Monia Ennajar

Jalloul Bouajila

Ahmed Lebrihi

Florence Mathieu

Manef Abderraba

Aly Raies

Mehrez Romdhane

Chemical Composition and Antimicrobial and Antioxidant Activities of Essential Oils and Various Extracts of Juniperus phoenicea L. (Cupressacees)

Keywords: antimicrobial activity, antioxidant activity, essential oil, GC-MS, Juniperus phoenicea L. (cupressacees)

GC-FID and GC-MS analysis of essential oils of Juniperus phoenicea resulted in the identification of 30 compounds, representing more than 98% of the total composition. α-pinene (55.7% and 80.7%), δ-3-carene (10.7% and 4.5%), and γ -cadinene (2.9% and 5.1%) were the main components, respectively, in leaves and berries essential oil. Extracts of J. phoenicea were obtained by different extraction solvents: methanol, ethanol, ethyl acetate, and dichloromethane and evaluated composition for polyphenols (gallic acid equivalent 52 to 217 g/kg), tannins (catechin equivalent 6.5 to 60.2 g/kg), antocyanins (cyanidin equivalent 84 to 373 mg/kg), and flavonoids (quercetin equivalent 6.4 to 29.3 g/kg). The samples (essential oils and extracts) were subjected to a screening for their antioxidant activity by using DPPH and ABTS assays; antimicrobial activity was tested with 6 bacteria (3 Gram-positive and 3 Gram-negative), 1 yeast, and 2 fungi. The strongest antioxidant activity was obtained by the methanolic extract (IC 50 = 6.5 ± 0.3 mg/L). Flavonoids are likely to contribute to the antifungal activity against Saccharomyces cerevisiae. Correlations were studied between chemical composition and antioxidant and antimicrobial activities.

Introduction

P hytochemicals are used as natural antimicrobial agents or "biocides" [START_REF] Smid | Natural antimicrobials for food preservation handbook of food preservation[END_REF]. There is growing interest in correlating phytochemical constituents of plant with its pharmacological activity [START_REF] Vaidya | New drugs from medicinal plants, opportunities and approaches[END_REF]. The antimicrobial and antioxidant properties of essential oils have been recognized for many years, and their preparations have found applications as naturally occurring antimicrobial and antioxidant agents in the field of pharmacology, pharmaceutical botany, phytopathology, medical, and clinical microbiology, food preservation, and so on. The essential oil and various extracts preparations that possess antimicrobial and/or antioxidant activities have been the subject of many investigations resulting in the screening of a wide variety of plant species, and have revealed structurally unique biologically active compounds. However, less attention was paid to the activity of their main components in essential oils tested. The main advantage of new natural agents is that they do not present the "antibiotic resistance," a phenomenon commonly encountered with the long-term use of antibiotics. The antimicrobial and antioxidant activities of essential oils are assigned to a number of small terpenoids and phenolic compounds (thymol, carvacrol, eugenol), which also in pure form demonstrate high antibacterial and antioxidant activities [START_REF] Conner | Naturally occurring compounds[END_REF]. For extracts, different solvent systems have been used for the extraction of polyphenols from plant material (Hernandez and others 2007). Extraction yield is dependent on the solvent and the method of extraction (Hayouni and others 2007).

Juniperus phoenicea L. (Cupressaceae) is a shrub or a small tree from the Mediterranean region [START_REF] Bonnier | La Grande Flore[END_REF]. The species present a very large geographical distribution ranging from Portugal to Saudi Arabia (Meloni and others 2006). It is also native to North Africa as well as the Canary Islands [START_REF] Adams | Junipers of the World: The Genus Juniperus[END_REF]Louriero and others 2007). This wide geographical range of the species makes it possible for a vast amount of genetic variability (Meloni and others 2006). In fact, many researchers reported the presence of intraspecific taxa based upon morphological, biochemical [START_REF] Lebreton | Sur une sous-espèce du genévrier de phénicie Juniperus phoenicea L définie à partir de critères biochimiques[END_REF], and molecular data (Le-Breton and Thivend 1981; Adams and others 2002;[START_REF] Adams | Junipers of the World: The Genus Juniperus[END_REF]Meloni and others 2006;Louriero and others 2007). Recently, on the basis of random amplified polymorphics DNAs fingerprinting, Adams and others (2006) have distinguished between J. phoenicea var phoenicea and J. phoenicea var. turbinata and affiliated populations.

The genus Juniperus is considered as an important medicinal plant largely used in traditional medicine. Its leaves are used in the form of decoction to treat diabetes, diarrhea, and rheumatism [START_REF] Bellakhder | La Pharmacopée Marocaine traditionnelle[END_REF]. The mixture of leaves and berries of this plant is used as an oral hypoglycaemic agent (Amer and others 1994), whereas the leaves are used against bronco-pulmonary disease and as a diuretic [START_REF] Bellakhder | La Pharmacopée Marocaine traditionnelle[END_REF].

Considering the medicinal interest of the species, several studies related to the chemical characterization of its essential oil were performed. However, these studies were related to only J. phoenicea grown in north Mediterranean basin (Rezzi and others 2001). On the J. phoenicea grown in south Mediterranean basin, a little information is available on its chemical composition (essential oils and extracts) and its infraspecific variation. In the literature, many papers report on the composition of the essential oil from berries and leaves of the Juniperus species (Guerra Hernandez and others 1987;Mastelic and others 2000) but few studies have investigated their antimicrobial activity (Stassi and others 1996;Angioni and others 2003). In fact, this study is the first to show the antioxidant activity of leaves and berries J. phoenicea essential oils from the south of Mediterranean basin.

Therefore, the objectives of this study were to (1) analyze the chemical composition of essential oils by a GC-FID and GC-MS system to determine essential oils chemotype of J. phoenicea plants collected from the south eastern region of Tunisia, (2) characterize polyphenols, tannins, flavonoids, and anthocyannins of their extracts, (3) investigate antimicrobial and antioxidant activities of essential oils and various extracts, and (4) the study of possible correlations between chemical compositions and antimicrobial and antioxidant activities.

Materials and Methods

Chemicals

All chemicals used were of analytical reagent grade. All reagents were purchased from Sigma-Aldrich-Fluka (Saint-Quentin, France): Sigma (sodium acetate

Collection of plant material

The leaves and berries of J. phoenicea were collected in October 2007 from the southeastern area of Tunisia, precisely in the surroundings of "Matmata" localized in 600 m of altitude, 37

Isolation of essential oils

A portion (100 g) of the dried aerial parts of J. phoenicea (leaves and berries) was submitted for 3 h to water distillation, using a clevenger-type apparatus according to the method recommended by the European Pharmacopoeia (1983). Yields are shown in Table 1. The obtained essential oils were dried by anhydrous sodium sulfate and after filtration stored at 4 • C until tested and analyzed.

Preparation of juniper extracts

The air-dried and finely ground leaves and berries (2.5 g) were extracted by stirring with 25 mL of solvent (methanol, ethanol, dichloromethane, or ethyl acetate) at room temperature for 30 min. Extracts were kept for 24 h at 4 • C then filtered through Whattman Millipore Filter paper (GF/A, 110 mm, 8 µm) and evaporated under vacuum. Yields are shown in Table 1.

Gas chromatography and gas chromatography-mass spectrometry

Quantitative and qualitative analysis of essential oils was carried out by gas chromatography-flame ionization detection (GC-FID) and gas chromatography/mass spectrometry (GC-MS). Gas chromatography analyses were carried out on a Varian Star 3400 Cx chromatograph (Les Ulis, France) fitted with a fused silica capillary DB-5MS column (5% phenylmethylpolysyloxane, 30 m × 0.25 mm, film thickness 0.25 µm). Chromatographic conditions were a 60 to 260 • C temperature rise with a gradient of 5 • C/min and 15 min isotherm at 260 • C. A 2nd gradient was applied to 340 • C at 40 • C/min. Total analysis time was 57 min. For analysis, essential oils were dissolved in petroleum ether. One microliter of sample was injected in the split mode ratio of 1 : 10. Helium (purity 99.999%) was used as carrier gas at 1 mL/min. The injector was operated at 200 • C. The mass spectrometer (Varian Saturn GC/MS/MS 4D) was adjusted for an emission current of 10 µA and electron multiplier voltage between 1400 and 1500 V. Trap temperature was 150 • C and that of the transfer line was 170 • C. Mass scanning was from 40 to 650 amu.

Compounds were identified by comparison of their KI (Kovats indices) relative to C5-C24 n-alkanes obtained on a nonpolar DB-5MS column, with those provided in the literature, by comparison of their mass spectra with those recorded in NIST 08 (Natl. Inst. of Standards and Technology) and reported in published articles and by co-injection of available reference compounds. The samples were analyzed in duplicate. The percentage composition of essential oils was computed by the normalization method from the GC peak areas. Results were calculated as mean values of 2 injections from each essential oil, without using correction factors. All determinations were performed in duplicate and averaged.

Total amount of phenolic compounds

The total phenolics of each extract were determined by the Folin-Ciocalteu method [START_REF] Folin | On tyrosine and tryptophane determination in proteins[END_REF]. Preparation of Folin and Ciocalteu reagent 2 N (Fluka): dissolve 10 g sodium tungstate and 2.5 g sodium molybdate in 70 mL water. Add The values are mean with standard deviations (SD) that did not exceed 5% for triplicate determinations.

5 mL 85% phosphoric acid and 10 mL concentrated hydrochloric acid. Reflux for 10 h. Add 15 g lithium sulfate, 5 mL water, and 1 drop bromine. Reflux for 15 min. Cool to room temperature and bring to 100 mL with water. Hexavalent phosphomolybdic/phosphotungstic acid complexes with the following structures are formed in solution.

The diluted aqueous solution of each extract (0.5 mL) was mixed with Folin-Ciocalteu reagent (0.2 N, 2.5 mL). This mixture was allowed to stand at room temperature for 5 min and then sodium carbonate solution (75 g/L in water, 2 mL) was added. After 1 h of incubation, the absorbances were measured at 765 nm against water blank using a Helios spectrophotometer (Unicam, Cambridge, U.K.). A standard calibration curve was plotted using gallic acid (0 to 300 mg/L). The results were expressed as gram of gallic acid equivalents (GAE)/kg of dry mass.

Condensed tannin content

Catechins and proanthocyanidins reactive to vanillin were analyzed by the vanillin method [START_REF] Broadhurst | Analysis of condensed tannins using acidified vanillin[END_REF]Naczk and others 2001). One milliliter (1 mL) of each extract solution was placed in a test tube and 2 mL of vanillin (1% in 7 M H 2 SO 4 ) in an ice bath and then incubated at 25 • C. After 15 min, the absorbance of the solution was read at 500 nm. Concentrations were calculated as gram catechin equivalents (CE)/kg dry mass from a calibration curve.

Total flavonoids determination

The total flavonoids were estimated according to the Dowd method as adapted by Arvouet-Grand and others (1994). A diluted methanolic solution (4 mL) of each extract was mixed with a solution (4 mL) of aluminum trichloride (AlCl 3 ) in methanol (2%). The absorbance was read at 415 nm after 15 min against a blank sample consisting of a methanol (4 mL) and extract (4 mL) without AlCl 3 . Quercetin was used as reference compound to produce the standard curve, and the results were expressed as gram of quercetin equivalents (QE)/kg of dry mass.

Determination of total anthocyanin content

Total anthocyanin content was measured with the pH differential absorbance method, as described by [START_REF] Cheng | Activity of phenylalanine ammonialyase (PAL) and concentrations of anthocyanins and phenolics in developing strawberry fruit[END_REF]. Briefly, absorbance of the extract was measured at 510 and 700 nm in buffers at pH 1 (hydrochloric acid-potassium chloride, 0.2 M) and 4.5 (acetic acid-sodium acetate, 1 M). The wavelength reading was performed after 15 min of incubation. Anthocyanin content was calculated using a molar extinction coefficient (ε) of 29600 (cyanidin-3-glucoside) and absorbance of A = [(A 510 -A 700 ) pH1.0 -(A 510 -A 700 ) pH4.5 ]. Results were expressed as milligram cyanidin-3glucoside equivalent (C3GE)/kg of dry mass.

Microbial strains

The different essential oils and extracts were individually tested against a panel of microorganisms. Six bacteria including 3 Gram-positive (Bacillus subtillis ATCC 6633, Staphylococcus aureus CIP7625, Listeria monocytogenes Scott A 724) and 3 Gram-negative (Pseudomonas aeruginosa CIPA22, Escherchia coli ATCC10536, Klebsiella pneumoniae CIP8291), 1 yeast (Saccharomyces cerevisiae ATCC 4226 A), and 2 fungi (Mucor ramamnianus ATCC 9314, Aspergillus westerdijkiae [ex: ochraceus]) were used.

Well-diffusion assay

Agar well-diffusion method [START_REF] Kalemba | Antibacterial and antifungal properties of essential oils[END_REF]Oke and others 2009) was employed for the determination for antimicrobial activity of essential oils and extracts. Briefly, a suspension of the tested microorganism (0.1 mL of 10 8 cells/mL) was spread on nutrient agar. Essential oils or extracts were serially diluted, using 10% Tween 80 in distilled sterile water. The wells with a diameter of 10 mm, were injected with 10 µL (9 µL of the essential oil + 1 µL of 10% Tween 80) of essential oils and with 100 µL (3 mg/mL) of extracts (300 µg/well) and placed on the inoculated plates. Ampicillin and nalidixic acid (30 µg/well) were used as positive reference standards to determine, respectively, the sensitivity of Gram-positive and Gram-negative bacterial. Nystatin (30 µg/well) was used as positive reference standard to determine the sensitivity of yeast and fungi species.

These plates, after staying at 4 • C for 2 h to allow dispersal, were incubated at 37 • C (bacteria) and at 30 • C (yeast and fungi) during 48 h. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organism. The diameter of the inhibition zones was measured in millimeters. Tests were carried out in triplicate.

Free radical scavenging activity DPPH test

Antioxidant scavenging activity was studied using 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) as described by [START_REF] Blois | Antioxidant determinations by the use of a stable free radical[END_REF] with some modifications; 1.5 mL of various dilutions of the test materials (pure antioxidants or plant extracts) were mixed with 1.5 mL of a 0.2 mM methanolic DPPH solution. After an incubation period of 30 min at 25 • C, the absorbance at 520 nm, the wavelength of maximum absorbance of DPPH, were recorded as A (sample) . A blank experiment was also carried out applying the same procedure to a solution without the test material and the absorbance was recorded as A (blank) . The free radical-scavenging activity of each solution was then calculated as percent inhibition according to the following equation:

% inhibition = 100 (A (blank) -A (sample) )/A (blank)
Antioxidant activity of essential oils or extracts was expressed as IC 50 , defined as the concentration of the test material required to cause a 50% decrease in initial DPPH concentration. Ascorbic acid was used as a standard. All measurements were performed in triplicate.

ABTS radical-scavenging assay

The radical scavenging capacity of the samples for the 2,2-AzinoBis-3-ethylbenzoThiazoline-6-Sulphonate radical cation (ABTS) was determined as described by Re and others (1999). ABTS was generated by mixing a 7 mM of ABTS at pH 7.4 (5 mM NaH 2 PO 4 , 5 mM Na 2 HPO 4 , and 154 mM NaCl) with 2.5 mM potassium persulfate (final concentration) followed by storage in the dark at room temperature for 16 h before use. The mixture was diluted with ethanol to give an absorbance of 0.70 ± 0.02 units at 734 nm using spectrophotometer. For each sample, diluted methanol solution of the sample (100 µL) was allowed to react with fresh ABTS solution (900 µL), and then the absorbance was measured 6 min after initial mixing. Ascorbic acid was used as a standard and the capacity of free radical scavenging was expressed by IC 50 (mg/L) values calculated denote the concentration required to scavenge 50% of ABTS radicals. The capacity of free radical scavenging IC 50 was determined using the same previously used equation for the DPPH method. All measurements were performed in triplicate.

Statistical analysis

All data were expressed as means ± standard deviations of triplicate measurements. The confidence limits were set at P < 0.05. Correlation coefficients (r) to determine the relationship between chemical composition and antioxidant or biological activity were calculated using MS Excel software (CORREL statistical function).

Results and Discussion

Chemical composition

Chemical composition of essential oils. Essential oils presented a white-yellow color. By hydrodistillation of leaves and berries of J. phoenicea, yields (relative to dry weight material) of 0.90% (w/w) and 3.95% (w/w), respectively, were obtained. El-Sawi and others (2007) obtained a berry's yield superior to that of leaf (0.36% and 1.96%, respectively), during its study of Egyptian juniper. Tunisian J. phoenicea has a yield at least twice higher than the Egyptian. Barrero and others (2006) have obtained yield 0.70% of Morocco J. phoenicea leaves.

The results of analyses of essential oils by GC-FID and GC-MS are given in Table 2, where the compounds were listed according to their KI. Twenty-nine constituents, representing 97.9% of the total components in the essential oil from the leaves of J. phoenicea, were identified. Monoterpenes (75.9%) represent the main fraction of the essential oil; α-pinene (55.7%) and δ-3-carene (10.7%) were the main components of this fraction. Also, α-terpineol (2.2%) and β-myrcene (2.1%) were present in reasonable amounts. A similar result was obtained by Barrero and others (2006), in his study of Marcocain J. phoenicea when the fresh leaves were treated by steam distillation and found that the largest group of constituents in the essential oil was the monoterpenes (71.1%), the major components were α-pinene (45.5%) and δ-3-carene (13.0%). It is clear that the Tunisian juniper was the highest content in different groups. The sesquiterpene compounds constituted 20% of the total essential oil (Table 2). γ -cadinene (2.9%), germacrene B (2.0%), β-caryophyllene (1.9%), and α-cubebene (1.5%) were the main components of the sesquiterpenes, which accounted for 20% of the essential oil. Also, in Morocain leaves essential oil, β-caryophyllene (3.4%) was the major component followed by γ -elemene (2.7%), which in the Tunisian leaves oil did not exceed 1.3%.

The chemical composition of the essential oil from J. phoenicea berries is also shown in Table 2. Nine compounds were identified, which present 98.8% of the total essential oil composition. The monoterpenes represent the major fraction, in which α-pinene (80.7%) was the main component. γ -cadinene (5.1%) represented the major compound of sesquiterpenes. There was a large difference between J. phoenicea leaves and berries essential oils. The 2 types of essential oils revealed a wide variation in chemical composition, their contents and the number of identified compounds.

Although leaves essential oil is richer in compounds than berries but it has a lower content in α-pinene. A similar result was found by Angioni and others (2003) when α-pinene of berries essential oil was at 87.5% but in leaves essential oil the percentage was 48.9%. In contrast with Afifi and others (1992), α-pinene was the major constituent of both essential oils recording a little difference by 39.3% for berries and 38.22% for leaves. β-pinene was present in not negligible amounts in both essential oils (2.45% and 0.48% in berries and leaves essential oils, respectively) compared with our study (0% and 1.1% in berries and leaves essential oils, respectively). The results were in agreement with those reported on the analysis of other juniper oils (Adams and others 1996) in which pinene derivatives are more common. Data reported by Adams and others (1996) on the chemical composition of the essential oil of Spanish J. phoenicea ssp. turbinata leaves showed that it was dominated by α-pinene (28.3%), βphellandrene (25.3%), β-myrcene (7.2%), and α-phellandrene (4.1%); while limonene and δ-3-carene were absent. In our study, we note the absence of β-phellandrene and presence of a high content of δ-3-carene (10.7%) as the 2nd major component; nevertheless, β-myrcene and α-phellandrene were in a lower value (2.1% and 0.8%, respectively). This important variation in chemical composition could be due to geographical and bioclimatic factors of the region.

Rezzi and others ( 2001) reported the composition of leaves essential oil of J. phoenicea from Corsica. They found 2 different main compositions, identified as Cluster I and II, with the former rich in α-pinene (70%) and the latter rich in α-pinene (33.0%), β-phellandrene (21.1%), and α-terpinyl acetate (8.2%). In the essential oil from J. phoenicea leaves from Portugal, Cavaleiro and others (2001) have recognized 3 clusters based on the α-pinene/ β-phellandrene ratio. We observed absence of β-phellandrene and α-terpinyl acetate in our study but contrary to this, δ-3-carene was the 2nd major compound (10.7%) in this work and it was low in study of Cavaleiro and others (2001). The variability in essential oil composition was present even in J. phoenicea and these variations, sufficient to allow the distinction of different specimens, were the result of an adaptative process to particular ecologic and geographic conditions.

Chemical composition of extracts. According to similarity and miscibility, 4 solvents having different polarities, methanol, ethanol, ethyl acetate, and dichloromethane, were used to extract the different secondary metabolites of J. phoenicea. Yields of different extracts obtained from leaves and berries are presented in Table 1. The highest yield, for leaves and berries, was achieved by the ethanol (35.4% and 35.8%, respectively), followed by methanol (26.2% and 16.4%, respectively) and ethyl acetate (18% and 6.4%, respectively), and the lowest was dichloromethane (1.1% and 0.8%, respectively). Variation in the yields of various extracts was attributed to polarities of different compounds in the leaves and berries, such differences have been reported in the literature concerning berries (Jayaprakasha and others 2001).

The amounts of the total polyphenols, tannins, flavonoids, and antocyanins in the extracts fractions of leaves and berries of J. phoenicea are shown in Table 3. The amount of total phenolics, in leaves and berries, varied in the different extracts and ranged from 52 ± 1 to 217 ± 2 g GAE/kg of dry material. The leaves of J. phoenicea were richer in polyphenols than berries. The highest amount of polyphenols was in methanolic leaves extract followed by ethanol extract. Also, Hayouni and others (2007) approved that the highest yield of berries J. phoenicea was 36.2% and the important amount of polyphenolics was 202 ± 0.43 g GAE/kg of dry ma-terial. In addition, for tannins and flavonoids, the ethanolic and methanolic leaves extracts are richer than berries extracts. While for antocyanins, berries are richer than the leaves and the highest amount was obtained by ethyl acetate extract (373 ± 13 mg C3GE/kg of dry material). Among these compounds, anthocyanins was at the lowest rate 84 to 373 mg C3GE/kg of dry mass compared with the amounts of polyphenols, flavonoids, and tanins, which were expressed in g/kg of dry mass. To the best of our knowledge, this is the 1st study reporting the amount of flavonoids, tanins, and anthocyanins of J. phoenicea leaves and berries extracts. Hence, it could be used as an important descriptor to characterize the extracts of J. phoenicea and consequently this endemic species itself.

Antimicrobial activity

The in vitro antimicrobial potential of J. phoenicea essential oils and extracts against microorganisms was quantitatively assessed by the presence or absence of inhibition zones (Table 4).

Antimicrobial activity of essential oils. As can be seen in Table 4, essential oils of leaves and berries showed reasonable in vitro antimicrobial activity compared with references against all the tested microorganisms including Gram-positive, Gram-negative, fungi, and yeast with diameter zones of inhibition 11 to 18 mm. The leaves essential oil has an antimicrobial activity greater than that of berries (except M. ramamnianus) that may be due to the wealth of leaves essential oil by oxygenated compounds that are totally absent in the berries essential oil. In addition, the amount of sesquiterpenes in the leaves essential oil was higher than in berries essential oil.

Essential oils of many Juniperus species were known to exhibit antimicrobial activity against several microorganisms (Stassi and others 1996;Cavaleiro and others 2001;Angioni and others 2003). The antibacterial activity of the essential oil of J. phoenicea buds could, in part, be associated with major constituents such as α-pinene, δ-3-carene, α-terpineol, β-myrcene, limonene, γterpinene, and α-amorphene. These components have been reported to display antibacterial effects (Cosentino and others 1999;Alessandra and others 2005). Terpenes were active against bacteria (Cosentino and others 1999).

As described previously by other researchers, essential oils containing terpenoids are more active against Gram-positive bacteria than against Gram-negative ones (Djoukeng and others 2005). In addition, the components in lower amount may also contribute to antibacterial activity of the essential oil, involving probably some type of synergism with other active compounds. Stassi and others (1996) have tested the antimicrobial activity of essential oils of 4 Juniperus spices against Gram-positive and Gram-negative bacteria. They approved that essential oils of the berries generally appear more active than essential oils of the leaves. Essential oils of the berries inhibit the growth of E. coli, P. aeruginosa, and S. aureus, while essential oils of the leaves do not. The same results were observed with our essential oils but not with E. coli. Essential oils of both the leaves and the berries inhibit the growth of B. subtulis at the same concentration. Furthermore, in our study, the most sensitive microorganism for the 2 essential oils was B. subtilus. Nevertheless, for the fungi, it turned out that the berries essential oil was inactive against A. westerdijkiae while leaves essential oil was active.

The activity of the essential oil varies with its concentration and the bacteria. These differences in the susceptibility of the test organisms to the essential oil could be attributed to variation in the rate of the essential oil constituents penetration through the cell wall and cell membrane structures. The ability of the essential oil to disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control are the most likely reasons for its lethal action.

Antimicrobial activity of extracts. The antibacterial activity of the different extracts of J. phoenicea (methanol, ethanol, ethyl acetate, and dichloromethane) was determined against 9 microbial strains, considered as among the common foodborne microorganisms. The inhibition zone, measured in millimeters, including the diameter of the well, was used as the criterion for measuring the antimicrobial activity of J. phoenicea extracts. Table 4 shows that the tested extracts could be classified according to their activity. The diameter of inhibition zones was obtained in the range of 11 to 34 mm. Methanol extracts of leaves and berries showed the highest activity. For Gram-positive, the most sensitive microorganism was the B. subtilis followed by S. aureus while L. monocytogenes was proved resistant against all samples. On the other hand, the most sensitive bacteria in Gram-negative was P. aeruginosa with a 23 mm inhibition obtained with ethyl acetate extract berries. K. pneumoniae and E. coli are completely resistant against the different samples. Our results are in agreement with the those of Hayouni and others (2007) when E. coli and L. monocytogenes were not inhibited by acetone extracts of J. phoenicea fruits. In the case of fungi, the highest activity of ethanol extract berries was against M. ramamnianus (22 mm), while all extracts were inactive against A. westerdijkiae. Finally, dichloromethane extracts had the lowest activity compared to all extracts (Table 4); this activity was due to compounds that presented a low polarity of secondary metabolites of J. phoenicea.

These data indicate that Gram-positive bacteria are the most sensitive tested strain to the different extracts. The tendency of polyphenols, flavonoids, tannins, and anthocyanins could be explained by that, the structures of cell envelope, including cytoplasmic membrane and cell-wall component, are different between Gram-positive and Gram-negative bacteria. Gram-negative bacteria possess an outer membrane surrounding the cell wall, which restricts diffusion of hydrophobic compounds through its lipopolysaccharide covering. Without outer membrane, the cell wall of Gram-positive bacteria can be permeated more easily and can disturb the cytoplasmic membrane, disrupt the proton motive force, electron flow, active transport, and coagulation of cell contents [START_REF] Burt | Essential oils: their antibacterial properties and potential applications in foods-a review[END_REF]. Therefore, the structural difference of bacteria plays an important role in their susceptibility.

Furthermore, the content of flavonoids in extracts correlates with their antifungal activity, confirming that flavonoids are likely to contribute to the antifungal activity against S. cerevisiae (r = 0.67). Similar results were reported for different plants by various studies (Rauha and others 2000;Sohn and others 2004) when they proved that there is a relationship between the content of flavonoids and antifungal activity; flavonoids cloud creased the activity of some extracts of Finnish and Korean plants against S. cerevisiae.

Although the concentrations of essential oils and extracts were generally about 10 times higher than those of the standard antibiotics, they showed marked antibacterial and antifungal activities. This difference can be explained of the fact that the active components in essential oils and extracts comprise only an active fraction. Therefore, the concentration of the active components could be much lower than the standard antibiotics used. It is important to note that, if the active components were isolated and purified, they would probably show higher antimicrobial activity.

Antioxidant activity

Antioxidant activity of essential oils and subfractions of the various extracts of J. phoenicea leaves and berries has been determined by 2 different test systems, namely, DPPH and ABTS assays. All of the data are presented in Table 5.

DPPH assay. In the present study, radical-scavenging activity by DPPH was exhibited by essential oils of leaves and berries (IC 50 value 5364 ± 121 and 14716 ± 411 mg/L, respectively). The low reactivity was explained by the high concentration of terpenes hydrocarbons of essential oils (91.0% for leaves and 98.3% for berries).

For extracts, antioxidant activity of leaves methanol extract was superior to all samples tested with an IC 50 value of 8.5 ± 0.3 mg/L, followed by leaves ethanolic extract (49.1 ± 0.6 mg/L). On the other hand, the leaves extracts prepared with ethyl acetate or dichloromethane have exhibited moderate radical-scavenging activity (273 ± 4 mg/L and 1052 ± 12 mg/L, respectively). For the berries, the major activity was detected in ethanol extract (54 ± 1 mg/L) followed by ethyl acetate and methanolic (642 ± 7 and 664 ± 7 mg/L, respectively). We can deduce that the antioxidant activity of J. phoenicea leaves (the essential oil and extracts) was better than the essential oil and extracts of berries. Methanol leaves extract showed a comparable activity to vitamin C (4.4 ± 0.2 mg/L). As can be seen in Table 5 that extracts prepared by different solvents exhibited varying degrees of antioxidant activity; this change in solvent polarity alters its ability to dissolve a group of antioxidant compounds and influences activity estimation (Hayouni and others 2007).

ABTS assay. According ABTS assay, the essential oil of berries has a more important activity than leaves (IC 50 = 87 ± 3 and 189 ± 5 mg/L, respectively). This result was opposite with the DPPH assay. In fact, this important activity compared to other samples was due to the specificity of ABTS radical cation.

For extracts, methanolic leaves presented a good antioxidant activity value, proving its capacity to scavenge the ABTS. However, compared with values of DPPH activity, those of ABTS assay exhibited more antioxidant activity whatever the extracting solvent.

We observed that the content of phenolics in the extracts correlates with their antioxidant activity, the correlation coefficients (r) between data of DPPH and ABTS assays and total phenolic compounds are 0.70 and 0.71, respectively. This confirms that phenolic compounds contribute to the radical scavenging activity of J. phoenicea extracts. In general, these properties depend on the ability to donate hydrogen or electron to a free radical. In Juniperus, a similar correlation was reported by Djeridane and others (2006) who found r = 0.79 between antioxidant activity and total phenolics but in 70% (v/v) aqueous ethanol extract of J. oxycedrus.

The highest correlation was detected between tannins against ABTS assay with r = 0.76. Falleh and others (2008) found the same behavior between tannins and DPPH assay (r = 0.96) in their study of phenolic compounds of Cynara cardunculus L. In the best of our knowledge, our study is the 1st research inducing the relationship between tannins and ABTS antioxidant assay. The high correlation explains the important antioxidant power of tannins of Tunisian J. phoenicea extracts.

The results indicated that methanol extract presented higher total phenolic quantity (217 ± 2 g GAE/kg of dry material) between the different extracts. Phenolic compounds, biologically active components, are the main agents that can donate hydrogen to free radicals. Essential oils had a weak antioxidant activity. The results indicated that methanolic extracts were effective antioxidants in different in vitro assays including DPPH radical scavenging and ABTS activity. Besides, ethanolic leaves extract also exhibited strong scavenging effect on DPPH free radical.

Conclusions

T he structure/antioxidant activity relationship evaluation of these compounds suggested that the number of hydroxyl groups was the most important factor in determining the antioxidant activity of the phenolic compounds. Therefore, from the above-mentioned results, we can conclude that essential oils and methanolic extracts derived from J. phoenicea leaves and berries could be considered as potential alternatives for synthetic bactericides and natural antioxidants for use in the food industry along with their possible applications in the pharmaceutical industry for the prevention or treatment caused by microorganisms and free radicals.

Further studies are necessary to assess the methanolic extraction of leaves of the compounds responsible for the antioxidant activity which could have far more capacity effective than vitamin C and might be used as a new antioxidant and alternative to synthetic antioxidants.

  Authors Ennajar and Abderraba are with Laboratoire de Physicochimie des Matériaux, IPEST, BP51, La Marsa 2070, Tunisia. Authors Ennajar and Romdhane are with Unite ´ de Modélisation, Analyse et commande des systèmes, ENIG, 6029 Gabés, Tunisia. Author Bouajila is with SPCMIB, UMR CNRS 5068, Univ. Paul-Sabatier, 118 route de Narbonne, F-31062 Toulouse, France. Authors Lebrihi and Mathieu are with LGC UMR 5503 (CNRS/INPT/UPS), EN-SAT/ INPT, 1, Ave. de l'Agrobiopole, BP 32 607, Auzeville-Tolosane, F-31326 Castanet-Tolosan, France. Author Raies is with LMBA, Faculte ´ des Sciences de Tunis, Campus Univ. 2092 El Manar II, Tunisia. Direct inquiries to au-thor Bouajila (E-mail: bouajila@cict.fr).

  • 19 ′ in the north and 8 • 66 ′ in the east of longitude. Matmata average rainfall was 78 mm for 2007, 6 mm in October 2007, the average temperature was 22.6 • C in October 2007 and humidity was 65% in October 2007 (Pluviometric Directory of Tunisia, 2007-2008, Head Office of the Water Resources, Ministry for Tunisian Agriculture, the Environment and the Hydraulic Resources). Specimens were identified by Dr. Nadia Ben Brahim at the Dept. of Botany,

	Natl. Inst. of Agronomic Research (INRAT, Tunis) and voucher spec-
	imens were deposited at the Herbarium of the Dept. of Botany in
	the cited institute.

Table 1 -

 1 --Extraction yields (percent) of essential oils and various extracts of J. phoenicea.

		Water
		distillation
		(essential oil)	Methanol	Ethanol	Ethyl acetate	Dichloromethane
	Leaves	0.90	26.2	35.4	18.0	1.1
	Berries	3.95	16.4	35.8	6.4	0.8

Table 2 -

 2 --Chemical composition (percent) of J. phoenicea essential oils.

		KI	M (g/mol)	Compounds	Leaves	Berries
	1	936	136	α-pinene	55.7	80.7
	2	974	136	β-pinene	1.1	ND
	3	985	136	β-myrcene	2.1	1.8
	4	1011	136	δ-3-carene	10.7	4.5
	5	1028	136	limonene	0.7	ND
	6	1030	136	α-phellandrene	0.8	ND
	7	1057	136	γ -terpinene	0.7	1.3
	8	1086	136	α-terpinolene	0.7	ND
	9	1185	154	α-terpineol	2.2	ND
	10	1237	148	cuminaldehyde	0.6	ND
	11	1272	196	isopulegyl acetate	0.6	ND
	12	1345	204	α-cubebene	1.5	ND
	13	1415	204	β-caryophyllene	1.9	2.6
	14	1428	204	γ -elemene	1.3	ND
	15	1432	204	β-gurjunene	0.7	ND
	16	1450	204	α-himachalene	0.8	0.8
	17	1454	204	α-humulene	0.5	ND
	18	1460	204	allo-aromadendrene	0.8	ND
	19	1480	204	γ -muurolene	0.8	ND
	20	1482	204	β-selinene	2.0	ND
	21	1488	204	α-amorphene	0.7	ND
	22	1502	204	α-chamigrene	0.7	ND
	23	1513	204	γ -cadinene	2.9	5.1
	24	1514	222	cubenol	1.5	ND
	25	1524	204	δ-cadinene	1.9	ND
	26	1540	222	α-calacorene	0.8	ND
	27	1554	204	germacrene B	2.0	1.5
	28	1960	272	13-isopimaradiene	ND	0.5
	29	2328	286	trans-ferruginol	0.6	ND
	30	nd	136	heptane 2,4-dimethyl a	0.6	ND
				Total	97.9	98.8
				Monoterpene hydrocarbons	72.5	88.3
				Monoterpenes oxygenated	3.4	0
				Sesquiterpenes hydrocarbons	18.5	10.0
				Sesquiterpenes oxygenated	1.5	0
				Others	2.0	0.5

a Tentatively identified supported by good match of MS. nd = not determined. ND = not detected.

Table 3 -

 3 --Chemical composition of J. phoenicea extracts.

		Samples	Polyphenols (GAE) a	Tannins (CE) a	Flavonoids (QE) a	Anthocyanins (C3GE) b
	Leaves	Ethyl acetate	116 ± 2	18.4 ± 0.7	24.6 ± 0.7	218 ± 10
		Dichloromethane	56 ± 1	9.3 ± 0.4	16.7 ± 0.7	219 ± 11
		Ethanol	169 ± 2	60.2 ± 2.8	29.3 ± 1.3	249 ± 12
		Methanol	217 ± 2	57.2 ± 2.8	20.9 ± 1.0	84 ± 4
	Berries	Ethyl acetate	79 ± 1	13.3 ± 0.5	15.0 ± 0.7	373 ± 13
		Dichloromethane	93 ± 1	20.3 ± 0.6	6.5 ± 0.2	275 ± 11
		Ethanol	96 ± 1	6.5 ± 0.2	6.4 ± 0.3	186 ± 8
		Methanol	52 ± 1	14.7 ± 0.4	8.6 ± 0.4	278 ± 11

a g/kg dry mass; b mg/kg dry mass. Standard deviations (SD) did not exceed 5%.

Table 4 -

 4 --Antimicrobial activity (diameter of the inhibition zones in mm) of essential oils and extracts of J. phoenicea.

		A. westerdijkiae
	Fungi and yeast	M. ramamnianus
		S. cerevisiae
		E. coli
	Gram-negative bacteria	P. aeruginosa K. pneumoniae
		S. aureus
	Gram-positive bacteria	L. monocytogenes
		B. subtillis
		Samples
		Essential oils

Table 5 -

 5 --Free radical scavenging capacities of essential oils and extracts of J. phoenicea.

			DPPH IC 50	ABTS IC 50
		Samples	(mg/L)	(mg/L)
	Essential oils	Leaves	5364 ± 121	189 ± 5
		Berries	14716 ± 411	87 ± 3
	Leaves extracts	Ethyl acetate	273 ± 4	30 ± 1
		Dichloromethane	1052 ± 12	16.0 ± 0.5
		Ethanol	49.1 ± 0.6	9.1 ± 0.3
		Methanol	8.5 ± 0.3	6.5 ± 0.3
	Berries extracts	Ethyl acetate	642 ± 7	35 ± 1
		Dichloromethane	2120 ± 24	131 ± 4
		Ethanol	54 ± 1	100 ± 4
		Methanol	664 ± 7	81 ± 4
	Reference	Vitamin C	4.4 ± 0.2	1.9 ± 0.1
	standard
	Standard deviations (SD) did not exceed 5%.


"The Role of Non-Invasive Brain Oximetry in Adult Critically ill Patients without Primary Non-Anoxic Brain Injury" -NIRS, a gadget or a must for critically ill patients? Minerva Anestesiologica

Pierre Simeone [START_REF] Zheng | Cerebral Near-Infrared Spectroscopy (NIRS) Monitoring and Neurologic Outcomes in Adult Cardiac Surgery Patients and Neurologic Outcomes: A Systematic Review[END_REF][START_REF] Badenes | The Role of Non-Invasive Brain Oximetry in Adult Critically ill Patients without Primary Non-Anoxic Brain Injury[END_REF] , Nicolas Bruder Words : 1020/1000

References : 13/20

Conflicts of interest : None

If we were able to define a common base for medical practice among neurointensivists, maintaining cerebral oxygenation would certainly be a major goal in neuro-intensive care patients. Several monitoring techniques have been developed to optimize cerebral oxygenation, from the less invasive (transcranial Doppler as a surrogate of cerebral blood flow) to the more invasive (intracranial pressure for monitoring cerebral perfusion pressure and brain tissue oxygenation probes). Each of these methods has its advantages and inconveniences, and the objective is probably to collect arguments to take the best possible treatment options. Among all these techniques, Near-infrared spectroscopy (NIRS) monitoring is particularly attractive because this monitoring technique is non-invasive, easy to use and gives continuous values that can be challenged against therapeutic changes. However, it is important to situate its place in the monitoring arsenal.

In year 2013, a systematic review suggested with a low-level evidence, that regional oxygen saturation index (rSO2) could be used to predict postoperative neurologic complications after cardiac surgery but data were insufficient to conclude that interventions that could improve rSO2 desaturation would be able to prevent stroke or postoperative cognitive dysfunction. [START_REF] Zheng | Cerebral Near-Infrared Spectroscopy (NIRS) Monitoring and Neurologic Outcomes in Adult Cardiac Surgery Patients and Neurologic Outcomes: A Systematic Review[END_REF] Eight years later what is the evidence on the use of NIRS to monitor the brain in the ICU ? The review by Badenes et al. [START_REF] Badenes | The Role of Non-Invasive Brain Oximetry in Adult Critically ill Patients without Primary Non-Anoxic Brain Injury[END_REF] in this issue of the journal gives precise answers and perspectives on this technique. First, all monitoring techniques are prone to errors and it is especially important to fully understand the limitations of NIRS monitoring to extract relevant information for clinical practice. A major problem is that different monitors give different values that cannot be compared. Several studies showed highly significant differences in local cerebral tissue oxygenation levels from one device to the other, making interpretation of studies with different devices difficult and hazardous. [START_REF] Schneider | Comparison of four near-infrared spectroscopy devices shows that they are only suitable for monitoring cerebral oxygenation trends in preterm infants[END_REF] In addition, extracranial contamination of the signal (despite unpublished algorithms used to minimize it) has been shown from the beginning of the monitoring technique and has long been reported as a limitation. [START_REF] Davie | Impact of Extracranial Contamination on Regional Cerebral Oxygen Saturation: A Comparison of Three Cerebral Oximetry Technologies[END_REF] Also, significant bias and large variation in reading errors were found in healthy subjects submitted to hypoxia. This has been related to a fixed ratio of arterial and venous cerebral blood in the algorithms assumed by the manufacturers which is probably not true in human physiology during hypoxia. [START_REF] Bickler | Factors Affecting the Performance of 5 Cerebral Oximeters During Hypoxia in Healthy Volunteers[END_REF] Likewise, more than twenty years ago, the use of vasopressors as norepinephrine in traumatic brain injury patients was associated with a spurious decrease in rSO2 despite an increase in cerebral perfusion pressure and did not adequately reflect changes in jugular venous oxygen saturation. [START_REF] Minassian | Correlation between Cerebral Oxygen Saturation Measured by Near-infrared Spectroscopy and Jugular Oxygen Saturation in Patients with Severe Closed Head Injury[END_REF] Some studies have been done in the ICU to determine if there was a correlation between rSO2 and brain tissue oxygen pressure (PbtiO2) for neuro-intensive care patients. [START_REF] Leal-Noval | Invasive and noninvasive assessment of cerebral oxygenation in patients with severe traumatic brain injury[END_REF] Even if several statistical approaches demonstrated direct correlation between rSO2 and PbtiO2, the diagnostic accuracy of rSO2 to detect cerebral hypoxia was limited showing that NIRS is not a substitute of direct PbtiO2 monitoring. Moreover, a work using 15 oxygen positron emission tomography data showed that ischemia in TBI patient was common but not systematically associated with intracranial hypertension and inconsistently detected by jugular or brain tissue oximetry. [START_REF] Launey | Spatial and Temporal Pattern of Ischemia and Abnormal Vascular Function Following Traumatic Brain Injury[END_REF] The high between-and within-patient cerebral circulation heterogeneity possibly reflecting abnormalities in brain flow-metabolism coupling makes clinical reasoning challenging in routine clinical practice.

Although there is no doubt that adequate cerebral oxygenation is important for preventing ischemia, the pathophysiology of brain ischemia is much more complex than oxygenation alone. Multiple other mechanisms leading to brain ischemia, not directly related to oxygenation, have been described. For example, cortical spreading depolarization, inflammation and apoptosis, microvascular injury, reperfusion injury and oxidative stress, blood-brain barrier disruption can all contribute to brain damage without involving alterations in rSO2. Finally, the heterogeneity of cerebral circulation changes and the multiple metabolic pathways leading to brain ischemia in most brain-injured patients explain that the interpretation of any brain monitoring technique, including NIRS, is not straightforward. NIRS is probably easier to interpret in cardiac surgery patients who have a non-injured brain before surgery.

All these limitations surely explain why majority of studies showing strong clinical application are in cardiac surgery, where the pathophysiological model of cerebral hypoxia is more pure and less complex.

However, a practical attitude can be extracted from all this literature. As no clear absolute threshold for ischemia is identified, NIRS monitoring should be used as a trend monitor with the assumption that there is no "baseline" value in critically ill patients. In any case, it is essential to keep in mind that it is needed to look at changes over time. A good example has been demonstrated in a recent study in post cardiac-surgical patients which showed a progressive postoperative decline in rSO2 from the day of surgery until day 7 with 97% of patients below baseline value at discharge. [START_REF] Cioccari | Prolonged postoperative cerebral oxygen desaturation after cardiac surgery: A prospective observational study[END_REF] A given value at one time point was not associated with clinical deterioration. Hence, it seems appropriate to set an alarm value on NIRS to look at the determinants of brain oxygenation in a dynamic manner. However, acute changes in rSO2 may give important information at the bedside. For instance, two studies showed the interest of using NIRS to detect intracranial hematoma in head trauma making it possible to manage this complication before cerebral herniation occurs. [START_REF] Gopinath | Early detection of delayed traumatic intracranial hematomas using near-infrared spectroscopy[END_REF][START_REF] Brogan | Near-infrared spectroscopy (NIRS) to detect traumatic intracranial haematoma: A systematic review and meta-analysis[END_REF] Another example well explained by Badenes et al. is the use of NIRS to show dysfunctional cerebral autoregulation associated with delirium and outcomes in ICU patients. [START_REF] Lee | Dysfunctional cerebral autoregulation is associated with delirium in critically ill adults[END_REF] For this purpose, NIRS seems easier to use than methods based on transcranial Doppler monitoring.

One of the lessons we can learn from the literature is that we certainly expect too much of our monitoring techniques. For example, a Cochrane database review did not find any clinical benefit of pulse oximetry in 22 992 patients enrolled in several studies with a good methodology. This does not mean we should not use pulse oximetry, but rather that it is difficult nowadays to demonstrate the benefit of a monitoring technique. [START_REF] Pedersen | Pulse oximetry for perioperative monitoring[END_REF] In this regard, the review from Badenes et al. gives updated information on how to use NIRS in critically ill patients, not as one more gimmick but as an added information to raise our attention on the patient's brain before it is too late.

1 1

 1 Aix Marseille University, AP-HM, Department of Anesthesiology and Critical Care Medicine, University Hospital Timone, Marseille, France ; 2 Aix Marseille University, CNRS, Institut des Neurosciences de la Timone, UMR7289, Marseille, France.


Yousef Swesi

Amaury Gillet

A Guerin

Marie-Line Zanota

Frédéric Bornette

Régis Philippe
email: regis.philippe@lgpc.cpe.fr

Valérie Meille
email: valerie.meille@ircelyon.univ-lyon1.fr

Alexandre Guérin

Comparison of Structured Reactors

  L'archive ouverte pluridisciplinaire

Introduction

At high altitudes, the ozone concentration can reach high values and ozone converters are needed to reduce this concentration before air enters the aircraft's cabin for partial renewal. [START_REF] Weisel | Ozone and Ozone Byproducts in the Cabins of Commercial Aircraft[END_REF][START_REF] Bhangar | Ozone Levels in Passenger Cabins of Commercial Aircraft on North American and Transoceanic Routes[END_REF] Structured reactors are now classically used in such air treatment applications, not only for exhaust car converters [START_REF] Dudák | Prediction of diffusivity and conversion of n-decane and CO in coated Pt/γ-Al 2 O 3 catalyst depending on porous layer morphology[END_REF][START_REF] Gálvez | Catalytic filters for the simultaneous removal of soot and NOx: Influence of the alumina precursor on monolith washcoating and catalytic activity[END_REF][START_REF] Torkashvand | On the Challenges and Constrains of Ultra-Low Emission Limits: Formaldehyde Oxidation in Catalytic Sinusoidal-Shaped Channels[END_REF] but also for aircraft ozone abaters. [START_REF] Limberg | Environmental Control Systems[END_REF][START_REF] Fisher | Manganese Ozone Decomposition Catalysts and Process for Its Preparation[END_REF][START_REF] Collins | Augmented Catalytic Heat Exchanger System[END_REF][START_REF] Galligan | Catalyst Systems and Methods for Treating Aircraft Cabin Air[END_REF][START_REF] Wu | Catalytic removal of ozone and design of an ozone converter for the bleeding air purification of aircraft cabin[END_REF][START_REF] Wu | Catalytic Removal of Ozone by Pd/ACFs and Optimal Design of Ozone Converter for Air Purification in Aircraft Cabin[END_REF] Their use offers a high surface to volume ratio, with a reduced pressure drop compared to dense particulate packed beds. As recalled by Farrauto et al., [START_REF] Farrauto | Moving from discovery to real applications for your catalyst[END_REF] the ozone conversion is strongly limited by masstransfer. Different options can be studied to decrease the boundary layer on the catalyst: using several slices of monoliths separated by empty spaces to induce some "turbulence" [START_REF] Heck | Catalytic Air Pollution Control: Commercial Technology[END_REF] or using a different geometry of structured objects. [START_REF] Moini | Corrugated metal substrate and coated product for ozone conversion[END_REF][START_REF] Kalluri | Comparative heterogeneous contacting efficiency in fixed bed reactors: Opportunities for new microstructured systems[END_REF][START_REF] Gu | Comparison of packed beds, wash-coated monoliths and microfibrous entrapped catalysts for ozone decomposition at high volumetric flow rates in pressurized systems[END_REF] A compromise has to be determined between mass transfer improvement and the inevitably induced pressure drop increase. This bottleneck is regularly encountered with catalytic structured internals operating in gas phase for various pollutant removal applications. [START_REF] Mo | Novel Insight into VOC Removal Performance of Photocatalytic Oxidation Reactors[END_REF][START_REF] Twigg | Progress and future challenges in controlling automotive exhaust gas emissions[END_REF][START_REF] Lucci | On the Catalytic Performance of Open Cell Structures versus Honeycombs[END_REF][START_REF] Giani | Mass-Transfer Characterization of Metallic Foams as Supports for Structured Catalysts[END_REF][START_REF] Patcas | CO oxidation over structured carriers: A comparison of ceramic foams, honeycombs and beads[END_REF] The choice depends often on a criterion combining mass transfer properties and corresponding generated pressure drop but also on more practical criteria like mechanical resistance properties, compactness and overall system costs.

At present, the ozone conversion is performed at 200 • C, a temperature at which homogeneous thermal conversion is important in the gas phase and where catalysts are very active for a parallel heterogeneous conversion reaction, making the conversion target of > 95% an easily achievable goal, even at very high GHSV of several hundreds h -1 . As future aircrafts will be more electrical, the heat availability to heat up the converter will be drastically reduced and catalytic converters able to work at 120 • C or even lower temperature need to be developed [START_REF] Alphonse | System for Treating Air, in Particular at a Low Temperature, for an Aircraft[END_REF] . Contrarily to what occurs at 200 • C, the homogeneous thermal ozone decomposition in the gas phase will be negligible at 120 • C, but a predominantly mass-transfer limited regime will still prevail. Thus, improving the mass transfer performances of the converters is more than ever before an important lever knowing that the acceptable pressure drop will be more than ever highly constrained in future aircrafts.

In this paper, different kinds of substrates able to host a layer of catalyst were evaluated: ceramic monoliths, metallic monoliths and metallic foams. Their channel/cell size was also varied. The objectives were to characterize their pressure drop behaviour, to evaluate the possible impact of a typical Pd/Al 2 O 3 catalyst coating on its value, and to characterize the mass transfer performance under reactive conditions. All these objectives contribute to find a trade-off between pressure drop and mass transfer performances.

Experimental

The ozone conversion was studied with a commercial 5 wt% Pd/Al 2 O 3 catalyst (sold under reduced form by Alfa Aesar, ref 11713, batch X12E021) coated on the different structured substrates of the study. 300, 400 and 600 CPSI ceramic monoliths (named MC300, MC400 and MC600), in the form of 1" (diam) x 2 cm (L) cartridges, were provided by ALSYS group. 300, 400 and 600 CPSI FeCrAl monoliths (named MM300, MM400 and MM600), in the form of 1" (diam) x 1" (L) cartridges, were purchased at Bersy company (Italy). Foamet FeCrAl foams (pore mean size 2 mm and 3.8 mm, named FM2mm and FM3.8mm) were purchased at Hollomet (Germany). Figure 1 depicts one example of 3D X-ray tomography reconstructed image for each kind of object. Due to heterogeneous, approximate and insufficient geometrical data obtained by the different providers, the different objects were fully analyzed by X-ray tomography (GE Phoenix v|tome|x s, RX tube of 160 kV with a focal point of up to 1 µm) with a range of spatial resolution between 6 and 20 µm. The tomographic data were processed with Matlab Image Processing toolbox and iMorph software [START_REF] Brun | iMorph : A 3D morphological tool to fully analyze all kind of cellular materials[END_REF] providing illustrative 3D images of the samples and a precise and statistical quantification of the structures. These characterizations allowed to provide the values of the geometric surface-to-volume ratio (a S ) and the porosity ( ) used in the following but also cell and windows size distributions for foams as well as hydraulic diameter and wall thickness distribution for monoliths. Concerning the foams, the cell diameter was estimated from the diameter of the biggest sphere inscribed in the cell.

The coating with the commercial Alfa Aesar powder catalyst was carried out according to a previously established procedure [START_REF] Meille | Reproducibility in the preparation of alumina slurries for washcoat application -Role of temperature and particle size distribution[END_REF] and resulted in the deposition of 0.2 to 1 g of catalyst per 1"x1" cartridge depending on its nature. The catalytic phase finally followed a thermal treatment consisting in a heating up to 450 • C with a ramp of 2 • C • min -1 under a flow rate of 5 L • min -1 of air, followed by a plateau of 4 h at this final temperature. Finally, cooling was made under flow with the same flow rate to room temperature. For each type of structure, 4 cartridges were coated with the Pd/alumina catalyst.

The set-up presented in Figure 2 is used to test the structured catalytic substrates. It consists of a tube, with a diameter of 32 mm, filled with 1 to 4 cylindrical pieces (1" x 1"). Upstream, a compressed air delivering system is able to deliver till 20 Nm 3 • h -1 of pure dry air (dew point of -60 • C). A fraction of the preheated air flow is used to generate ozone by corona arc discharge in an ozone generator (Ozone service, Mimaud équipements, France).

The ozone generator was able to produce nominally 3 g • h -1 and the excess of ozone was catalytically destroyed in an auxiliary guard bed. The hot air and ozone mixture (from 2 to 18 Nm 3 • h -1 with a nominal ozone content of 1.9 ± 0.1 ppmv) is homogenized through a static mixer and then enters the catalytic zone. This ozone concentration is representative of the one existing outside the aircraft at cruise altitude. The GHSV range is [4

× 10 4 -1 × 10 6 ] h -1
with the GHSV definition recalled in Equation 1.

GHSV = Q G /V Reactor ( 1
)
The temperature is set at 120 • C, a temperature at which NOx adsorption is not competitive (NOx can be formed during ozone generation [START_REF] Touati | Unexpected Role of NOx during Catalytic Ozone Abatement at Low Temperature[END_REF] ) and thermal decomposition appears negligible. The ozone content is measured upstream and downstream of the catalytic zone thanks to a BMT932 analyzer (BMT-Berlin, Germany) based on the UV photometric method at 254 nm. The pressure drop is measured using a Fuji Electric™ FCX-AIII differential pressure transmitter with a high precision (6 Pa).

Results and discussion

Preliminary observations

Blank experiments conducted at a flow rate of 4.8 Nm 3 • h -1 with the different uncoated structures (2 cartridges per test) helped to check that conversion levels remain under 5% at 120 • C. It was true for all substrates except for FM2mm at the lower flow rates for which it was 12%. No clear explanation is proposed to explain the result obtained with this foam. However, as the structures will be covered by a catalyst layer, the conversion that can arise from the contact with the metal of the structure itself will be neglected. The thermal decomposition was considered as negligible at this temperature in the following parts.

Characterization of the structures

The image analysis of the X-Ray tomography data allowed to estimate different morphological parameters which are summarized in Table 1.

Pressure drop

Coated structures

The pressure drop was measured at different temperatures and over a wide range of air flow rates. Figure 3 presents the variation of the pressure drop per unit length with the gas flow rate at 120 • C for the different coated structures presented in Table 2. The graph is of the results obtained for a single cartridge; they have a standard deviation of ± 5% when using 2 or 4 cartridges. Regarding the 6 different monoliths, a linear relationship is observed between the pressure drop and the gas superficial velocity. This was expected due to the laminar flow occurring inside these small channels and pressure drop can be modelled by a Darcy's law. [START_REF] Darcy | In Les fontaines publiques de la ville de Dijon : exposition et application des principes à suivre et des formules à employer dans les questions de distribution d[END_REF] In the 2 classes of monoliths, the linear pressure drop increases with the increase in cell density. It is logically linked to the increase in contact surface exposed to the gas flow for a roughly identical porosity. MC300 behave differently with a higher pressure drop compared to MC400. This is easily explained by the thicker walls employed for this structure and thus by a resulting lower porosity of 63.6% after coating. Note that MC400 also presents a much higher catalyst loading that could also explain the ranking but this explanation is not favoured because the same is not observed for MM400, also highly loaded. Still linked to a significantly different level of porosity (83.6% vs. 70%) due to thinner walls, metallic monoliths present logically lower pressure drops compared to their ceramic counterparts. In the case of opencell foams, the linear pressure drop relationship with gas superficial velocity disappears. The pressure drop tends to obey to a more parabolic behaviour that is classically modelled by a Darcy-Forchheimer law (Equation 2). This law introduces an inertial term to the viscous one of the initial Darcy's law. It is interesting to notice here that even with higher porosities and quite smaller geometrical specific surface area, the foam objects present higher pressure drops than monoliths due to this non negligible inertial term.

∆P/L = 1 k 1 µu + 1 k 2 ρ G u 2 (2)
In this equation, µ is the gas viscosity, ρ G the density, u the gas superficial velocity (m/s), k 1 the structure permeability (m 2 ) and k 2 passivity (m), the latter often being referred to as inertial permeability.

The use of Equation 2 with the experimental results obtained for the different structures allows to regress permeabilities and passivities (when necessary, for foams). This was made using the correlation of Chung et al. [START_REF] Chung | Applications of kinetic gas theories and multiparameter correlation for prediction of dilute gas viscosity and thermal conductivity[END_REF] to estimate the air viscosity at 120 • C according to a least squares optimization approach. For foams, the fitting was done in two steps according to Clavier et al. [START_REF] Clavier | Experimental investigation on singlephase pressure losses in nuclear debris beds: Identification of flow regimes and effective diameter[END_REF] in order to avoid reaching any local minimum in the optimization:

• a first fitting of k 1 only for the very low flow-rates;

• a second fitting of k 2 only for the remaining ones.

Table 3 summarizes the obtained values for these coated objects.

Influence of the coating

To check the possible influence of the coating on the linear pressure drop behaviour, two monoliths both presenting 400CPSI were coated with different amounts of catalyst (these 2 samples were not used for the mass transfer study and are not reported in Table 2).

The ceramic monolith was covered by 35 µm of catalyst while the metallic one had only 9 µm of catalytic material layer. The pressure drop measurements made with or without these 2 coatings indicate that with only 9 µm of catalyst, the pressure drop appears not impacted while with the 35 µm, a drop of 20% is observed (Figure 4). Note that this series of experiments was performed at 20 • C as preliminary experiments but the message remains valid at 120 • C.

These observations lead us to conclude that pressure drop measurements should be carried out with the coated objects as they will be used in the intended application.

Mass transfer

A series of catalytic tests was performed with 1 to 4 stacked catalytic cartridges, to vary the catalytic bed length (L). For each kind of structuration, the catalyst content was the same for the 4 elements. The ozone conversion (X) was recorded at various superficial flow velocities (u) under fixed conditions of ozone concentration (1.9 ± 0.1 ppmv), temperature (120 • C) and pressure (atmospheric). Typical results are depicted on figure 5 for the MM400 structure. It is clearly observed that for a given contact time (Equation 3), multiple conversion levels are encountered due to the superficial velocities used and the corresponding bed length. This is a signature of a strong external mass transfer limitation as expected.

tc = L/u (3)
Assuming that the intrinsic kinetic model published by Kalluri et al. [START_REF] Kalluri | Comparative heterogeneous contacting efficiency in fixed bed reactors: Opportunities for new microstructured systems[END_REF] is applicable for the catalyst used in this work, a comparison of the overall resistance (1/k ov ) with the contribution of the chemical one (1/ηkρ c ) can be done easily using the additivity of resistances in series depicted in Equation 4.

1 k ov = 1 k s a s + 1 ηkρ c (4)
In the whole experimental domain considered in this study and for all structured objects, the chemical resistance was found to account for 0.04 to 0.75 % of the overall effective one.

This helps to support the assumption that in the following, it will be considered that k ov = k s a s directly.

The obtained results were thus used to determine the effective mass transfer coefficients, except those with a conversion level higher than 95% in order to be insensitive to hydrodynamic non idealities. A 1-D heterogeneous isothermal isobaric steady state plug flow model was used to recover the effective mass transfer coefficient k s a s . It is made under the assumption of an instantaneous reaction on the solid surface and the gas phase mass balance equation reduces to Equation 5. The effective mass transfer coefficients k s a s obtained by this method for the different studied structures are plotted in Figure 6 versus the linear pressure drop. With this approach, the most attractive structures -with high mass transfer and low pressure drop -appear in the top left corner. First of all, open cell foams appear as the worst structures with either a very low effective mass transfer coefficient k s a s and a reasonable linear pressure drop or a reasonable mass transfer coefficient with prohibitive pressure drops. This result is consistent with the publications of Giani et al. [START_REF] Giani | Mass-Transfer Characterization of Metallic Foams as Supports for Structured Catalysts[END_REF] and Patcas et al. [START_REF] Patcas | CO oxidation over structured carriers: A comparison of ceramic foams, honeycombs and beads[END_REF] Then, for a similar CPSI, metallic monoliths appear more interesting than their ceramic counterparts. It is linked to the highest void fraction of the metallic structures that generates a lower pressure rate and surface area.

ln(1 -X) = -k s a s (L/u) (5)
The coarser 300 CPSI structures are less interesting that the 400-600 ones which are more or less equivalent. This last result may appear counter-intuitive but the 600 CPSI structures generate more pressure drop when washcoated. Finally, it appears quite clear that MM400 appears as the most attractive structure for this application. Note that metal monoliths are more mechanically stable against vibrations and other perturbations experienced during landings, etc. They tend nevertheless to be more expensive than ceramic ones.

To complete this analysis, knowing the specific surface area a s through the geometric characterizations made by X-ray tomography, a second comparison of the structures is provided in terms of k s only in Figure 7. This complementary figure demonstrates that foams generate the highest mass transfer coefficients k s in comparison to monoliths which fall close to each others in this representation. It is easily explained thanks to the better gaseous mixing obtained in the tortuous paths of the foams that contributes to induce some convective mixing contributions in all the directions and to reduce significantly the effective diffusion layer in comparison to monoliths. A trade-off index (I t.o. ), according to Giani et al., [START_REF] Giani | Mass-Transfer Characterization of Metallic Foams as Supports for Structured Catalysts[END_REF] can also be calculated (see Equation 6).

I t.o. = ln(1 -X) ∆P/ρu 2 (6)
This index, representing the ratio between the apparent conversion rate and the pressure drop observed to ensure such a performance, has to be maximized.

The indexes for all the structures are plotted in Figure 8. The plots correspond to data collected for 1 to 4 stacked objects, at various gas velocities. It is again very clear that the metallic monoliths allow to reach a better compromise between mass transfer and pressure drop. According to Giani et al., [START_REF] Giani | Mass-Transfer Characterization of Metallic Foams as Supports for Structured Catalysts[END_REF] two different Reynolds numbers were used, depending on the structure: a first was based on the foam strut diameter, d S , for foams (Equation 7), while the the other one was based on the channel hydraulic diameter, d h , for monoliths (Equation 8). It can be noticed that experimental points remains more scattered around the correlation with foam objects . It may comes from the irregular geometry of the foam and its numerous defects. It also indicates that foam geometrical description remains probably not sufficient with a single characteristic length (d cell for our work and d S for the previous ones) involved in the mass transfer correlations. In addition, these correlations may still need further improvements to properly capture the involved phenomena.

Re (d S ) = ρ G u (4(1 -)/a s ) µ (7)
Re (d h ) = ρ G u d h µ (8)
Figure 9a confronts the experimental values obtained in this study for foam objects to published correlations of Giani et al. [START_REF] Giani | Mass-Transfer Characterization of Metallic Foams as Supports for Structured Catalysts[END_REF] and Richardson et al. [START_REF] Richardson | Properties of ceramic foam catalyst supports: mass and heat transfer[END_REF] and more recently Bracconi et al [START_REF] Bracconi | A Fundamental Investigation of Gas/Solid Mass Transfer in Open-Cell Foams Using a Combined Experimental and CFD Approach[END_REF] . The evolution of Sh/Sc 1/3 with Re (d S ) are similar and the absolute values lies in between the ones predicted by correlations of both research groups. The more important differences with Giani et al. [START_REF] Giani | Mass-Transfer Characterization of Metallic Foams as Supports for Structured Catalysts[END_REF] and Richardson et al. [START_REF] Richardson | Properties of ceramic foam catalyst supports: mass and heat transfer[END_REF] can be explained by the less quantitative morphological characterizations available when these pioneering papers were realized. to reference published correlations dedicated to square or triangle monolithic channels. [START_REF] Uberoi | External Mass Transfer Coefficients for Monolith Catalysts[END_REF][START_REF] Wibulswas | Laminar-Flow Heat-Transfer in Non-Circular Ducts[END_REF][START_REF] Maestri | Comparison among structured and packed-bed reactors for the catalytic partial oxidation of CH4 at short contact times[END_REF][START_REF] Hawthorn | Effect of heat and mass transfer between gas and catalyst surface[END_REF][START_REF] Holmgren | Enhanced Mass Transfer in Monolith Catalysts with Bumps on the Channel Walls[END_REF] Note that the correlation used by Maestri et al. [START_REF] Maestri | Comparison among structured and packed-bed reactors for the catalytic partial oxidation of CH4 at short contact times[END_REF] comes directly from an analogy with a heat transfer correlation coming from the reference book of Shah and London. [START_REF] Shah | Laminar Flow Forced Convection in Ducts[END_REF] The experimental values appear, in most of the cases, similar or lower than the ones predicted by the various correlations. ) has been derived through least square regression (Equation 12)

with a mean relative deviation of 16% (Figure 10a). This sounds a more direct estimation with a proper geometrical data (today easily measurable through X-ray microtomography)

than the use of a theoretical model of equivalent spheres.

Figure 1 :

 1 Figure 1: Example of one ceramic monolith (MC400), one metallic monolith (MM400) and one metallic foam (FM3.8mm)

Figure 2 :

 2 Figure 2: Set-up used

Figure 3 :

 3 Figure 3: Pressure drop per unit length for different coated structures at 120 • C

Figure 4 :

 4 Figure 4: Effect of a coating on the pressure drop per unit length -experiments at 20 • C

Figure 5 :

 5 Figure 5: ozone conversion vs. contact time at 120 • C for different reactor configurations using MM400 coated cartridges

1 )

 1 Linear pressure drop, ∆P/L (Pa/m)

Figure 6 :

 6 Figure 6: Mass transfer vs. pressure drop for the tested structures at 120 • C

Figure 7 :

 7 Figure 7: Mass transfer coefficients vs. pressure drop for the tested structures at 120 • C

Figure 8 :

 8 Figure 8: Trade-off index obtained for all the structures under various gas flow rates and with 1 to 4 cartridges

Figure 9 :

 9 Figure 9: Comparison of structures performances with the literature data -a) foams, b) monoliths with square channels, c) monoliths with triangular channels

Sh

9 )

 9 (d h ) = 3.01(1 + 0.072 Re (d h ) Sc d h /L) 0.45 (Sh (d h ) = 2.16(1 + 0.081 Re (d h ) Sc d h /L)

Figure 10 : 11 )

 1011 Figure 10: Comparison of data with new correlations -a) foams, b) monoliths with square channels, c) monoliths with triangular channels

Sh (d cell) = 1 .

 1 38 Re 0.43 (d cell) Sc 1/3 (12) Conclusion Eight different structured objects were coated by a Pd/Al 2 O 3 layer and used at high GHSV as ozone converters. As the reaction was strongly mass transfer limited, the effort was focused on finding a good trade-off between mass transfer and pressure drop. The best identified structure was a metallic monolith, with an intermediate number of channels (400 CPSI), higher cell densities being really detrimental to the pressure drop in the presence of catalytic layers. The performance of the structures showed acceptable agreement with predicted standard correlations, even though predictions sometimes differ widely. New correlations were adapted to better represent this new set of data. Nomenclature a s = Volumetric surface area (m 2 • m -3 ) CPSI = Cells Per Square Inch d cell = Foam cell diameter (m) d h = Hydraulic diameter (m) d S = Foam strut diameter (m) e w = Wall thickness (m) ρ G = Gas density (kg • m -3 ) ρ c = Catalyst density (kg • m -3 ) µ = Gas dynamic viscosity (Pa.s)

Table 1 :

 1 Geometrical characteristics of pristine structures determined through X-ray tomography and volumetric image analysis algorithms

		Porosity Spec. surf. area wall thickness Charac. length
	REF	(%)	a )
	MC300	65 ± 0.8	2250 ± 100	265 ± 20	1155 ± 200
	MC400	77.6 ± 0.2	2750 ± 150	178 ± 7	1168 ± 77
	MC600	78.3 ± 0.1	3380 ± 100	111 ± 15	938 ± 71
	MM300	85 ± 0.5	3250 ± 300	70 ± 10	1150 ± 150
	MM400	88.7 ± 0.6	3700 ± 500	50 ± 10	1017 ± 177
	MM600	85.1 ± 0.1	4400 ± 600	51 ± 8	850 ± 150
	FM2mm	89 ± 0.5	895 ± 50	-	2143 ± 183
	FM3.8mm 90 ± 0.5	433 ± 25	-	4510 ± 293

s (m 2 • m -3 ) e w (µm) d h , d cell (µmBecause catalyst coating can affect some final geometrical data of the structures, Table 2 depicts some additional characteristics and a residual porosity. This former parameter was estimated assuming a homogeneous coating and a coating apparent density of 1000 kg • m -3 . The catalytic material loading of the different structures varies significantly

Table 2 :

 2 Mean mass of catalyst and porosity modification of the structures cata /L reactor for the different structures. This is explained by several factors such as very different specific surface areas, a part of dip-coating procedure reproducibility, different geometries and different materials. Foams, presenting a very low specific surface area, can only host 6 to 13 g cata /L reactor . Monoliths classically host 15-20 g cata /L reactor , corresponding to a mean catalytic layer of ca. 5 µm, but 2 samples were out of this range, with a huge loading, probably due to insufficient blowing during the coating process. Note that the reported mass value is the average value between 2 to 4 samples; the uncertainty corresponds to the experimental deviation of the mass of different cartridges of the same batch.

	REF	Mass cata /V reactor (g/L) New porosity (%) Mean coating thickness (µm)
	MC300	17 ± 3	63.3 ± 0.3	6 ± 1
	MC400	63 ± 2	71.3 ± 0.2	23 ± 1
	MC600	18 ± 1	76.5 ± 0.1	5 ± 1
	MM300	17 ± 3	83.3 ± 0.3	5 ± 1
	MM400	51 ± 1	83.6 ± 0.1	13 ± 1
	MM600	15 ± 4	83.6 ± 0.4	3 ± 1
	FM2mm	13 ± 6	87.7 ± 0.6	14 ± 7
	FM3.8mm	6 ± 1	89.4 ± 0.1	13 ± 2
	from 6 to 63 g

Table 3

 3

	: k 1 and k 2 values for the studied coated objects
	REF	k 1 (m 2 )	k 2 (m)
	MC300	2.90.10 -8	-
	MC400	2.39.10 -8	-
	MC600	2.16.10 -8	-
	MM300	3.66.10 -8	-
	MM400	3.10.10 -8	-
	MM600	2.66.10 -8	-
	FM2mm 7.29.10 -8 5.69.10 -3
	FM3.8mm 2.63.10 -7 1.34.10 -2

  The evolution with Re d h /L is conform to what predict these correlations and confirms the interest of the functional form adopted by Hawthorn et al. and other younger works. 34 Despite the dispersion of the different correlations, this new set of results appears acceptable. Experimental mass transfer coefficients (k s ) obtained in the different monolithic structures of this study are satisfactorily represented through 2 new correlations adopting the Hawthorn's formalism with fitted parameters using least squares regression (Equations 9 and 10). A first correlation is specific of the metallic monoliths triangular channels with a mean relative deviation of 16.5% and a second one for the more classical ceramic monoliths with square channels with a mean relative deviation of 15.5%. The matching of each correlation with the experimental data is shown on Figures 10.

Acknowledgement

The authors thank BPI France for the funding of the FUI AURA and AIRCLEAN projects, Fabrice Campoli for his help for the experimental set-up design and ALSYS for providing ceramic monoliths.

FM = Metallic foam

GHSV = Gas Hourly Space velocity (h -1 )