
1480
0.55±0.02
0.69±0.01
0.50±0.01
0.55±0.02
90.61
94.99
93.25
92.86
TOTAL
a
c
b
When we increase the irradiation dose, the changes are even more pronounced. Thus, for 20 kGy, tree compounds (α-terpinene, p-cymene and α-humulene) increased in irradiated samples whereas for 30 kGy four compounds (α-terpinene, carvacrol, Oacetylthymol and β-humulene) increased including the main compound (carvacrol). With respect to the effect of gamma irradiation on chemical composition of M. pulegium EOs, we note five main compounds showed in non- irradiated samples and the irradiated sample. When the major compound (pulegone) was not affected by gamma irradiation at any studied dose, the other compounds changed according the intensity of the dose. For 10 kGy, four main compounds (limonene, menthone, piperitone oxide and piperitenone) were modified. Above this dose, four compounds were modified, limonene that decrease from 1.59 to 1.33 % %, Menthone increases from 1.37 to 2.36 %, Piperitone oxide decreases from 1.82 to 0.82 and Piperitenone increases from 6.54 to 7.97 %. Our results agree with those obtained by Machhour et al. (2011). In fact these authors working by Mentha peperita have shown quantitative but not qualitative changes with doses ranging from 0.5 to 2.66 kGy. Antonelli et al. (1998) have also shown that gamma irradiation increased significantly the content of linalool and estragole in the irradiated basil.
On the other hand, our results disagree with dose obtained by Haddad et al. (2007). These authors did not show any effect of gamma irradiation on Thymus, Eucayptus and Lavandula up to 25 kGy. Seo et al. (2007) have also demonstrated that chemical composition of Nigella EOs did not change after irradiation treatment from 1 to 20 kGy. A Similar trend has been observed by Chatterjee et al. (2000) on the volatile oils compounds of Turmeric and by Variyar et al. (1997) on Ginger. Hadda et al. (2007), in an attempt to explain differences obtained between authors, they have concluded that the effects of gamma irradiation on chemical composition of plants extracts depend on irradiation doses, time exposure, plant type and type of extraction. The same authors suggested that the same compound can be affected differently according to the type plant. 3.2. Antimicrobial activity Tables 3 and 4 show the effect of gamma irradiation at 10, 20 and 30 kGy on antimicrobial activity of T. vulgaris and Mentha pulegium EOs respectively. According to these results, T. vulgaris EO showed a strong activity on all tested bacterial strains based on the inhibition diameters obtained between 30.7 and 36.7 mm. The largest antimicrobial activity was observed against L. monocytogenes (36.7 mm) and the weakest against E. coli (30.7 mm). M. pulegium EO showed lower antibacterial activity compared to the EO of T. vulgaris (table 4). The inhibition diameters ranged from 9 mm to 12.7 mm. The largest is obtained with Escherichia coli (12.7 mm) and the smallest with Salmonella Senftenberg (9 mm).
Table 3. Zones of growth inhibition (mm) for Thymus vulgaris EOs extracted from unirradiated and irradiated plants against a selection of Gram-positive and Gram-negative bacteria; disk diameter 6.0 mm. Strain
Growth inhibition diameter (mm)
Escherichia coli Gram -
Salmonella Senftenberg Listeria monocytogenes
Control
10 kGy
20 kGy
30 kGy
34.7a±1.5
37.3b±0.6
38.3b±0.6
38.7b±0.6
34.3a±3.2
37.3ab±2.5
38.0b±0.6
40.3b±0.6
36.7a±3.2
38.0a±1.0
38.3a±3.8
36.3a±1.5
35.0a±1.0
37.3a±0.6
36.3a±3.5
37.0a±3
Gram + Staphylococcus aureus a,b means
on the same line followed by the same letters are not significantly different (P>0.05).
Table 4. Zones of growth inhibition (mm) for Mentha pulegium EOs extracted from unirradiated and irradiated plants against a selection of Gram-positive and Gram-negative bacteria; disk diameter 6.0 mm. Growth inhibition diameter (mm) Strain
Escherichia coli Gram -
Salmonella Senftenberg Listeria monocytogenes
Control
10 kGy
20 kGy
30 kGy
9.3a±0,6
13.3d±0.6
12.0c±1
10.7b±0,6
9.0a±1
11.3b±0,6
11.0b±1
11.7b±0,6
10.3a±0,6
10.7a±1,2
11.0a±1
10.3a±0,6
10.7a±0,6
11.3ab±0,6
12.3bc±0,6
12.7c±0.6
Gram + Staphylococcus aureus a,b means
on the same line followed by the same letters are not significantly different (P>0.05).
Diameters of zone inhibition of EOs obtained from irradiated T. vulgaris (table 3) are ranged from 36.3 and 40.3 mm. Gamma irradiation affected significantly the antimicrobial activity against the two gram negative bacteria studied, while gram positive bacteria was not affected. We also noted that the increase in antimicrobial activity was more pronounced at higher doses. The high sensibility of gram negative bacteria after gamma irradiation could be due to the high level of carvacrol that increases from 81.29 to 84 %. Indeed, antimicrobial activity of carvacrol is well demonstrated (Gutierrez et al. 2008).
EOs extracted from M. pulegium has also been affected by gamma irradiation. In fact, antimicrobial activity increases significantly against gram negative bacteria. Thus, zones of inhibition diameter increase from 9 mm to 12.5 mm. Like in T. vulgaris, gram positive bacteria were more resistant and changes were observed only from the dose of 20 kGy with S. aureus. Studies evaluating the effect of gamma irradiation on antimicrobial activity are very scarce. To the best of our knowledge, this is the first time when the effect of gamma irradiation on the antimicrobial activity is studied against a panel of gram positive and gram negative bacteria. The only works that appear in scientific literature (Khattak, 2012 ) have shown that irradiation did not change the antimicrobial activity of Fagonia arabiga and tee, despite that the maximal dose tested did not exceed 10 kGy. On the other hand, our results agree with those obtained by Khattak and Simpson, 2010 who showed that irradiation up to 25 kGy induce enhancement in antimicrobial acitivity of Glyccerihza glabra extract against Micrococcus luteus. 3.3. Antioxidant activity The effect of irradiation on antioxidant activity is one of the most parameter studied on plants and species. In fact, they are the most important source of antioxidant. The synthetic DPPH radical has been used to study the effect of irradiation on the antioxidant activity of T. vulgaris and M. pulegium essential oils (Figure 1 and 2). While the antioxidant activity increased with concentration increase for M. pulegium, for T. vulgaris this increase was only observed up to 15 uL/mL. Beyond 15μL/mL, it remained constant. As we can see, T. vulgaris EO showed a greater antioxidant activity than M. pulegium EO. Antioxidant activity of T. vulgaris increased with increasing concentration and it was comparable to the antioxidant effect of BHT at a concentration of 15 µL/mL. Our results show that gamma irradiation up to a dose of 30 kGy did not affect the antioxidant activity of T. vulgaris and M. pulegium. These results show that the modifications caused by irradiation up to 30 kGy were not sufficient to induce a modification in the antimicrobial activity of M. pulegium and T. vulgaris essential oils. Our results agree with those obtained by Pérez et al. (2011) who showed that irradiation up to a dose of 30 kGy did not change antioxidant activity of methanolic extracts of Sage and Oregane. On the contrary, they differ as to what Pérez et al. (2007) and Khattak et al, (2008) reached. Khattak et al. (2008), have demonstrated that the effect of irradiation on the antioxidant activity
depend on the nature of the solvent used in the extraction. In fact, antioxidant activity increase in methanolic and acetone extracts. However, it does not change from aqueous extract. Pérez et al. (2007), have observed that irradiation improve antioxidant activity of methanolic and ethanolic activities. In the same way, Adamo et al. (2004) explain why antioxidant activity increases after irradiation. The increased antioxidant activity may be due to the degradation of molecules to other phenolic molecules or because of the changes in the conformation of molecules that contribute in antioxidant activity. On the other hand, Oufedjikh et al. (2000) explained that this augmentation is due to the enzymatic activity of de phenylalanine ammonia-lyase (PAL) after the irradiation.
Figure 1. Scavenging activity of Thymus vulgaris essential oil extracted from unirradiated and irradiated plant at 10, 20 and 30 kGy.
Figure 2. Scavenging activity of Mentha pulegium essential oil extracted from unirradiated and irradiated plant at 10, 20 and 30 kGy. 4. Conclusion The study of the effect of irradiation on the chemical composition, and biological activities of plants and species is decisive before adopting this decontamination technique. During this study conducted on T. vulgaris and M. pulegium up to 30 kGy, we have shown that the modifications observed in the chemical composition of the two plants on which the research is carried out allow in the extreme cases either to maintain or improve antimicrobial and antioxidant activity of the two studied AMPs. In fact, the improvement of biologic properties of essential oils of AMPs after irradiation would only reinforce the possibility of using this technique in decontamination of AMPs Other studies are necessary to conduct a research on the effect of gamma irradiation on the EOs antifungal properties. To conclude, it can be said that there is not a universal effect of irradiation on plants. Effects depend on the dose, plant, the nature of the extract, type of microorganism. This means that for each plant we can conduct an isolated study. However, according to the present available information in the international literature, effects of irradiation are positive, as in all cases, do not expose the biological activities of the plant to risk.
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Kume, T., Furata, M., Todoriki, S., Uenoyama, N., Kobayashi, Y., 2009. Status of food irradiation in the world. Radiat Phy Chem. 78 (3), 222-226. Lacroix, M., Ouattara, B., 2000. Combined industrial process with irradiation to assure innocuity and preservation of food products – A rewiew. Food Res Int. 33, 719-724. Legnani, P.P., Leoni, E., Righi, F., Zarabini, L.A., 2001. Effect of microwave heating and gamma irradiation on microbiological quality of spices and herbs. Italian J Food Sci. 3, 337-345. Loaharanu, P., 1990. Role of irradiation to facilitate international trade in pepper. In: Paper presented at the 15th Peppertech and 2nd Pepperexim Meet. Convened by the international Pepper community (IPC), London, UK, 21-25 May, p. 19. Machhour, H., El Hadrami, I., Imziln, B., Mouhib, M., Mahrouz, M., 2011. Microbial decontamination by low dose gamma irradiation and its impact on the physicochemical quality of peppermint (Mentha piperita). Radiat Phy Chem. 80, 604-607. Machhour, H., Mahrouz, M., Imziln, B., Hadra, I., Idliman, A., 2008. Décontamination de la menthe poivrée par un traitement combiné thermochimique. Revue des énergies renouvelables. 203-206. Mclafferty, F.W., Stauffer, D.B., 1989. Registry of mass spectral data. 5th ed. Wiley: New York, p. 453. Oufedjikh, H., Mahrouz, M., Aliot, M.J., Lacroix, M., 2000. Effect of gamma irradiation on phenolic compounds and phenylalanine ammonia-lyase activity during storage in relation to peel injury from peel of Citrus clementina Hort. Ex. Tanaka. J Agr Food Chem. 48 (2), 559-565. Pérez, M.B., Banek, S.A. Croci, C.A., 2011. Retention of antioxydant activity in gamma irradiated argentinian sage and oregano. Food Chem. 126, 121-126. Pérez, M.B., Calderon, N.L., Croci, C.A., 2007. Radiation-induced enhancement of antioxydant activity in extracts of rosemary (Rosmarinus officinalis L.). Food Chem. 104, 585-592. Pezzutti, A., Marucci, P.L. Sica, M.G., Matzkin, M.R., Croci, C.A., 2005. Gamma-ray sanitization of Argentinian dehydrated garlic (Allium sativum L) and onion (Allium cepa L) products. Food Res Int. 38 (7), 797-802. Raso, J., Barbosa-Canovas, G. V., 2003. Nonthermal preservation of foods using combined processing techniques. Crit Rev Food Sci Nutr. 43 (3), 265-285. Seo, H.Y., Kim, J.H., Song, H.P., Kim, D.H., Byun, M.W., Kwon, J.H., Kim, K.S., 2007. Effects of gamma irradiation on the yields of volatile extracts of Angelica gigas Nakai. Radiat Phy Chem. 76, 1869-1874. Variyar, P.S., Gholap, A.S., Thomas, P., 1997. Effect of gamma irradiation on the volatile oil constituents of fresh ginger (Zingeber officinalis) rhizome. Food Res Int. 30, 41-43.
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highlights The irradiation affects quantitatively the chemical composition of EO of AMPs; The irradiation affected significantly the antimicrobial activity; The antimicrobial activity was more pronounced at higher doses for gram -; The irradiation up to a dose of 30 kGy did not affect the antioxidant activities; The irradiation at sterilising doses did not compromise the biological activities.
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S0969-806X(15)00187-5 http://dx.doi.org/10.1016/j.radphyschem.2015.05.019 RPC6816
To appear in: Radiation Physics and Chemistry Received date: 25 February 2015 Revised date: 27 April 2015 Accepted date: 9 May 2015 Cite this article as: Said Zantar, Rachid Haouzi, Mohammed Chabbi, Amin Laglaoui, Mohammed Mouhib, Boujnah Mohammed, Mohammed Bakkali and Mounir Hassani Zerrouk, Effect Of Gamma Irradiation On Chemical Composition,Antimicrobial And Antioxidant Activities Of Thymus Vulgarisand Mentha Pulegium Essential Oils, Radiation Physics and Chemistry, http://dx.doi.org/10.1016/j.radphyschem.2015.05.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
EFFECT OF GAMMA IRRADIATION ON CHEMICAL COMPOSITION, ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES OF THYMUS VULGARIS AND MENTHA PULEGIUM ESSENTIAL OILS AUTHORS: Said Zantara*, Rachid Haouzib, Mohammed Chabbib, Amin Laglaouid, Mohammed Mouhiba, Boujnah Mohammeda , Mohammed Bakkalid, Mounir Hassani Zerrouke a Irradiation
plant of Tangier, National Institute of Agronomic Research (INRA), Tangier, Morocco. b Departement of Chemistry, Faculty of Science and Technology, Tangier, Morocco. d Departement of Biology, Faculty of Science and Technology, Tangier, Morocco. e Departement of Biology, Polydisciplinary Faculty of Larache, Morocco.
*Corresponding author: Said Zantar Address: Centre Régional de la Recherche Agronomique de Tanger.78 Avenue Sidi. Mohamed Ben Abdellah, Tanger 90010 Morocco. Phone number: +212 539938033 Fax Number: +212 539394523 E-mail: [email protected] Abstract: The effects of gamma irradiation doses (10, 20 and 30 KGy) on chemical composition, antimicrobial and antioxidant activities of T. vulgaris and M. pulegium essential oils (EOs) have been studied. The chromatographic analysis showed that the studied EOs were constituted mainly by carvacrol for T. vulgaris and pulegone for M. pulegium. Gamma irradiation on the studied doses, affects quantitatively and not qualitatively some components of the investigated oils. This effect was dose dependent. While the antioxidant activity remains stable at any dose applied for the plants studied, the antimicrobial activity increased in the irradiated samples for gram negative bacteria and did not change for gram + bacteria. This study supports that gamma irradiation employed at sterilising doses did not compromise the biological activities of medicinal and aromatic plants. Keywords: essential oils, gamma radiation, chemical composition, antimicrobial activity, antioxidant activity
1. INTRODUCTION The importance of aromatic and medicinal plants (AMPs) and their extracts have been demonstrated all over the world for years. In fact, they are used for their nutraceutical, therapeutic, and cosmetic benefits. For this reason, they are subject to a high demand. In a globalized context and free trade, hygienic quality is primordial for a large commercialization of these products. Like foods, AMPs are susceptible to be
contaminated during their collection, treatment, transport, and storage by a several number of pathogen microorganisms and chemical contaminants like pesticides, heavy metals and dioxins (Machhour et al., 2008; Legnani et al., 2001; Idu et al., 2008). According to the World Health Organization, the numbers of intoxications are due to an incorrect utilization of medicinal plants which are in continuous increase (WHO, 2003). Logically, the situation is more alarming in developing countries. The Anti-poison and pharmacology Centre of Morocco, published that in 2012, 12 persons died due to an improper use of medicinal plants. One of the more important intoxication causes is the plant hygienic states that contain habitually a high number of microorganisms (Abu-dounia, 2008). The problem is more dramatic in the sense that the medicinal plants are in many cases used craw without any pretreatment. (Beuchat, 1996). In such a situation, the conformity of the international standards in terms of hygiene and safety is crucial for a wide commercialization of these plants. In addition to the good manufacturing practices, establishment of techniques for a good decontamination of AMPs is of a big importance. Two techniques of plants decontamination are actually used. The thermal processes like steam and ohmic heating and non-thermal processes like fumigation by ethylene oxide and high hydrostatic pressure (Machhour et al. 2008). When the thermal processes are destructive and affect the quality of plants, fumigation uses chemical products that are in most cases dangerous for public health (Machhour et al., 2008; Loaharanu et al., 1990). Another non-thermal technique decontamination that has proved its efficacy for microbial inactivation is irradiation (Raso and Barbosa-Canobvas, 2003). This technique, which is widely used for food preservation, has been proposed for plants decontamination by several authors (Pezzutti et al., 2005; Kume et al., 2009). Although irradiation efficacy for microbial inactivation has been widely demonstrated (Lacroix and Ouattara, 2000 ; Hong et al., 2008), the effects on chemical composition and biological activities of medicinal plants raised the interest of several authors in last past years (Khattak et al., 2008; Khattak, 2012; Pérez et al., 2011). From these works it can be concluded that biological activities of medicinal plants are affected by gamma irradiation which can be modified depending on several factors like plants species, irradiation doses, time of exposition and the solvent used.
Despite that the fact that the effect of gamma irradiation on the biological activities of aqueous and organic extracts has been already studied, surprisingly, the effect on essential oils has not been well studied while they represent one of the most important fraction in medicinal plants. In food and pharmaceutical sector, essential oils are used for their antimicrobial and antioxidant activities (Burt, 2004). Preservation of these activities is very important to design and validate the decontamination process of a plant. T. vulgaris and M. pulegium are two spontaneous AMPs belonging to the Lamiaceae family that grow in several regions of Morocco. They have an undeniable commercial interest, too. However, to the best of our knowledge, there are no studies on scientific literature that evaluate the effect of gamma irradiation on chemical composition, and biological activities of their essential oils. The main objective of this work is to evaluate the effect of gamma irradiation at very high doses on the chemical composition, antioxidant and antimicrobial activity of T. vulgaris and M. pulegium essential oils.
2. MATERIALS AND METHODS 2.1. Plant material The studied plants (T. vulgaris and M. Pulegium) were collected during the period of March 2011 to June 2011. T. vulgaris was harvested in the region of Beni Idder in the Northeast of Morocco and M. pulegium from Bougedour in the North. The plants were dried from 24 to 48 hours at a temperature of 40°C under ventilation. Leaves and flowers were used for the extraction of EOs. 2.2. Irradiation
For gamma irradiation, the dried leaves and flowers (100 g) were packaged in plastic bags and then exposed to gamma irradiation for 10, 20 and 30 kGy at ambient temperature (20°C) in the Boukhalef Ionization Plant (Tangier, Morocco), using a cobalt 60 radio-isotopic source . A dosimetry work has been done using samples in same packaging and position to have dose rate and dose uniformity. For this we used Gamma Chrome dosimeter batch YR3. The read out of result has been done using a spectrophotometer under 530 nm Wavelength after 2 hours for stabilization of dosimeters. Five dosimeters were used in bags of product to determine dose rate and
doses Uniformity. We have got a minimum dose rate of 1.17 Gy/min and dose uniformity of 1.06. Using this result we irradiated specified samples to 10, 20 and 30 kGy according the time of irradiation issue to dosimetry result and corrected with daily decay factor of cobalt60. Dosimeters Amber batch 3042K for 10 kGy and red Perspex batch 4034CV for 20 and 30 kGy were used for control in order to be in the best result of calibration curve of dosimeters and have maximum precision to be within 3% given in calibration curve. After irradiation, essential oils were immediately extracted and stored at -18°C until their use. 2.3. Essential oils extraction EOs extraction was carried out by steam distillation for the two plants. 100 g of dried leaves and flowers of T. vulgaris (50 g for M. pulegium) were introduced into flask with distilled water; the mixture was boiled for 3 hours. 2.4.
Essential oils analysis
Gas chromatography/mass spectrometry (GC/MS) analysis: The GC/MS analyses were carried out using a gas chromatograph coupled with Mass Spectrophotometer (MS) (GC-MS Trace GC ultra - ITQ900, Thermo Scientific, USA) operating in electron-impact (70 eV, m/z 40-450) mode. The capillary column used was 1MS (30 m x 0.25 mm x 0.25 µm film thickness). Analytical conditions were: injector and transfer line temperatures 250 and 300°C, respectively, oven temperature programmed from 50 to 200°C at 10°C/min and from 200 to 290°C at 35°C/min, carrier gas helium, at 1mL/min, injection of 1 µl (10% hexane solution), the split ratio was 1:20. The identification of the EOs compounds was performed by comparing their mass spectra with data bank (NIST MS search V.2.0) and homemade library mass spectra built up from pure substances and components of known essential oils and MS literature data (Mclafferty and Stauffer, 1989; Adams, 2001), and by coinjection with an authentic sample. Also the constituents of essential oils were identified based on their Kovats Index, calculated in relation to the retention time of a series of alkanes (C4- C28) as reference products, in comparison with those of the chemical compounds gathered by Adams Table (Adams, 2001).
2.5.
Antimicrobial activity
Antimicrobial activity was determined by paper disc agar plates. Microbial strains used were provided by the Spanish Collection of Type Cultures (CECT). Two Gram negative bacteria Escherichia coli (SCTC 471), Salmonella Senftenberg 775W (ATCC 43845) and two Gram-positive Listeria monocytogenes (SCTC 4031), Staphylococcus aureus (SCTC 976) were used. A volume of 10 mL of Muller Hinton Agar medium (Liofilchem, Roseto degli Abruzzi, Italy) (MHA) was poured into Petri plate. 100 μL of culture bacteria are plated at approximately final concentration of 106 bacteria/mL. After 15 min, a paper disc Whatman No. 1 of 6 mm (Whatman International L7d Maidstone, England), impregnated with 10μl of the EO was put to the surface of the agar. The plates were incubated in an incubator at 37°C for 24h and the inhibition diameter was measured. 2.6.
Antioxydant activity
DPPH method: 1 mL of a solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (sigma Aldrich INC .P.O box14508 St Louis, MO 63178 USA) radical at 1 mmol/L in ethanol was transferred into tubes containing 3 mL of the diluted EO in ethanol. After agitation, the tubes were placed in the dark in a temperature room for 30 min. For each concentration, the tests were repeated three times. The negative control was composed by 1 mL of ethanol DPPH solution and 3 mL of ethanol. The positive control was represented by a standard solution of an antioxidant butylated hydroxytoluene (BHT) (Fluka, Sigma-Aldrich Chemie GmbH, CH-9471 Buchs, Spain). Reading is performed by measuring the absorbance at 517 nm by a spectrophotometer. Results expression The antioxidant activity "AA%" is given by the following formula: AA% = [(Ac –Ap) /Ac] x 100 Ac: absorbance of the control reaction (containing all reagents except the tested essential oil).
Ap: absorbance with essential oil. 2.7.
Statistical analysis
Analyses of variance were performed by Statgraphics Plus 4.0 statistical software. All experiments were performed in triplicate. The results were presented as the mean with its standard deviation (n = 3) in each case. Differences were considered significant at P <0.05. 3. RESULTS AND DISCUSSION Although gamma irradiation has proved its efficacy for microbial inactivation in plants and species, the study of side effects is actually the goal of many works. In this paper, we have decided to investigate the effect of high irradiation doses (until 30 kGy) on the chemical composition, antimicrobial and antioxidant activities of essential oils from two AMPs widely used in food and pharmaceutical sectors (T. vulgaris and M. pulegium). Most studies that have evaluated the effect of gamma irradiation on medicinal plants and species were conducted on aqueous or organic extracts when essential oils are one of the most important fractions in plants. 3.1. Chemical composition The study of the effect of gamma irradiation on chemical composition of T. vulgaris and M. pulegium has been carried out, firstly, because the biological activities of plants extract are essentially due to their wealth in chemical compounds and, secondly, to understand the results obtained in antimicrobial and antioxidant posteriorly. The effect of gamma irradiation at 10, 20 and 30 kGy doses on chemical composition of T. vulgaris and M. pulegium essential oils have been analyzed by GC/MS chromatography. Briefly, chromatographic analysis has highlighted five main components for T. vulgaris that represent more than 1% levels. Carvacrol was the major component with a content ranging from 78.33 to 81.49% accompanied with other components at relatively low levels: -Terpinene (0.7 to 0.8%), p-cymene (3.9 to 6.7%), δ-terpinene (2.8 to 4.0 %), β-humulene (2.0 to 2.5%). With respect to M. pulegium EO, four major compounds were found. Pulegone was the major constituent with a content of 80 %, accompanied by small amounts of limonene (1.3 to 1.5 %),
menthone (1.3 to 2.3 %) and periperitone (6,5 to 8.16 %). Tables 1 and 2 show that gamma irradiation process affects quantitatively but not qualitatively the chemical composition of T. vulgaris and M. pulegium EOs. The effect was dose dependent. With respect to T. vulgaris (table 1), gamma irradiation with 10 kGy, caused the increase in concentration of one component (p-cymene) that raise from 3.89 to 4.31 %, the content of other compounds was similar to the control. Table 1. Chemical composition of Thymus vulgaris essential oils extracted from unirradiated and irradiated plant at 10, 20 and 30 kGy. Retention
Kovats
time (min)
Index
α-thujene
5.16
α-pinene
Compounds
0 kGy
10 kGy
20 kGy
30 kGy
930
0.82b±0.09
0.82b±0.08
0.78b±0.06
0.55a±0.06
5.27
939
0.32±0.04
0.35±0.08
0.38±0.04
0.3±0.02
δ-terpinene
6.5
1021
0.75a±0.05
0.76a±0.02
0.86b±0.06
0.79ab±0.06
p-cymene
6.64
1030
3.89a±0.04
4.31b±0.13
-terpinene
7.14
1064
2.77a±0.07
2.51a±0.13
2.54a±0.08
2.49b±0.06
carvacrol
10.67
1308
81.29a±0.25
82.47ab±1.38
82.86ab±1.99
84b±0.32
O-acetylthymol
11.61
1380
0.28a±0.02
0.33a±0.02
0.34ab±0.02
0.41b±0.08
β-humulene
12.38
1440
2.07a±0.05
2.04a±0.04
3.07b±0.08
3.35c±0.13
92.19%
93.59%
95.66%
TOTAL a,b, means
4.83c±0.15
4.1ab±0.45
95.99%
on the same line followed by the same letters are not significantly different (P>0.05).
Table 2. Chemical composition of Mentha pulegium essential oils extracted from unirradiated and irradiated plant at 10, 20 and 30 kGy.
a,b,c means
on the same line followed by the same letters are not significantly different (P>0.05).
Compounds α-pinene β-pinene
Retention Kovats time index (min) 5.28 939 5.95
985
0 kGy
10 kGy
20 kGy
30 kGy
0.37a±0.02
0.42b±0.01
0.35a±0.02
0.36a±0.01
0.20±0.01
0.28±0.03
0.25±0.01
0.20±0.01
3-octanol
6.13
997
0.23 ±0.01
0.33 ±0.01
0.29 ±0.02
0.32c±0.01
limonene
6.70
1035
1.59c±0.01
1.33a±0.06
1.42b±0.04
1.30a±0.04
p-mentha 3,8 diene
7.33
1076
0.10a±0.01
0.23c±0.02
0.14b±0.01
0.16b±0.01
menthone
8.52
1157
1.37a±0.04
2.36c±0.13
1.93b±0.08
1.90b±0.07
isomenthol
8.96
1187
0.43a±0.03
0.66b±0.02
0.69c±0.01
0.70c±0.03
pulegone
9.88
1252
77.16±0.25
79.51±1.05
78.98±0.53
78.03±0.40
piperitone oxide
10.85
1277
1.82c±0.04
0.84b±0.02
0.52a±0.01
0.60a±0.01
piperitenone
11.31
1375
6.54a±0.04
7.97b±0.07
8.06c±0.14
8.16c±0.20
β-humulene
12.38
1440
0.25b±0.01
0.37c±0.02
0.12a±0.02
0.14a±0.02
Germacrene D
12.88
1480
0.55±0.02
0.69±0.01
0.50±0.01
0.55±0.02
90.61
94.99
93.25
92.86
TOTAL
a
c
b
When we increase the irradiation dose, the changes are even more pronounced. Thus, for 20 kGy, tree compounds (α-terpinene, p-cymene and α-humulene) increased in irradiated samples whereas for 30 kGy four compounds (α-terpinene, carvacrol, Oacetylthymol and β-humulene) increased including the main compound (carvacrol). With respect to the effect of gamma irradiation on chemical composition of M. pulegium EOs, we note five main compounds showed in non- irradiated samples and the irradiated sample. When the major compound (pulegone) was not affected by gamma irradiation at any studied dose, the other compounds changed according the intensity of the dose. For 10 kGy, four main compounds (limonene, menthone, piperitone oxide and piperitenone) were modified. Above this dose, four compounds were modified, limonene that decrease from 1.59 to 1.33 % %, Menthone increases from 1.37 to 2.36 %, Piperitone oxide decreases from 1.82 to 0.82 and Piperitenone increases from 6.54 to 7.97 %. Our results agree with those obtained by Machhour et al. (2011). In fact these authors working by Mentha peperita have shown quantitative but not qualitative changes with doses ranging from 0.5 to 2.66 kGy. Antonelli et al. (1998) have also shown that gamma irradiation increased significantly the content of linalool and estragole in the irradiated basil.
On the other hand, our results disagree with dose obtained by Haddad et al. (2007). These authors did not show any effect of gamma irradiation on Thymus, Eucayptus and Lavandula up to 25 kGy. Seo et al. (2007) have also demonstrated that chemical composition of Nigella EOs did not change after irradiation treatment from 1 to 20 kGy. A Similar trend has been observed by Chatterjee et al. (2000) on the volatile oils compounds of Turmeric and by Variyar et al. (1997) on Ginger. Hadda et al. (2007), in an attempt to explain differences obtained between authors, they have concluded that the effects of gamma irradiation on chemical composition of plants extracts depend on irradiation doses, time exposure, plant type and type of extraction. The same authors suggested that the same compound can be affected differently according to the type plant. 3.2. Antimicrobial activity Tables 3 and 4 show the effect of gamma irradiation at 10, 20 and 30 kGy on antimicrobial activity of T. vulgaris and Mentha pulegium EOs respectively. According to these results, T. vulgaris EO showed a strong activity on all tested bacterial strains based on the inhibition diameters obtained between 30.7 and 36.7 mm. The largest antimicrobial activity was observed against L. monocytogenes (36.7 mm) and the weakest against E. coli (30.7 mm). M. pulegium EO showed lower antibacterial activity compared to the EO of T. vulgaris (table 4). The inhibition diameters ranged from 9 mm to 12.7 mm. The largest is obtained with Escherichia coli (12.7 mm) and the smallest with Salmonella Senftenberg (9 mm).
Table 3. Zones of growth inhibition (mm) for Thymus vulgaris EOs extracted from unirradiated and irradiated plants against a selection of Gram-positive and Gram-negative bacteria; disk diameter 6.0 mm. Strain
Growth inhibition diameter (mm)
Escherichia coli Gram -
Salmonella Senftenberg Listeria monocytogenes
Control
10 kGy
20 kGy
30 kGy
34.7a±1.5
37.3b±0.6
38.3b±0.6
38.7b±0.6
34.3a±3.2
37.3ab±2.5
38.0b±0.6
40.3b±0.6
36.7a±3.2
38.0a±1.0
38.3a±3.8
36.3a±1.5
35.0a±1.0
37.3a±0.6
36.3a±3.5
37.0a±3
Gram + Staphylococcus aureus a,b means
on the same line followed by the same letters are not significantly different (P>0.05).
Table 4. Zones of growth inhibition (mm) for Mentha pulegium EOs extracted from unirradiated and irradiated plants against a selection of Gram-positive and Gram-negative bacteria; disk diameter 6.0 mm. Growth inhibition diameter (mm) Strain
Escherichia coli Gram -
Salmonella Senftenberg Listeria monocytogenes
Control
10 kGy
20 kGy
30 kGy
9.3a±0,6
13.3d±0.6
12.0c±1
10.7b±0,6
9.0a±1
11.3b±0,6
11.0b±1
11.7b±0,6
10.3a±0,6
10.7a±1,2
11.0a±1
10.3a±0,6
10.7a±0,6
11.3ab±0,6
12.3bc±0,6
12.7c±0.6
Gram + Staphylococcus aureus a,b means
on the same line followed by the same letters are not significantly different (P>0.05).
Diameters of zone inhibition of EOs obtained from irradiated T. vulgaris (table 3) are ranged from 36.3 and 40.3 mm. Gamma irradiation affected significantly the antimicrobial activity against the two gram negative bacteria studied, while gram positive bacteria was not affected. We also noted that the increase in antimicrobial activity was more pronounced at higher doses. The high sensibility of gram negative bacteria after gamma irradiation could be due to the high level of carvacrol that increases from 81.29 to 84 %. Indeed, antimicrobial activity of carvacrol is well demonstrated (Gutierrez et al. 2008).
EOs extracted from M. pulegium has also been affected by gamma irradiation. In fact, antimicrobial activity increases significantly against gram negative bacteria. Thus, zones of inhibition diameter increase from 9 mm to 12.5 mm. Like in T. vulgaris, gram positive bacteria were more resistant and changes were observed only from the dose of 20 kGy with S. aureus. Studies evaluating the effect of gamma irradiation on antimicrobial activity are very scarce. To the best of our knowledge, this is the first time when the effect of gamma irradiation on the antimicrobial activity is studied against a panel of gram positive and gram negative bacteria. The only works that appear in scientific literature (Khattak, 2012 ) have shown that irradiation did not change the antimicrobial activity of Fagonia arabiga and tee, despite that the maximal dose tested did not exceed 10 kGy. On the other hand, our results agree with those obtained by Khattak and Simpson, 2010 who showed that irradiation up to 25 kGy induce enhancement in antimicrobial acitivity of Glyccerihza glabra extract against Micrococcus luteus. 3.3. Antioxidant activity The effect of irradiation on antioxidant activity is one of the most parameter studied on plants and species. In fact, they are the most important source of antioxidant. The synthetic DPPH radical has been used to study the effect of irradiation on the antioxidant activity of T. vulgaris and M. pulegium essential oils (Figure 1 and 2). While the antioxidant activity increased with concentration increase for M. pulegium, for T. vulgaris this increase was only observed up to 15 uL/mL. Beyond 15μL/mL, it remained constant. As we can see, T. vulgaris EO showed a greater antioxidant activity than M. pulegium EO. Antioxidant activity of T. vulgaris increased with increasing concentration and it was comparable to the antioxidant effect of BHT at a concentration of 15 µL/mL. Our results show that gamma irradiation up to a dose of 30 kGy did not affect the antioxidant activity of T. vulgaris and M. pulegium. These results show that the modifications caused by irradiation up to 30 kGy were not sufficient to induce a modification in the antimicrobial activity of M. pulegium and T. vulgaris essential oils. Our results agree with those obtained by Pérez et al. (2011) who showed that irradiation up to a dose of 30 kGy did not change antioxidant activity of methanolic extracts of Sage and Oregane. On the contrary, they differ as to what Pérez et al. (2007) and Khattak et al, (2008) reached. Khattak et al. (2008), have demonstrated that the effect of irradiation on the antioxidant activity
depend on the nature of the solvent used in the extraction. In fact, antioxidant activity increase in methanolic and acetone extracts. However, it does not change from aqueous extract. Pérez et al. (2007), have observed that irradiation improve antioxidant activity of methanolic and ethanolic activities. In the same way, Adamo et al. (2004) explain why antioxidant activity increases after irradiation. The increased antioxidant activity may be due to the degradation of molecules to other phenolic molecules or because of the changes in the conformation of molecules that contribute in antioxidant activity. On the other hand, Oufedjikh et al. (2000) explained that this augmentation is due to the enzymatic activity of de phenylalanine ammonia-lyase (PAL) after the irradiation.
Figure 1. Scavenging activity of Thymus vulgaris essential oil extracted from unirradiated and irradiated plant at 10, 20 and 30 kGy.
Figure 2. Scavenging activity of Mentha pulegium essential oil extracted from unirradiated and irradiated plant at 10, 20 and 30 kGy. 4. Conclusion The study of the effect of irradiation on the chemical composition, and biological activities of plants and species is decisive before adopting this decontamination technique. During this study conducted on T. vulgaris and M. pulegium up to 30 kGy, we have shown that the modifications observed in the chemical composition of the two plants on which the research is carried out allow in the extreme cases either to maintain or improve antimicrobial and antioxidant activity of the two studied AMPs. In fact, the improvement of biologic properties of essential oils of AMPs after irradiation would only reinforce the possibility of using this technique in decontamination of AMPs Other studies are necessary to conduct a research on the effect of gamma irradiation on the EOs antifungal properties. To conclude, it can be said that there is not a universal effect of irradiation on plants. Effects depend on the dose, plant, the nature of the extract, type of microorganism. This means that for each plant we can conduct an isolated study. However, according to the present available information in the international literature, effects of irradiation are positive, as in all cases, do not expose the biological activities of the plant to risk.
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highlights The irradiation affects quantitatively the chemical composition of EO of AMPs; The irradiation affected significantly the antimicrobial activity; The antimicrobial activity was more pronounced at higher doses for gram -; The irradiation up to a dose of 30 kGy did not affect the antioxidant activities; The irradiation at sterilising doses did not compromise the biological activities.