Marwa Drira
email: marwa.drira@cbs.rnrt.tn

Fatma Elleuch
email: fatma.elleuch@ymail.com

Jihen Elleuch
email: jihen.elleuch@gmail.com

Riadh Drira
email: riadh.drira@gmail.com

Florent Boissou
email: florent.boissou@biosedev.com

Julien Souquet-Grumey
email: julien.souquet.grumey@biosedev.com

Sophie Drouillard
email: sophie.drouillard@cermav.cnrs.fr

Didier Le Cerf
email: didier.lecerf@univ-rouen.fr

Philippe Michaud
email: philippe.michaud@uca.fr

Slim Abdelkafi
email: slim.abdelkafi@enis.tn

Imen Fendri
email: imen.fendri@fss.usf.tn

Oligoalginates from Padina pavonica obtained by chemical and mechanical fractionation differentially induce resistance of date palm vitroplants to biotic and abiotic stresses

Keywords: Alginate, ball milling, oligoalginates, elicitor, Date palm, stress, Padina

Oligoalginates from Padina pavonica obtained by chemical and mechanical fractionation differentially induce resistance of date palm vitroplants to biotic and abiotic stresses

Introduction

Micropropagation has been usually used to produce commercially a large number of plants from a single specimen [START_REF] Smith | Current applications of tissue culture in plant propagation and improvement[END_REF][START_REF] Lone | Plant Propagation through Tissue Culture -A Biotechnological Intervention[END_REF]. However, two main problems hampered tissue culture technology. Firstly, microbial contamination that can dominate plant tissues, affects growth and shoot proliferation [START_REF] Romadanova | Effect of plant preservative mixture TM on endophytic bacteria eradication from in vitro-grown apple shoots[END_REF]. Secondly, poor survival plants are observed after transfer to soil due to the sudden changes in light, temperature and relative humidity [START_REF] Chandra | Acclimatization of tissue cultured plantlets: From laboratory to land[END_REF]. To cope with these J o u r n a l P r e -p r o o f Journal Pre-proof harmful factors, such resistance can be activated by plant application with various agents. PGPR, hormones, minerals, proteins or polysaccharides are widely reported to stimulate plant immunity [START_REF] Walters | Controlling crop diseases using induced resistance: Challenges for the future[END_REF]. Algal polysaccharides such as carrageenan, fucan, laminarin and alginate derived from macroalgae were successfully proved as a plant's protector as well as growthpromoting agents [START_REF] Drira | Optimization of exopolysaccharides production by Porphyridium sordidum and their potential to induce defense responses in Arabidopsis thaliana against Fusarium oxysporum[END_REF][START_REF] Drira | Influence of the sulfate content of the exopolysaccharides from Porphyridium sordidum on their elicitor activities on date palm vitroplants[END_REF][START_REF] Zhang | Preparation of alginate oligosaccharides and their biological activities in plants: A review[END_REF][START_REF] Riseh | Alginate-induced disease resistance in plants[END_REF][START_REF] Moenne | Chitosan-, alginate-carrageenan-derived oligosaccharides stimulate defense against biotic and abiotic stresses, and growth in plants: A historical perspective[END_REF]. Alginates are heteropolysaccharides from brown macroalgae.

They are defined based on β-D-mannuronic (M) and α-L-guluronic (G) acids [START_REF] Smith | Current applications of tissue culture in plant propagation and improvement[END_REF][START_REF] Chandra | Acclimatization of tissue cultured plantlets: From laboratory to land[END_REF] linked.

These monomers can be arranged on homogenous (MM or GG) and heterogeneous (M/G) blocks. The proportion of these blocks determines the biological and physicochemical proprieties of these anionic polysaccharides [START_REF] Hentati | Production, extraction and characterization of alginates from seaweeds, Handbook of Algal Technologies and Phytochemicals: Volume I: Food[END_REF]. Thanks to their nontoxicity and biodegradability, alginates or oligoalginates have been demonstrated as plant growth stimulators and found to enhance seed germination and seedling growth of maize [START_REF] Hu | Promotive effects of alginate-derived oligosaccharide on maize seed germination[END_REF] and wheat [START_REF] He | Effects of calcium alginate submicroparticles on seed germination and seedling growth of wheat (Triticum aestivum L.)[END_REF]. Moreover, they were reported to promote root and shoot growth of several plants such as barley [START_REF] Yang | Growth stimulation activity of alginatederived oligosaccharides with different molecular weights and mannuronate/guluronate ratio on Hordeum vulgare L[END_REF], rice and peanut [START_REF] Hien | Growth-promotion of plants with depolymerized alginates by irradiation[END_REF]. Besides, alginate-derived polysaccharides were proved to mitigate the negative effects of abiotic stress including heavy metals [START_REF] Li | An alginate-derived oligosaccharide enhanced wheat tolerance to cadmium stress[END_REF], high temperature [START_REF] Yang | Alginate oligosaccharides alleviate the damage of rice leaves caused by acid rain and high temperature[END_REF], drought [START_REF] Li | Alginate-derived oligosaccharides promote water stress tolerance in cucumber (Cucumis sativus L.)[END_REF][START_REF] Liu | Alginate oligosaccharides enhanced Triticum aestivum L. tolerance to drought stress[END_REF] and salinity [START_REF] Salachna | Oligo-alginate with low molecular mass improves growth and physiological activity of Eucomis autumnalis under salinity stress[END_REF][START_REF] Golkar | Effects of sodium alginate elicitation on secondary metabolites and antioxidant activity of safflower genotypes under in vitro salinity stress[END_REF]. Further research demonstrated that those biopolymers are effective elicitors that improve plant resistance to several pathogens by stimulating the expression of the genes involved in salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) dependent signaling pathways [START_REF] Riseh | Alginate-induced disease resistance in plants[END_REF]. A foliar spray containing oligoalginates was applied to Arabidopsis thaliana leading to a decrease of disease symptoms provoked by Pst DC3000 by inducing the early accumulation of nitric oxide (NO), ROS and the transcript level of PR1 [START_REF] Zhang | Alginate oligosaccharide (AOS) induced resistance to Pst DC3000 via salicylic acid-mediated signaling pathway in Arabidopsis thaliana[END_REF]. Similarly, Dey et al., [START_REF] Dey | Sodium alginate potentiates antioxidant defense and PR proteins against early blight disease caused by Alternaria solani in Solanum lycopersicum Linn[END_REF] demonstrated that tomato leaves sprayed with oligoalginates showed higher resistance to Alternaria solani by regulating the antioxidant enzymes and PR proteins.

Date palm tissue culture for large-scale multiplication has gained industrial relevance [START_REF] Mazri | Micropropagation of date palm: A review[END_REF].

Bacterial (e.g. Bacillus, Pseudomonas, Staphylococcus, Proteus) and fungal (e.g. Alternaria

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Journal Pre-proof alternate, Aspergillus niger, Penicillium spp. contaminations of cultures [START_REF] Mazri | Micropropagation of date palm: A review[END_REF][START_REF] Benjama | Control of Bacillus contaminating date palm tissue in micropropagation using antibiotics[END_REF] and high mortality during acclimatization [START_REF] Pospóšilová | Acclimatization of micropropagated plants to ex vitro conditions[END_REF] are still unresolved obstacles affecting the subsequent steps of date palm cultivation.

To evaluate the elicitor effect of oligoalginates from Padina pavonica, a brown macroalgae from Tunisian coats, oligomers were produced with acid and mechanocatalytic hydrolysis and then tested on date palm vitroplants subjected to abiotic and biotic stresses.

Materials and methods

Extraction of alginate from Padina pavonica (SAPP)

Padina pavonica was collected from Mahdia-Tunisia seawater in (35.410995, 11.041932) in August 2021. Algae were washed with tap water before sun-drying at ambient temperature.

The dry algal biomass was then ground using an electric blender. Alginates were extracted according to Hentati et al., [START_REF] Hentati | Structural characterization and antioxidant activity of water-soluble polysaccharides from the Tunisian brown seaweed Cystoseira compressa[END_REF]. Fifty grams of algae powder were soaked twice in 700 mL of acetone and then twice with 300 mL of methanol during 24 h at room temperature under constant agitation. Subsequently, biomass was dried in oven at 50°C for 24h. Twenty-five grams of depigmented powder were treated twice with HCl (0.1 M, pH: 2, 500 mL) for 2h at 60°C under constant agitation. The insoluble fraction was collected by centrifugation (10,000×g, 10 min, 20°C) and washed with ultra-pure water before extraction with sodium carbonate (3% (w/v), pH = 11, 500 mL) at 60 °C for 2 h under stirring. After centrifugation (10,000×g, 15 min, 4°C) polysaccharides were precipitated by three volumes of cold ethanol 96%. Sodium alginate collected by centrifugation (12,000×g, 30 min, 4°C) was dissolved in ultra-pure water and acidified by HCl (6M) at pH value comprised between 1.5 and 3. Precipitated alginic acid collected by centrifugation (5,000×g, 20 min, 4°C) was solubilized in ultra-pure water and neutralized with NaOH (1 M). It was then precipitated again with three volumes of cold ethanol 96% and collected by centrifugation (12,000×g, 30 min, 4°C).

Finally, sodium alginate (SAPP) was solubilized in ultra-pure water and then freeze-dried.

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Partial acid depolymerization of Padina pavonica alginate was performed according to Fenoradosoa et al., [START_REF] Fenoradosoa | Extraction and characterization of an alginate from the brown seaweed Sargassum turbinarioides Grunow[END_REF]. Briefly one gram of sodium alginate was dissolved in 100 mL of ultra-pure water and then heated at reflux with 3 mL of HCl 3 M for 20 min. After centrifugation (3,000×g, 20 min, 20°C), the supernatant was neutralized with NaOH (1M) and subsequently 100 mL of ethanol 96% were added. The precipitate was collected by centrifugation (10,000×g, 20 min, 20°C), dissolved in ultra-pure water and then freeze-dried (fraction G+M). The pellet of the first centrifugation was dissolved in 100 mL of HCl 0.3M and heated at reflux for 2h. The suspension was centrifuged (10,000×g, 20 min, 20°C) and the insoluble fraction was neutralized and then the pH was adjusted to 2.85. After centrifugation (10,000×g, 20 min, 20°C), the soluble fraction was neutralized, dialyzed against ultra-pure water and finally freeze-dried. (Fraction M). The pellet was dissolved in ultra-pure water, neutralized, dialyzed and freeze-dried (Fraction G).

Acid-Assisted Ball Milling depolymerization of Padina pavonica and its alginate

Acid-assisted ball milling depolymerization of dried Padina pavonica powder (PPP) or of SAPP was performed according to Boissou et al., [START_REF] Boissou | Acid-assisted ball milling of cellulose as an efficient pretreatment process for the production of butyl glycosides[END_REF]. Samples were impregnated with acid.

Typically, 1 g of PPP or SAPP was suspended in 15 mL of acetone containing 4 wt % (0,45mmol) H 2 SO 4 . The suspension was shaken for 1 hour. The solvent was then removed under vacuum at 40°C, affording a powder. The acid impregnated PPP or SAPP was immediately introduced into a planetary ball mill (Retsch PM 100) and was milled for the desired time. The two obtained samples were named hereafter PPBM and OABM for acidassisted ball milling depolymerization of PPP and SAPP respectively.

Hydrolysis Yield

To determine the yield of the degradation process, the weight of the used alginate and obtained hydrolysis product were determined and the yield of the process was calculated Where Mn and Mw are the number-average molecular mass and the weight-average molecular mass of alginate or oligoalginates respectively and 198 g/mol represents the repeat unit in the sodium salt form.

NMR spectroscopy analyses of SAPP, OABM, M, G and G+M fractions

Samples were dissolved in D 2 O and lyophilized to replace exchangeable protons with deuterium (two times). 13 C and 1 H NMR spectra were recorded with a Bruker Avance 400 spectrometer operating at a frequency of 100.618 MHz for 13 C and 400.13 MHz for 1 H. Samples were solubilized in D 2 O at a temperature of 353 K for the polysaccharide and

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Journal Pre-proof residual signal of the solvent was used as internal standard: HOD at 4.25 ppm at 353 K. 13 C spectra were recorded using 90° pulses, 20,000 Hz spectral width, 65,536 data points, 1.638 s acquisition time, 1 s relaxation delay and between 8192 and 16,834 scans. 1 H spectra were recorded with a 4006 Hz spectral width, 32,768 data points, 4.089 s acquisition times, 0.1 s relaxation delays and 16 scans.

On the bases of Grasdalen method [START_REF] Grasdalen | High-field, 1H-n.M.R. Spectroscopy of alginate: Sequential structure and linkage conformations[END_REF], the areas of A, B and C signals were used to calculate the individual blocks of mannuronic and guluronic acids (FG and FM), the homopolymeric (FGG and FMM) and heteropolymeric blocks (FGM and FMG) as reported by equations ( 3) and ( 4).

FTIR analyses

The IR spectra were recorded using VERTEX 70 FTIR instrument with ATR A225 diamante (Bruker, Palaiseau, France) at room temperature in the wavenumber range of 600-4,000 cm -1 at 4 cm -1 resolution. The acquisition and the processing of spectra have been carried out using the "Spectrum" software.

Biochemical characterization of SAPP and PPBM

Total carbohydrate content was determined by the phenol and sulfuric acid assays with glucose as standard [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF]. Neutral sugar and uronic acid contents were quantified using

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Journal Pre-proof respectively the sulfuric resorcinol method with glucose as standard [START_REF] Monsigny | Colorimetric determination of neutral sugars by a J o u r n a l P r e -p r o o f Journal Pre-proof resorcinol sulphuric acid micromethod[END_REF] and the mhydroxydiphenyl (MHDP) assay with glucuronic acid as standard [START_REF] Blumenkrantz | New method for quantitative determination of uronic acids[END_REF]. Protein concentrations were determined according to Bradford method with bovine serum albumin as standard [START_REF] Bradford | A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF].

Elicitor treatment

Date palm vitroplants var. Khnizi at the end of rooting stage were obtained by in vitro vegetative multiplication and kindly provided by the commercial laboratory "Green Biotechnology", Sfax, Tunisia. For elicitor treatments SAPP, OABM, PPBM, G+M, G or M fractions at final concentration of 1 mg/mL were added to the ½ MS medium [START_REF] Murashige | A revised medium for rapid growth and bioassays with tobacco tissue cultures[END_REF]. After 48 h vitroplants were harvested and stored at -80°C for further uses.

Extraction of RNA, synthesis of cDNA and Real-time quantitative PCR

Total RNA extraction was performed on the fresh vitroplant of date palm (100 mg) with the plant-RNA mini kit (Qiagen, USA) in accordance with the manufacturer's instruction.

Five µg of total RNA were used to synthesize cDNA using PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Kyoto, Japan) according to the manufacturer's procedure. The real-time qPCR was conducted for PAL (phenylalanine ammonia-lyase), PR1 (Pathogenesis-related protein 1) SOD (superoxydes dismutases) and DHN (dehydrin) (Table 1). The amplification reactions were performed in 10 μL final volumes containing 5 μL 2x TB green (Takara), 0.4 μL dye, 1 μL primer-pair mix (0.5/ 0.5 μM for forward and reverse primers), 1 μL cDNA, and 3 μL RNase-free water. Reactions were carried out in StepOnePlus™ PCR cycler (Applied Biosystems, Foster City, USA).

Thermal cycling conditions were performed as described by Drira et al., [START_REF] Drira | Optimization of exopolysaccharides production by Porphyridium sordidum and their potential to induce defense responses in Arabidopsis thaliana against Fusarium oxysporum[END_REF]. Transcript abundance of each selected gene was normalized to the housekeeping gene EF-1a and was determined with the 2 -ΔΔCt method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆CT method[END_REF].

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Table 1: Specific primers used for qRT-PCR.

Gene

Stress treatments

After 48 of elicitor treatments, vitroplants were transferred in liquid MS medium supplemented with 200 mM NaCl for salt stress, 200 mM mannitol for drought stress or inoculated with 10 6 of Aspergillus niger for biotic stress and incubated for 4 additional weeks.

Measurement of chlorophyll content

Fresh leaves (100 mg) of untreated and treated date palm with OABM were collected and chlorophyll was extracted using 80% acetone. After centrifugation (10,000xg, 10 min, 4°C).

The A 645 and A 663 of the supernatants were measured and the total chlorophyll content in each sample, were calculated according to Drira et al. [START_REF] Drira | Improvement of Arabidopsis thaliana salt tolerance using a polysaccharidic extract from the brown algae Padina pavonica[END_REF].

Leaf disease severity measurement

The leaf disease severity (LDS) of vitroplants infected by Aspergellus niger was estimated using the ImageJ software (https://imagej.net/ij/) and calculated as following (equation 6) : The extraction yield of SAPP (around 10.8% ± 0.9 (w/w)) is near to those achieved in different species such as Sargassum turbinarioides (10%) [START_REF] Fenoradosoa | Extraction and characterization of an alginate from the brown seaweed Sargassum turbinarioides Grunow[END_REF], Cystoseira sedoides (11%)

and Fucus guiryi (13.6%) [START_REF] Belattmania | Isolation and FTIR-ATR and 1H NMR characterization of alginates from the main alginophyte species of the Atlantic Coast of Morocco[END_REF]. Since alginates are polyuronic polymers [START_REF] Hentati | Production, extraction and characterization of alginates from seaweeds, Handbook of Algal Technologies and Phytochemicals: Volume I: Food[END_REF], the total sugars (56.68%) is mainly composed by uronic acids (48.18%) with minors amount of proteins (0.73%).

FTIR spectroscopy was used to partially characterize the functional groups of SAPP. Fig. 1, showed clearly the fingerprint of alginates that is observed in many studies [START_REF] Hentati | Structural characterization and antioxidant activity of water-soluble polysaccharides from the Tunisian brown seaweed Cystoseira compressa[END_REF][START_REF] Belattmania | Isolation and FTIR-ATR and 1H NMR characterization of alginates from the main alginophyte species of the Atlantic Coast of Morocco[END_REF][START_REF] Rashedy | Evaluation and characterization of alginate extracted from brown seaweed collected in the Red Sea[END_REF]. In fact, SAPP displayed several characteristic signals at 3274, 1600, 1317, 1123, 1083, 1025, 946, 902 and 808 cm -1 . According to Fenoradosoa et al., [START_REF] Fenoradosoa | Extraction and characterization of an alginate from the brown seaweed Sargassum turbinarioides Grunow[END_REF], Hentati et al, [START_REF] Hentati | Structural characterization and antioxidant activity of water-soluble polysaccharides from the Tunisian brown seaweed Cystoseira compressa[END_REF] and Hentati et al., [START_REF] Hentati | Production, extraction and characterization of alginates from seaweeds, Handbook of Algal Technologies and Phytochemicals: Volume I: Food[END_REF], the band at 3274 cm -1 was assigned to hydrogen bond O-H stretching vibrations.

The signal observed at 1600 cm -1 could be attributed to carboxylate O-C-O asymmetric stretching vibrations. Peaks at 1317, 1123 cm -1 could be assigned respectively to the deformation of (O-C-H) groups and to the elongation vibration of the (C-O) groups of pyranose rings. In addition, the bands at 1083 and 1025 cm -1 correspond to the units of guluronic and mannuronic acids respectively. The signal at 946 cm -1 may be attributed to the C-O stretching vibration of uronic acids. More peaks at 902 and 808 cm -1 could be assigned to C-O stretching vibrations of alginate backbone (Fig. 1).

J o u r n a l P r e -p r o o f Mw values ranging from 106 and 177 kDa [START_REF] Rioux | Characterization of polysaccharides extracted from brown seaweeds[END_REF].

For detailed block structure of the SAPP, 1 H-NMR spectroscopy was used and presented in Fig. 2. The values of FG, FGG, FM, FMM and FGM or FMG were respectively 0.554, 0.573, 0.446, 0.427 and 0.019. Moreover, SAPP had a low M/G ratio of 0.805 (<1) indicating that G units were abundant in the alginate polymer. Thus, the parameter η = 0.08 (<1) demonstrated a high quantity of GG homopolymeric blocks. By contrast, the M/G ratio of some other Padina alginates such as Padina gymnospora and Padina antillarum were 1.75(>1) and 1.85(>1) respectively [START_REF] Rhein-Knudsen | Characterization of alginates from Ghanaian brown seaweeds: Sargassum spp. and Padina spp[END_REF]. The variability of data obtained from the same species could be explained by the influence of environment on the composition of alginates [START_REF] Rhein-Knudsen | Characterization of alginates from Ghanaian brown seaweeds: Sargassum spp. and Padina spp[END_REF].

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Structural features of oligoalginates (OABM) obtained by acid assisted ball milling

Ball milling in the presence of an acid catalyst (H 2 SO 4 ) was considered as a promising tool for particle size reduction of many bioorganic material such as polysaccharides especially cellulose [START_REF] Boissou | Acid-assisted ball milling of cellulose as an efficient pretreatment process for the production of butyl glycosides[END_REF] and chitin [START_REF] Zhong | The influence of pre-fibrillation via planetary ball milling on the extraction and properties of chitin nanofibers[END_REF]. However, mechanical treatment of alginate was never demonstrated. Alginate acid-assisted ball milling depolymerization of SAPP was verified by J o u r n a l P r e -p r o o f Journal Pre-proof anomeric region, corresponding to the C-1 of mannuronic and guluronic acids were assigned at 101.85 ppm (M1) and 101.32 ppm (G1) respectively. Other chemical shifts are listed in supplemental table [START_REF] Grasdalen | High-field, 1H-n.M.R. Spectroscopy of alginate: Sequential structure and linkage conformations[END_REF]. It's worth to note that chemical shifts at 94.15, 69.14 and 74.28 ppm could be attributed to C1, C5 and C3 reducing end respectively [START_REF] Yang | Expression and characterization of a new polyg-specific alginate lyase from marine bacterium microbulbifer sp. Q7[END_REF].

OABM was structurally characterized by the ratio M/G (0.33) < 1 indicating that mechanical depredated alginate exhibited higher amounts of monomer G (FG=0.75) and diad GG (FGG=0.72) than the monomer M (FM= 0.25) and diad MM (FMM=0.22). The value of FMG or FGM was 0.03. Interestingly, we note that the acid-assisted ball milling depolymerization gives higher values for the G block and lower values for MG blocks (75% and 3% respectively) compared to the native alginate (60 % and 19 % respectively). It seems that the mechanical hydrolyses may affect the MG blocks.

The parameter η (0.18<1) which completes the description of the OABM sequence reveals the abundance of homopolymeric blocks.

Interestingly, based on the SEC-MALLS analyses, OABM was characterized by a low Mw and Mn (4.3 ± 0.28 and 3.43 ± 0.47 kDa respectively). A low polydisperse character was deduced by a polydispersity index < 2 (Ð =1.25). In addition, the calculated degree of polymerization (DPn= 17.32) confirms that ball milling could be an effective method for alginate depolymerization which produces homogeneous hydrolysates composed of oligomers with short chain lengths.

Physicochemical characterization of PPBM

After ball milling treatment of Padina pavonica powder, the chemical analysis of PPBM demonstrated its richness in total sugar (31% (w/w)) mainly composed by neutral carbohydrates (26.15 (w/w)) and lower content of uronic acids (12.29 % (w/w)) with the presence of minor amount of proteins (1% (w/w)). Those results were close to those obtained with untreated Padina pavonica [START_REF] Drira | Improvement of Arabidopsis thaliana salt tolerance using a polysaccharidic extract from the brown algae Padina pavonica[END_REF]. Moreover, it can be seen in Fig. 4 that the general

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Journal Pre-proof profile FTIR spectra of untreated and treated algae powder was similar. The two bands at 1080 and 876 cm -1 are more prominent in the PPBM spectra which are characteristic to the guluronic and mannuronic acids respectively. This might be attributed to the alginate depolymerization after mechanocatalytic treatment of the dried algae.

1 H-NMR analysis of G+M, G and M fractions

Compared to the 1 H-NMR spectrum of the native alginate SAPP (Fig. 2), Fig. 6 (signal A) and HG-5 (signal C) respectively (Fig. 6). Similar 1 H-NMR spectra profiles were obtained after chemical hydrolysates alginates extracted from Cystoseira trinode, Cystoseira myrica, Sargassum dentifolium, Sargassum asperifolium and Sargassum latifolium [START_REF] Larsen | r n a l P r e -p r o o f Journal Pre-proof Characterization of the alginates from algae harvested at the Egyptian Red Sea coast[END_REF].

J o u r n a l P r e -p r o o f The results of the chemical fractionation for the SAPP are summarized in Table 3. In agreement with the FTIR analysis (Fig. 5), the obtained fractions varied in terms of their monomeric block distributions. In fact, fraction M showed the highest MM content, while the highest content in GG was observed in G fractions. The measured M/G values confirmed that G+M and G fractions were rich with G units (M/G < 1), whereas in the M fraction (M/G >1) the M units were abundant. Moreover, the block distribution of M and G fractions were homogenous with calculating η parameter (<1) 0.45 and 0.15 respectively. In the case of M+G fraction the parameter η (>1) demonstrates a high distribution of heteropolymeric blocks (Table 3).

J o u r n a l P r e -p r o o f the fraction obtained by acid hydrolysis, OABM has the much higher yield (71.5 %) after mechanical degradation. This suggests that ball milling assisted depolymerization could be an effective method for potential industrial use.

Effect of SAPP, OABM, PPBM, G+M, G and M treatments on the induction of plant defense gene expression

To investigate whether SAPP and their derivatives activated date palm vitroplants resistance to diverse environmental stresses, we the expression levels of PAL, SOD, PR1 and DHN was monitored. PAL is considered as an efficient marker, which involved in the plant cells defense response to ROS generated under environmental stresses [START_REF] Macdonald | A modern view of phenylalanine ammonia lyase[END_REF]. SOD is the first important line of antioxidant enzyme defense systems against ROS [START_REF] Zelko | Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression[END_REF]. DHN play key role in plant adaptation to abiotic and biotic stresses [START_REF] Drira | Comparison of full-length and conserved segments of wheat dehydrin DHN-5 overexpressed in Arabidopsis thaliana showed different responses to abiotic and biotic stress[END_REF] and PR1 serves as molecular indicator in the case of pathogen attack [START_REF] Sudisha | Pathogenesis related proteins in plant defense response[END_REF].

The relative expression levels of all the analyzed genes were normalized with the elongation factor (EF-a) gene. Compared to those of untreated date palm vitroplants, all the studied genes were activated differentially in regard to the elicitor treatment. Interestingly, the highest transcription levels were observed in the vitroplants treated with oligoalginates J o u r n a l P r e -p r o o f

Journal Pre-proof obtained with mechanical depolymerization (OABM) followed by those obtained by chemical hydrolysis. The up-regulated transcription levels were similar in both PPBM and SAPP treated plants (Fig. 7).

A large number of studies support that deferential elicitor responses of alginates and their derivatives may be associated with their structural characteristics. In fact, the molecular weight, the M/G ratio and the degree of polymerization were key factors affecting their biological activities [START_REF] Zhang | Preparation of alginate oligosaccharides and their biological activities in plants: A review[END_REF][START_REF] González | Seaweed oligosaccharides stimulate plant growth by enhancing carbon and nitrogen assimilation, basal metabolism, and cell division[END_REF]. For example, oligoalginates fractions obtained by γ-irradiation with the lowest M W (1-3 kDa) exhibited the highest increased leaf biomass of flower vitroplants [START_REF] Luan | Biological effect of radiation-degraded alginate on flower plants in tissue culture[END_REF]. Likewise, oligoalginates with the lower degree of polymerization values obtained with an alginate lyase, mitigate the negative effects of salt stress in Brassica campestris by the stimulation of the superoxide dismutase (SOD) and guaiacol peroxidase (POD) activities [START_REF] Tang | Characterization of alginase and elicitor-active J o u r n a l P r e -p r o o f Journal Pre-proof oligosaccharides from Gracilibacillus A7 in alleviating salt stress for Brassica campestris L[END_REF]. In contrast with our results, M fraction obtained with chemical depolymerization was proved to be more active than G one to stimulate defense response against tobacco mosaic virus in tobacco [START_REF] Laporte | Structurally unrelated algal oligosaccharides differentially stimulate growth and defense against tobacco mosaic virus in tobacco plants[END_REF]. Results from this study, revealed clearly that there is a close relationship between oligosaccharides structure and the elicitor activity. The structural modification of SAPP by reducing the Mw and DP may improve their functionality. As suggested by many authors, the low M W and DP may help the oligosaccharides to bind plant cell receptors and facilitate the stimulation of certain signal transduction pathways [START_REF] Naeem | Radiation-Processed Polysaccharides: Emerging Roles in Agriculture[END_REF].

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Phenotypic and physiological analyzes of vitroplants treated by OABM under abiotic and biotic stresses

Beside transcriptional analysis, tolerance of vitroplants treated with oligoalginates (OABM) was investigated under salt, drought and fungal contaminations. In the absence of stress, similar phenotypes, fresh weight, root length and chlorophyll content were observed between control and treated vitroplants under normal conditions (MS), indicating that OABM had no detrimental effect on growth (Fig. 8a, Fig. 9). Four weeks of salt stress applications led to the shoot and root growth inhibition and development of chlorotic leaves in control plants, whereas growth inhibition and chlorophyll content were less pronounced in treated J o u r n a l P r e -p r o o f vitroplants (Fig. 8b, Fig. 9). Likewise, Salachna et al [START_REF] Salachna | Oligo-alginate with low molecular mass improves growth and physiological activity of Eucomis autumnalis under salinity stress[END_REF] reported that oligoalginates improve morphological features, physiological and biochemical parameters of Eucomis autumnalis under salt stress application. In fact, those authors suggested that oligoalginates may activate plant stress adaptation mechanisms via the stimulation of antioxidants such as a high accumulation of L-ascorbic acid.

Under drought stress, treated OABM plants revealed phenotypic tolerance associated with the apparition of secondary roots (Fig. 8b). In addition, fresh weight, root length and chlorophyll content values were strongly decreased in control vitroplants, whereas these values were less pronounced in OABM treated vitroplants (Fig. 9). Those results were similar to those observed by Liu et al., [START_REF] Liu | Alginate oligosaccharides enhanced Triticum aestivum L. tolerance to drought stress[END_REF] which demonstrated that application of oligoalginates in barley plants confers an increased root length and fresh weight compared to the PEG plants under dehydration status.

Fungal contamination is one of the major causes of in vitro culture losses that led finally to the death of tissues [START_REF] Benjama | Control of Bacillus contaminating date palm tissue in micropropagation using antibiotics[END_REF]. The genus Aspergillus is the most abundant contaminant which is identified in date palm tissue culture growth media [START_REF] Mohammed | A PCR ITS-RFLP method for identifying fungal contamination of date palm (Phoenix dactylifera L.) tissue cultures[END_REF]. The J o u r n a l P r e -p r o o f and G + M fractions) in order to compare their elicitor activity on date palm with the native alginate (SAPP). We demonstrated that OABM with homogenous distribution of blocks, low Mw and DP showed the highest capacity to induce defense genes expression levels.

Moreover, OABM treated vitroplants were able to mitigate the negative effects of salt, drought and fungal contamination. Based on those results, the products of ball milling depolymerization of alginate oligosaccharides could act as novel environment-friendly elicitors agents that can efficiently protect vitroplants of date palm against abiotic and biotic stresses for successful micropropagation process.

2 . 5 .

 25 J o u r n a l P r e -p r o o f Journal Pre-proof using the following equation (1) (1) % 𝑦𝑖𝑒𝑙𝑑 = 𝑊𝑝 Wa with Wp : weight of the obtained hydrolysate and Wa : weight of used alginate Molecular Weight Analysis by HPSEC-MALLS High-pressure Average molecular weights of SAPP, OABM, M, G and G+M blocks were determined by high pressure size exclusion chromatography (HPSEC) comprising on-line detectors including a multi-angle laser light scattering (MALS) filled with a He-Ne laser at 690 nm and a K5 cell (50 µl) (HELEOSII, Wyatt Technology Corp., USA) equipped with 18 measuringdiodes and calibrated with Toluene and BSA, and a differential refractive index (DRI) (RID10 A Shimadzu, Japan). Columns (OHPAK 804 and 806 HQ columns (Shodex)) were eluted with LiNO 3 (0.1M) at 0.5 ml.min -1 . Samples (10 g/L) were solubilized in 0.1 M LiNO 3 for 24h at room temperature under stirring. Solutions were then filtrated (0.45 µm filter) and injected in a 100 µL full loop. The collected results were analyzed on the basis of a dn/dc of 0.15 mL/g. The collected data were analyzed using the Astra 6.1 software package. The number average degree of polymerization (DPn) and the weight-average degree of polymerization (DPw) values were estimated using equation (2)[START_REF] Andriamanantoanina | Relationship between the molecular structure of alginates and their gelation in acidic conditions[END_REF].

1 ( 4 )

 14 𝐹𝐺𝑀 = 𝐹𝑀𝐺 = 𝐹𝐺 -𝐹𝐺𝐺, 𝐹𝑀𝑀 = 𝐹𝑀 -𝐹𝑀𝐺 Where A, B and C are the relative areas at 4.96-5.18 ppm (H-1 of G), 4.57-4.82 ppm (H-5 of G and H-1 of M) and 4.38-4.55 ppm (H-5 of G) respectively. The block distributions in alginate or oligoalginate were estimated by the calculation of the parameter η (equation 5)

1 .

 1 Structural features of sodium alginates of Padina pavonica (SAPP)Extraction yield of alginate depend on the algae species, age, season and ecological habitat.

Figure 1 :

 1 Figure 1: FT-IR spectra of SAPP

Figure 2 :

 2 Figure 2: 1 H-NMR spectra of SAPP

1 H

 1 -NMR. There were some different chemical shifts of signals compared to the 1 H-NMR profile of native alginate (Fig 2and 3a). An increase in signal intensities of the terminal reducing end was observed at around 5.27 ppm and 4.95 ppm which represent M1-redα and G1-redβ respectively (Fig3a). 13C-NMR was also performed to get insight about the monomer composition and the structure of the chain ends of OABM (Fig.3b).

Figure 3 :

 3 Figure 3: 1 H-NMR (a) and 13 C-NMR (b) spectra of OABM

Figure 4 : 3 . 4 . 3 . 4 . 1 .

 434341 Figure 4: FTIR spectra of untreated (Bleu spectra) and ball milling treated (Pink spectra)

Figure 5 : 3 . 4 . 2 .

 5342 Figure 5: FTIR analysis of the fractions G+M (black spectra), G (red spectra) and M (Green

  showed an increase in signals intensities for G reducing end (G-1 redβ) at 4.85 and 4.95 ppm for G and G+M fractions. Only a slight increase in the M reducing end (M-1 redβ) at around 4.95 ppm for the G+M fraction was detected. In the case of M fraction, an increase in signals intensities for M reducing end M-1 redα and M-1 redβ was observed at around 5.25 and 4.95 ppm respectively. Moreover, remarkable decline in internal H M -1+H GM -5 (signal B) and H G -5 (signal C) for G and G+M fractions respectively was measureded. The 1 H-NMR spectrum of M fraction showed an important decrease in the both internal signal intensities of HG-1

Figure 6 :

 6 Figure 6: 1 H-NMR spectra of G fraction (green spectra), M fraction (red spectra) and G+M

Figure 7 :

 7 Figure 7: Relative expression level of defense-related genes PAL, PR1, SOD, WRKY in date

  Fig 8b demonstrated that the addition of OABM in MS medium enhanced the resistance of vitroplants to the Aspergillus niger expressed by a minor necrosis area in treated plant (LDS=10%) compared to the untreated ones (LDS=36%) (Fig 8a).

Figure 8 :

 8 Figure 8: Phenotypic analyzes of untreated date palm vitroplants (a) and OABM treated

Figure 9 :

 9 Figure 9: Physiological parameters in untreated and OABM treated plants date palm

J o u r

  n a l P r e -p r o o f Journal Pre-proof Conclusion Oligosaccharides were generated by using two different degradation methods of sodium alginate (SAPP): mechanocatalytic depolymerization (OABM) and hydrochloric acid (G , M

Table 2 :

 2 SEC-MALLS results of G+M, G and M fractions

	Fraction	Yields (%) Mn (kDA) Mw (KDa) Ð	DPn	DPw
	G+M	9.98±0.08	1.5±0.64	16.6±0.55	11.09
	G	51.72±0.49 5.92±0.45	9.87±0.25	1.67	29.89	49.84
	M	12.97±0.12 4.04±0.33	7.53±0.25	1.86	20.4	38.03

Table 3 :

 3 Distribution of monomers and dyads in G+M, G and M fractions

	Fraction	FG	FM	FGG	FMM	FMG	M/G	η
	G+M	0.9	0.1	0.0056	0.0	0.89	0.11	9.88
	G	0.98	0.02	0.95	0.0	0.03	0.02	0.15
	M	0.14	0.86	0.084	0.776	0.056	6.14	0.45
	Conclusively, the two different methods of alginate depolymerization generate alginate
	oligosaccharides with different blocks distribution, Mw and DP. Interestingly, compared to

Acknowledgments

This work was supported by Grants from the Ministry of Higher Education and Scientific

J o u r n a l P r e -p r o o f

Journal Pre-proof Research of Tunisia.

Funding

This research received no external funding.

Declaration of competing interest

The authors declare that there is no conflict of interest and give their informed consent. No financial or other interests influenced the outcome of the research.

Conflicts of Interest

The authors declare no conflict of interest


Milad Jamalzadeh

Yosra Rekik

Laurent Grisoni

The Effect of Attention Saturating Task on Eyes-free Gesture Production on Mobile Devices

come

Introduction

Gesture Features

This project has received funding from the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement No 860114.

Conclusion Methods

• 13 Right handed participants (7 males and 6 females) The results indicated that performing another task while drawing a gesture shortened the length and size of the gesture. Conversely, drawing a gesture with the left hand increased the length of the gesture. Additionally, engaging in another task while drawing the gesture reduced the duration of gesture entry, while the finger maintained the same speed across the touchscreen. Using the left hand to produce eyes-free gestures when performing an attention-saturating task is also more frustrating and demands more physical effort than when using the right hand or when there is no primary task.

(Gesture set) (Setup)

Acknowledgements


G6P: glucose 6-phosphate; PEP: phosphoenolpyruvate; 3PG: 3-phosphoglycerate; 3P-Pyr: 3-phosphohydroxypyruvate; 3P-Ser: 3-phosphoserine.

Figure 1 :

 1 Fig 1

Figure 2 :

 2 Figure 2: Chromatin bound MDM2 regulates an ATF-dependent transcriptional

Figure 3 :

 3 Figure 3: Serine/glycine deprivation regulates MDM2 recruitment to chromatin

Figure 5 :

 5 Figure 5: PKM2 and oxidative stress control MDM2 recruitment to chromatin

Figure 6 :

 6 Figure 6: MDM2 is a key regulator of metabolism to sustain cellular anti-oxidant


Camille Quenon
email: camille.quenon@icloud.com

Thierry Hennebelle
email: thierry.hennebelle@univ-lille.fr

Jean-François Butaud
email: jfbutaud@hotmail.com

Raimana Ho
email: raimana.ho@upf.pf

Jennifer Samaillie
email: jennifer.samaillie@univ-lille.fr

Christel Neut
email: christel.neut@univ-lille.fr

Tamatoa Lehartel
email: tamatoa.lehartel@upf.pf

Céline Rivière
email: celine.riviere@univ-lille.fr

Ali Siah
email: ali.siah@yncrea.fr

Natacha Bonneau
email: natacha.bonneau@univ-lille.fr

Sevser Sahpaz
email: sevser.sahpaz@univ-lille.fr

Sébastien Anthérieu
email: sebastien.antherieu@univ-lille.fr

Nicolas Lebegue
email: nicolas.lebegue@univ-lille.fr

Phila Raharivelomanana

Vincent Roumy
email: vincent.roumy@univ-lille.fr

Antimicrobial Properties of Compounds Isolated from Syzygium

Keywords: Society Islands, traditional medicine, phytochemical, antimicrobial, salicylic compounds, Syzygium malaccense

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

In 2011, the WHO (World Health Organization) asked for an increased search for new drugs as antibiotic resistance has increased dramatically, but only a few new molecules are in development. Plants can be a source of new antimicrobial drugs, hopefully safe both for human use and the environment [START_REF] Abreu | Plants as sources of new antimicrobials and resistance-modifying agents[END_REF]). An ethnopharmacological study performed in the Raiatea and Tahaa islands led to the collection of 30 of the most common plants and lichens among 63 species used to treat infectious diseases. In vitro assays revealed the antimicrobial activities of 13 plants and one lichen species with MIC ≤ 0.6 mg/mL for one or several of the 34 micro-organisms. Cytotoxic assays performed on HepG2 cells did not reveal growth inhibitions at MIC ≤ 0.4 mg/mL except for Curcuma longa (rhizome), Calophyllum inophyllum (bark and leaves) and Hibiscus tiliaceus (bark), which were known to have specific toxicity against hepatoma cell lines (HepG2) without any effects on healthy human cells [START_REF] Jaikumar | Anticancer activity of Calophyllum inophyllum[END_REF][START_REF] Salama | Hepatoprotective effect of ethanolic extract of Curcuma longa on thioacetamide induced liver cirrhosis in rats[END_REF].

Then, authors focussed on Syzygium malaccense, which was shown to be one of the most active of the tested plants and therefore deserves deeper investigation as it was little previously reported in literature.

S. malaccense (Myrtaceae) is a cultivated fruit tree from French Polynesia, the leaves of which are used in local medicine to treat infectious diseases like bronchitis, cystitis, furunculosis, oral thrush, and vaginal mycosis. Scientific references also report a traditional use against a wide variety of inflammatory conditions in Western Samoa [START_REF] Dunstan | Evaluation of some Samoan and Peruvian medicinal plants by prostaglandin biosynthesis and rat ear oedema assays[END_REF][START_REF] Savitha | In vitro antioxidant activities on leaf extracts of Syzygium malaccense (L.) merr and perry[END_REF]. Nonetheless, S. malaccense is still poorly studied regarding the bioactive compounds of its leaves [START_REF] Batista | Red-jambo (Syzygium malaccense): Bioactive compounds in fruits and leaves[END_REF][START_REF] Nunes | Physico-chemical characterization, bioactive compounds and antioxidant activity of Malay apple (Syzygium malaccense (L.) Merr. & L.M. Perry)[END_REF] and its antimicrobial activity [START_REF] Locher | Antimicrobial activity and anti-complement activity of extracts obtained from selected Hawaiian medicinal plants[END_REF].

This study reports the antimicrobial activity of methanolic extract of S. malaccense and of 29 other species used against infectious diseases. It also revealed the synergistic effect of the S. malaccense leaf extract with Curcuma longa rhizome extract.

Bio-guided isolation and identification of compounds were performed by various chromatographic methods and by spectroscopic analysis (UHPLC-UV-MS, HRMS, EI-MS, NMR). Seven antibacterial compounds (6-alkenyl or 6-alkyl salicylic acids) were isolated from S. malaccense methanolic leaf extract and were identified for the first time in this species (Figure 1). The antimicrobial activity of some of these compounds has already been briefly mentioned in other studies against Gram-positive bacteria-Staphylococcus aureus [START_REF] Hua | The antibacterial activity and mechanism of ginkgolic acid C15:1[END_REF], Bacillus subtilis, Brevibacterium ammoniagenes, Streptococcus mutans, Cutibacterium acnes [START_REF] Kubo | Structure-antibacterial activity relationships of anacardic acids[END_REF] and Porphyromonas gingivalis (Rivero-Cruz et al., 2011)-and a Gram-negative bacterium (Helicobacter pylori, [START_REF] Kubo | Anti-Helicobacter pylori agents from the Cashew Apple[END_REF], but not against most of the microorganisms cited in this study, which also allowed a discussion on the structure-antibacterial activity relationships.

Salicylic acids derivatives

Results and Discussion

Ethnopharmacological Survey

The survey was focussed on plants and remedies used against infectious diseases in the Raiatea and Tahaa islands during 2017-2018. This preliminary step led to the identification of 63 species belonging to 36 botanical families with the collection of the 30 most cited species by 18 healers (collected species with their traditional use are presented in Table 1 and other plant species were added in Supplementary data). Table 1. Medicinal plants most cited in the survey with their medicinal uses. Parts used: leaf (l.); fruit (f.); flower (fl.); bud (b.); bark (bk.); whole plant (w.p.); rhizome (rh.); root (r.); aerial part (a.p.); aerial root (a.r.); stem (st.); almond (alm.); latex of fig (l.f.). Route of administration-preparation: Per os (Po); Cataplasm (Cat); Gargle (Garg); Inhalation (Inh). Medicinal uses (abbreviations and specifications): V. Cutaneous symptoms (various symptoms: spots, rash, erysipelas, folliculitis), Pneumo. (bronchitis; cough; pneumonia); V. diseases (general infectious diseases), STD (Sexually transmitted disease, Uterus (care post-partum, "impure" menstruation), Burn (sunburn sometimes with headache and dehydration), Purge (diet between ra'au tahiti and conventional medicinal drug), "Crumpled lung" (crumpled lung provoking cough and pain), Umbilic. (care of umbilical cord).

Latin

S. malaccense leaves were usually used to treat "cystitis" (83.3% of uses) in association with C. longa rhizome, suggesting synergistic effects of plant extracts (Table 3).

Antimicrobial Activity and Cytotoxicity of Plant Methanolic Extracts

The antimicrobial activity of 33 extracts was evaluated against a panel of 34 pathogenic and multi-resistant microbes. As the authors are aware of the difficulties in ascribing any specific identified bacterial species to a so-called "traditional use", they decided to test plant extracts on a large panel of Gram-positive, Gram-negative bacteria and one yeast. According to previous publications concerning the anti-infective potential of natural products, plant extracts were considered to be interesting antimicrobial agents at MIC ≤ 0.30 mg/mL, as was observed eight 8 plant species: (Calophyllum inophyllum, Cordia subcordata, Curcuma longa, Psidium guajava, Syzygium cumini, Syzygium malaccense, Thespesia populnea, Torenia crustacea) [START_REF] Cos | Anti-infective potential of natural products: How to develop a stronger in vitro "proof-of-concept[END_REF][START_REF] Ríos | Medicinal plants and antimicrobial activity[END_REF].

The screening allowed the identification of 15 plant extracts, with antibacterial activities characterised by MIC ≤ 0.6 mg/mL (Tables 2 and3). The higher antifungal activities against the yeast Candida albicans were observed for the bark extract of Syzygium cumini and the aerial part extract of Torenia crustacea (MIC = 0.15 mg/mL).

Regarding antibacterial effects, the best activities were observed for Calophyllum inophyllum bark extract, mainly against Gram-positive bacteria belonging to the genus Staphylococcus (0.07 < MIC < 0.30 mg/mL), Streptococcus (MIC = 0.15 mg/mL) and the species Corynebacterium striatum (MIC = 0.15 mg/mL). Moderate antimicrobial activities against Gram-positive bacteria (0.15 ≤ MIC ≤ 0.30 mg/mL) were also observed for Thespesia populnea bark extract (against Staphylococcus sp and Streptococcus agalactiae), Cordia subcordata and Syzygium cumini bark extracts (against Staphylococcus sp.), and with Psidium guajava leaf extract (against Staphylococcus epidermidis).

An antibiotic control using standard compounds had been previously performed and were available for each strain (Table S2 of Supplementary data), but it is important to remember that antibiotics are pure active compounds that do not have a vegetal origin; therefore, it cannot be expected that plant extracts could have the same efficiency as antibiotics [START_REF] Das | Techniques for evaluation of medicinal plant products as antimicrobial agent: Current methods and future trends[END_REF]. Nevertheless, those tests were performed to obtain useful information for the choice of plant remedies against infectious diseases and to extend the scope of therapeutic applications. MIC: 0.6 mg/mL, MIC: 0.6 mg/mL ≥ MIC > 0.3 mg/mL, MIC ≤ 0.3 mg/mL; (-) no MIC or FIC determined at 1.2 mg/mL; (n = 3; with standard deviation for value: 1.2 ± 0.4 mg/mL; 0.6 ± 0.2 mg/mL; 0.3 ± 0.1 mg/mL; 0.15 ± 0.05 mg/mL; 0.07 ± 0.03 mg/mL).

No activities against tested pathogens were detected with the following plant extracts: Musa troglodytarum and M. paradiasiaca buds, Hibiscus tiliaceus leaves, Ficus tinctoria fruits and Ficus prolixa leaves, Centotheca lappacea and Cenchrus echinatus aerial parts, Fagraea berteroana flowers and leaves, Cordyline fruticosa buds, Davalia solida rhizoma and leaves, Commelina diffusa, Adenostemma viscosum and Rorippa sarmentosa aerial parts.

Cytotoxic assays performed on HepG2 cells did not reveal growth inhibitions at MIC ≤ 0.4 mg/mL except for C. longa (rhizome, IC50 = 33 ± 9 μg/mL), C. inophyllum (bark and leaves; IC50 =59 ± 4 and 76 ± 5 μg/mL) and H. tiliaceus (bark, IC50 = 83 ± 10 μg/mL). While this assay was intended as a preliminary screening of nonspecific cell toxicity, its results should not discourage the use of these three plants, as their toxicity seems to be specific against tumoral cells like hepatoma cell lines (HepG2) without having any effects on healthy human cells [START_REF] Jaikumar | Anticancer activity of Calophyllum inophyllum[END_REF][START_REF] Salama | Hepatoprotective effect of ethanolic extract of Curcuma longa on thioacetamide induced liver cirrhosis in rats[END_REF].

Antimicrobial Tests of Methanolic Extract from Syzygium malaccense and Curcuma longa

In vitro antimicrobial activities were characterized by the Minimal Inhibitory Concentration (MIC) and by the Fractional Inhibitory Concentration index (FIC index, Table 3).

)
no MIC or FIC determined at 1.2 mg/mL; (n = 3; with standard deviation for value: 1.2 ± 0.4 mg/mL; 0.6 ± 0.2 mg/mL; 0.3 ± 0.1 mg/mL; 0.15 ± 0.05 mg/mL; 0.07 ± 0.03 mg/mL) with FIC index of mixture.

Results exhibited important activities of Syzygium malaccense leaf methanolic extract against Candida albicans, Staphylococcus epidermidis, Streptococcus pyogenes and Pseudomonas aeruginosa with MIC ≤ 0.3 mg/mL. Lower activities were also observed against Staphylococcus aureus, Staphylococcus warneri, Staphylococcus pettenkoferi, Corynebacterium striatum and Proteus mirabilis (0.3 ≤ MIC ≤ 0.6 mg/mL). Regarding the rhizome of Curcuma longa, which is traditionally used in mixture with S. malaccense, good antimicrobial activities were also observed against Enterococcus faecalis, Staphylococcus spp., Streptococcus agalactiae and Corynebacterium striatum (0.15 ≤ MIC ≤ 0.30 mg/mL).

The FIC determination exhibited an additive effect of the mixture of Syzygium malaccense (leaves) and Curcuma longa (rhizome), characterized by an FIC index between 0.7 and 0.9, against Enterococcus faecalis (C159-6), Staphylococcus aureus (8146, 8241, ATCC 538, T28-1 et T17-4) and Corynebacterium striatum (T40A3). This mixture also exhibited a synergic effect against Pseudomonas aeruginosa 8129, characterized by an FIC index = 0.5.

These results suggested the presence of antimicrobial compounds in S. malaccense leaf methanolic extracts, which could have synergic effects with compounds from the rhizome of Curcuma longa such as curcumin, and reinforce the therapeutic choice of this plant mixture in traditional medicine.

Structural Identification

Activity-guided isolation of S. malaccense leaf extract led to the identification of seven 6-alkenyl or 6-alkyl-salicylic acids (anacardic and ginkgolic acids) that were described for the first time in this species (Figure 1). Nonetheless, these compounds have already been identified in other species such as Ginkgo biloba L. (Ginkgoaceae), Anacardium occidentale L., Spondias mombin L. (Anacardiaceae) and Knema laurina (Blume) Warb. (Myristicaceae).

The NMR spectra of these salicylic acid derivatives provided a common pattern of peaks characteristic of their aromatic structure (Table 4). In fact, the chemical shifts recorded for compound 1, at δH: 6.89 ppm (d, J = 8.4 Hz): H-3, δH: 7.38 ppm (dd, J = 7.8, 8.4 Hz): H-4, and δH: 6.79 ppm (d, J = 7.8 Hz): H-5, corresponding to the salicylic acid moiety, were similar to those obtained for the six other isolated compounds [START_REF] Choi | Quantitative analysis of ginkgolic acids from Ginkgo leaves and products using 1H-NMR[END_REF]. The HSQC and HMBC spectra indicated the presence of four non-protonated carbon atoms including three carbons associated with the aromatic ring (δC: 110.7, 147.5 and 163.5 ppm) and the carboxyl group of the salicylic acid at δC: 175.7 ppm. Bibliographical data and NMR heteronuclear correlations permitted the complete structural elucidation of this aromatic part.

Regarding the olefinic chain unsaturations of all isolated compounds, the determination of the cis configuration was afforded by 13 C-NMR spectra that exhibited specific chemical shifts at 25.5 ≤ δC ≤ 27.3 ppm for the allylic methylene carbons of a cis geometry, whereas a trans stereochemistry would be indicated by a downfield shift of approximately 10 ppm [START_REF] Coates | SB-202742, a novel β-lactamase inhibitor isolated from Spondias mombin[END_REF]; Lie Ken Jie, 1997). Moreover, IR spectra of an unsaturated chain of phenolic acids showed an important absorbance at 695-699 cm -1 characterizing the cis configuration, whereas no trans configuration-specific absorption was observed around 960-980 cm -1 [START_REF] Silverstein | Spectrometric Identification of Organic Compounds[END_REF].

Localisation of the double bound was supported by NMR data (2D-HMBC correlations, COSY or selective TOCSY), and by mass spectrometry analysis of the thiomethylated derivatives (from compounds: 2, 4, 6 and 7). The position of the double bonds in the alkyl side chains was afforded by the EI-MS fragments of the α,β-bis-(methylthio)-derivatives produced by the reaction of the compounds with dimethyl disulfide [START_REF] Mansour | High contents of 24:6(n 3) and 20:1(n 13) fatty acids in the brittle star Amphiura elandiformis from Tasmanian coastal sediments[END_REF]. The EI-MS spectra of the thiomethylated derivatives showed intense fragment ions [CnH2n+1S] + corresponding to the cleavage of the terminal aliphatic chain residues at the original site of the unsaturation. For example, the presence of a fragment ion at m/z: 117 corresponding to the cleavage of [C6H13S] + clearly revealed that the double bond on the alkyl side chain was originally located between the fifth and sixth carbons from the terminal methyl group, as observed for compounds 2, 4 and 7.

(Figure 1). Compound 1 was found to possess a molecular formula of C22H36O3 as derived from its HR EI-MS spectrum with m/z: 347.25840 [M -H] -. The mono and bidimensional CDCl3 500 MHz 1 HNMR spectra revealed a 6-alkyl-salicylic acid moiety as previously described. The bidimensional HSQC and HMBC spectra combined with the molecular formula also demonstrated the presence of a linear saturated carbon chain composed of 15 carbons linked by the C-6 of the aromatic ring, corresponding to the 6-pentadecyl salicylic acid. The comparison of spectroscopic data (UHPLC-MS and NMR) with the standard of the ginkgolic acid C15:0 indicated a total similarity of the compounds structure. This anacardic acid (15:0) was originally identified in Amphipterygium adstringens (Schltdl.) Schiede ex Standl. (Anacardiaceae) stem bark (Rivero-Cruz et al., 2010) and NMR chemical shift were also consistent with previous spectroscopic data provided by the G. biloba leaves phytochemical analysis [START_REF] Choi | Quantitative analysis of ginkgolic acids from Ginkgo leaves and products using 1H-NMR[END_REF]). The COSY and selective TOCSY spectra showed correlations of protons from the terminal methyl of the aliphatic chain (δH = 0.91 ppm) with the aliphatic protons δH = 1.20 to 1.40 ppm and the proton δH = 2.03 ppm (carried by an alpha unsaturated carbon at δC = 27.2 ppm). The HMBC spectrum of the terminal methyl (δH = 0.91 ppm) also indicated correlations with methylenic carbons (δC = 22.7 and 32.2 ppm) and with the alpha unsaturated carbon at 27.2 ppm, but this correlation did not permit the determination of the position of the double bond. The localisation of this unsaturation was achieved by the fragmentation of the thiomethylated derivatives by GC-EI-MS. The presence of a specific fragment ion at m/z = 117.1, corresponding to [C6H13S] + revealed that the double bond was located between the fifth and sixth carbons from the terminal methyl group, namely C10′-C11′: ω5. Other ions corresponding to the dehydrated, or decarboxylated, thiomethylated aromatic moiety (m/z = 305.2 and 277.1) supported this structural determination. This compound was originally isolated and identified in Knema elegans seed oil (Myristicaceae, [START_REF] Spencer | Alkyl and phenylalkyl anacardic acids from Knema elegans seed oil[END_REF] with similar spectrometric data. 2.4.3. Compound 3: Ginkgolic acid (C17:0) Compound 3 was found to possess a molecular formula of C24H40O3 suggested by its HR EI-MS spectrum with m/z: 375.28930 [M -H] -. NMR spectra exhibited a similar structure as observed for compound 1 with a 6-alkylsalicylic acid moiety substituted by a linear saturated carbon chain with two additional carbons (R: C17H35 instead of C15H31). This alkyl-salicylic acid derivative has already been isolated, and identified, from ginkgo leaves and cashew nuts with similar spectrometric data (Gellerman and Schlenk, 1968).

Compound 4: Ginkgolic Acid (C17:1, ω5)

HR EI-MS spectrum exhibited a molecular ion at m/z: 373.27316 ([M -H] -calculated for C24H38O3) that corresponded to compound 3 with an additional unsaturation. NMR data of the aliphatic chain (linked by the C-6 of the aromatic ring) also attested to the presence of 17 carbons with one unsaturation. The COSY and HMBC spectra did not show correlations of the protons from the terminal methyl of the aliphatic chain (δH = 0.90 ppm) with the ethylenic group, suggesting a distant double bond from the terminal methyl, but did not allow its location. The comparison of TOCSY long range NMR experiments at different time mixtures (tm = 60 or 80 ms) exhibited minor correlations between the methyl terminal (δH = 0.90 ppm) and the methylenic proton (δH = 5.36 ppm) at tm = 80 ms. This comparison permitted us to assume the location of the double bond between aliphatic carbons C11′ and C14′, but additional spectrometric mass analysis was required for an accurate determination. The EI-MS spectra of the thiomethylated compound 4 exhibited a fragment ion at m/z: 117.1 corresponding to [C6H13S] + and allowed the definitive location of the unsaturation between the fifth and sixth carbons from the terminal methyl group (ω5). Other fragment ions at m/z: 333.2 and 307.2 corresponding to the dehydrated and decarboxylated residual salicylic moiety afforded the location of this double bond at ω5. This compound has already been identified in plant species from various botanical families, such as Myristicaceae (Knema elegans), Anacardiaceae (Anacardium occidentale), Ginkgoaceae (Ginkgo biloba), Geraniaceae (Pelargonium hybridum) and their spectrometric data were in accordance with our results [START_REF] Gellerman | Methods for isolation and determination of anacardic acids[END_REF]Schlenk, 1968, Spencer et al., 1980;[START_REF] Walters | Geranium defensive agents. III. Structural determination and biosynthetic considerations of anacardic acids of geranium[END_REF]. The COSY and HMBC spectra showed correlations of protons from the methyl terminal of the aliphatic chain (δH = 0.99 ppm) with the protons δH = 2.0 ppm (COSY) and with δC = 20.6 ppm (alpha unsaturated carbon, HMBC) corresponding to the double bound in ω3. This localization of the unsaturation was afforded by GC-EI-MS analysis of the thiomethylated derivative, which exhibited the presence of a fragment ion at m/z: 89.2, characterizing a [C4H9S] + moiety and corresponding to an unsaturation localized at ω3. Moreover, the selective TOCSY experiment performed on the signal at δH = 0.99 ppm exhibited correlations with the protons at δH = 2.09 and 2.82 ppm from the methylenes of the allylic groups, affording the position of double bonds in ω3 and ω6. The ω9 unsaturation was deduced from the chemical shift of the two methylenic protons at δH = 2.82 (carried by the allylic methylene carbon at δC = 25.5 ppm), which is specific of a methylene group wedged between two double bonds (ω6 and ω9), as it was also measured for the ω3-ω6 unsaturations [START_REF] Coates | SB-202742, a novel β-lactamase inhibitor isolated from Spondias mombin[END_REF].

Bibliographical data exhibited a similar compound previously isolated from the species Spondias mombin within double bonds located at ω3, ω6 and ω9 [START_REF] Corthout | Antibacterial and molluscicidal phenolic acids from Spondias mombin[END_REF]. 2.4.6. Compound 6: Ginkgolic Acid (C17:2; ω6, ω9)

The spectral NMR and the molecular formula C24H36O3 deduced from the HR El-MS spectra data highlighted the presence of a 6-alkenylsalicylic acid with a C17 diunsaturated side chain. The aromatic ring and carbonyl group accounted for five of the seven degrees of unsaturation from the molecular formula and the remaining two were attributed to the two olefinic double bonds with their methine protons (δ: 5.38 ppm, 4H).

Similar chemical shifts and correlations to those observed for compound 5 were observed, except for the aliphatic chain ending that did not exhibit any double bond in ω3. This compound was already identified in the aerial parts of Spondias mombin and their spectrometric data were corroborated [START_REF] Corthout | Antibacterial and molluscicidal phenolic acids from Spondias mombin[END_REF].

2.4.7. Compound 7: Ginkgolic Acid (C19:1; ω5) Compound 7 was found to possess a molecular formula of C26H42O3 suggested by its HR El-MS spectrum with a molecular ion at m/z: 401.30478 [M -H] -. NMR spectra exhibited a similar structure to that observed for compound 4 with a 6-alkenylsalicylic acid moiety substituted by a linear mono-unsaturated carbon chain with two additional carbons (R: C19H37 instead of C17H33). Localisation of the olefinic unsaturation was allowed by the fragmentation of the thiomethylated derivatives by GC-EI-MS, providing a fragment ion at m/z = 117.1 corresponding to the cleavage of [C6H13S] + and highlighting a double bond in ω5. Other ions corresponding to the dehydrated or decarboxylated thiomethylated aromatic moiety (m/z = 361.2 and 335.3) supported this structural determination as observed for compound 4. This compound has already been identified in Amphipterygium adstringens bark (Rivero-Cruz et al., 2010).

Antimicrobical Activity of Compounds 1 to 7

MIC of isolated compounds and standard were determined on three strains: S. aureus 8241, S. pyogenes 19138 and P. aeruginosa 8129 (Table 5).

Synergistic Effects of Plant Extracts

The evaluation of the antibacterial synergy of curcumin (major compound from C. longa rhizome) with isolated compounds from S. malaccense were performed by a checkerboard method, but no particular effect was observed (in contrast with the synergy observed for the crude plant extracts in mixture), suggesting the presence of other bioactive compounds.

Structure-Activity Relationships

The best activities were observed against S. pyogenes. Bacteriological tests showed a decrease in the activity between C15:0 and C17:0 compounds. However, an improvement in the activity was observed between C15:0 and C15:1, and between C17:0, C17:1, C17:2 and C17:3, meaning an enhancement of antibacterial activity correlated with the number of unsaturations (the most unsaturated compound (5) was the most active against Grampositive bacteria). Moreover, the activity of compound C17:1 was comparable to those observed for C19:1 (CMI = 18.75 μg/mL). Therefore, experimental data underlined evidence of an optimal chain length and number of unsaturation, promoting the antibacterial activity against S. pyogenes. Similar observations were made for other Grampositive bacteria (S. aureus). Those results were in accordance with previous data regarding the importance of the number of unsaturation even if optimal activity were also previously recorded against Gram-positive or -negative bacteria for saturated alkyl chains ranged between 6 and 13 carbons [START_REF] Kubo | Structure-antibacterial activity relationships of anacardic acids[END_REF].

Nonetheless, no important variation in the activity against P. aeruginosa was observed for the different compounds, suggesting another pharmacological mechanisms against this Gram-negative bacterium (ginkgolic acids could restrict P. aeruginosa virulence by abolishing pyocyanin pigment production as described by [START_REF] Tahrioui | Membrane-interactive compounds from Pistacia lentiscus L. thwart Pseudomonas aeruginosa virulence[END_REF].

Those results attested the importance of the aliphatic chain length and its unsaturation number or location for an optimal antimicrobial activity of these salicylic derivatives against Gram-positive bacteria and permitted us to explain the antimicrobial plant extract activity.

Conclusions

This preliminary ethnopharmacological survey was the first performed in the Leeward Islands of Society archipelago (most of our previous works were focussed on the Windward Islands archipelago, [START_REF] Grand | Le Monde Polynésien des Soins Traditionnels; Au vent des iles Pirae[END_REF][START_REF] Hooper | Tahitian Folk Medicine, in: Rank and Status in Polynesia and Melanesia[END_REF][START_REF] Pétard | Tahiti: Plantes Médicinales Polynésiennes et Remèdes Tahitiens[END_REF] and led to the selection of antimicrobial plant extracts.

S. malaccense leaf methanolic extract was studied for its antimicrobial activities against Gram-positive bacteria (MIC < 0.6 mg/mL) with additive or synergistic effects with C. longa rhizome methanolic extract. no antibacterial synergy was observed between ginkgolic acids and curcumin, which are, respectively, major active compounds from these two plant extracts, suggesting the presence of other bioactive compounds. The 6-alkenyl or alkyl-salicylic acids (anacardic acids or ginkgolic acids C15:0, C15:1, C17:0, C17:1, C17:2, C17:3 and C19:1) were identified as the main antibacterial compounds from the methanolic extract of S. malaccense. The presence of these compounds can justify the medicinal uses of the plant species against infectious diseases and creates interesting antiinflammatory and complementary effects to those observed for similar salicylic derivatives [START_REF] Hemshekhar | Emerging roles of anacardic acid and its derivatives: A pharmacological overview[END_REF] and to those described in other traditional uses and experimental data [START_REF] Dunstan | Evaluation of some Samoan and Peruvian medicinal plants by prostaglandin biosynthesis and rat ear oedema assays[END_REF].

Antimicrobial activity of the seven isolated compounds from S. malaccense was determined against three strains. The present study showed a good antimicrobial activity of all compounds on S. pyogenes, followed by S. aureus, and low activity against P. aeruginosa. The structure-activity relationship analyses concluded to the importance of the length of the side chain of molecules as well as the number of their unsaturations.

The methanolic extract from S. malaccense appeared to have interesting antimicrobial activity against Gram-positive bacteria and did not show toxicity on hepG2 cells at 400 μg/mL. Nonetheless, it is important to point out that the potential toxicity of these compounds may induce dermatitis and hepatic or renal disorders [START_REF] Marks | Dermatitis from cashew nuts[END_REF][START_REF] Qian | Metabolic profiling of the hepatotoxicity and nephrotoxicity of ginkgolic acids in rats using ultra-performance liquid chromatography-highdefinition mass spectrometry[END_REF].

Materials and Methods

Ethnopharmacological Survey

The Tahaa and Raiatea islands are located in the Society archipelago (16°44′00″ S; 151°27′00″ W and 16°36′58″ S; 151°30′00″ W). The ethnopharmacological survey was carried out among the 18 traditional healers specialized in the treatment of "ma'i mau" or "real diseases" using semi-structured interviews. The study was focused on infectious diseases and was conducted with a rigorous ethical approach. The legal authorities of Polynesia were contacted and informed of the survey. The project was declared to the environment department of Polynesia (DIREN), with declaration to the Minister of Health and Social Services, and all informant consents were collected.

The scientific identification and biogeographical status of each plant species was checked by the botanist J.F. Butaud in accordance with regional floras [START_REF] Florence | Base de Données Botaniques Nadeaud de l'Herbier de la Polynésie Francaise (PAP)[END_REF] and traditional medicine literature [START_REF] Florence | Les Ressources Végétales Polynésiennes, Substances Naturelles en Polynésie Française: Stratégies de Valorisation, Expertise Collégiale[END_REF][START_REF] Girardi | Herbal medicine in the Marquesas Islands[END_REF][START_REF] Grand | Le Monde Polynésien des Soins Traditionnels; Au vent des iles Pirae[END_REF][START_REF] Hooper | Tahitian Folk Medicine, in: Rank and Status in Polynesia and Melanesia[END_REF][START_REF] Pétard | Tahiti: Plantes Médicinales Polynésiennes et Remèdes Tahitiens[END_REF]. Plant names have been checked with the "http://www.theplantlist.org" website.

Antimicrobial Activity

Plant Material and Preparation of Extract

The parts of the plants traditionally used were collected and dried in a dark room at 30 then ground and macerated in methanol during 12 h (1 g/10 mL). Extracts were filtered, and the solvent was removed by evaporation under reduced pressure.

MIC and FIC Index Measures

The antimicrobial test of methanolic extract was carried out on Petri dishes containing Mueller-Hinton Agar (MHA) mixed with plant extract to obtain final concentrations ranged from 1.25 to 0.07 mg/mL. According to previous publications about the anti-infective potential of natural products, plant extracts were considered as active at MIC ≤ 0.30 mg/mL [START_REF] Cos | Anti-infective potential of natural products: How to develop a stronger in vitro "proof-of-concept[END_REF][START_REF] Ríos | Medicinal plants and antimicrobial activity[END_REF].

Extracts from S. malaccense and C. longa were tested against 34 clinical microbial strains. A multi-headed inoculator seeded the bacterial strains on prepared Petri dishes at 10 5 CFU/mL (Colony Forming Unit/mL) in Cysteine Ringer (CR) solution (Merck). Minimal Inhibitory Concentrations (MIC) were visually determined after 24 h of incubation at 37 °C. MIC values of extracts and controls were recorded as the lowest concentrations of extracts, showing no growth of colonies. The results were obtained from the average of triplicate measurements.

Synergy was evaluated by a checkerboard method and determination of the FIC index (fractional inhibitory concentration). The final extract concentrations were ranged from 1.25 to 0.07 mg/mL both for vegetal extract or compounds (A and B). FIC index was calculated with FIC = (MIC of A in combination/MIC of A alone) + (MIC of B in combination / MIC of B alone). Results were interpreted as "synergistic activity" (FIC ≤ 0.5), "additional activity" (0.5 < FIC < 1), "indifferent activity" (1 ≤ FIC ≤ 2) and "antagonistic activity" (2 < FIC) (EUCAST, 2000).

MIC Determination of Isolated Compounds and Standard Molecules Using a Broth Microdilution Method

A serial dilution technique using 96-well microtiter plates was used to determine the MIC of the pure compounds against sensitive bacteria: Streptococcus pyogenes 19138, Staphylococcus aureus 8241 and Pseudomonas aeruginosa 8129. Nine concentrations of each compound, from 300 to 1.7 μg/mL, were used. They were serially twofold diluted with RC in nine wells. Two wells were represented as bacteria culture control (positive control) and medium sterility control (negative control). Then the wells were loaded with MH liquid medium and bacterial suspension (10 4 bacteria/mL), giving a final volume of 200 μL. The plates were incubated overnight at 37 °C. Bacterial growth was indicated visually and then by direct spray of 0.2 mg/mL INT to each well with incubation at 37 °C for 30 min. Bacterial growth was indicated by a reddish-pink colour. MIC values were determined as the lowest concentrations of compounds showing clear wells.

Cytotoxic Activity of Crude Plant Extracts

Hepatocellular carcinoma cell line (HepG2) and an MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay method were used to quantify cell viability. Cells were seeded into a 96-well microplate in a Gibco Dulbecco's Modified Eagles Medium (DMEM), with 10% FBS (Foetal Bovine Serum, ThermoFisher Scientific) and antibiotics (mixture penicillin/streptomycin 100 UI/mL, Sigma Aldrich). After two days at 37 °C (5% of CO2), wells were emptied by suction and the cells were treated with plant extracts diluted in the culture medium (with 0.2% DMSO, at concentrations from 400 μg/mL to 25 μg/mL), to obtain a final volume of 100 μL. After two days, the medium was discarded and replaced with DMEM containing 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). After 1 h 30 of incubation at 37 °C and 5% CO2, the water-insoluble formazan was dissolved in 100 μL DMSO and the absorbance was measured at 550 nm using a UV-spectrometer (Tecan, Spark 10M). The cytotoxicity of the crude extracts and standard (camptothecin: IC50: 0.9 ± 0.1 μg/mL) was determined by comparing the absorbance of treated cells with the absorbance of control cells cultured in 0.2% DMSO. Data were expressed as a percentage of inhibition calculated according to the formula (% Cell viability = Abs treated cells × 100/Abs control cells) and were analysed by linear regression using GraphPad Prism (5.01).

Bioautography and Isolation of Aliphatic-Salicylic Acids from Syzygium malaccense

Bioautography

Bioautography of Syzygium malaccense leaf methanolic extract against Streptococcus pyogenes, Streptococcus pyogenes and Staphylococcus aureus enabled the activity-guided isolation of antibacterial compounds. TLC was carried out on silica gel 60 F254 aluminium plates (Merck) submitted to Tol/AcOEt/MeOH (7:2:1; v/v) eluent with UV detection 254 and 366 nm. The plates were covered by MHA containing the bacterial strain suspension in Petri dishes. After incubation (24 h at 37 °C), growth was revealed by iodonitrotetrazolium chloride (INT, 2 mg/mL) and inhibition zones were visually localized on the TLC.

Purification of Salicylic Acids Derivatives

The methanolic dried extract (50 g), obtained from 600 g of powdered leaf of S. malaccense, was subjected to column chromatography on silica gel (MN Kieselgel 60, 0.063-0.2 mm) and successively eluted with n-Hex, Tol, AcOEt and MeOH to afford 46 fractions. Fractions F26 to F43 were subjected to various column chromatography on silica gel (MN Kieselgel 60, 0.063-0.2 mm) and successively eluted with Tol, AcOEt and MeOH for the purification of active compounds monitored by UHPLC-UV-MS.

Final purifications were performed by preparative-HPLC (U5SC18HQ 250 × 0.46) at a 17 mL/min flow rate; eluted with gradient H2O/ACN (5:95 with 0.1% formic acid) to yield compounds 1 (16 mg), 2 (3.4 mg), 3 (21 mg), 4 (94 mg), 5 (40 mg), 6 (13.2 mg) and 7 (158 mg). Compounds from S. malaccense and standards (ginkgolic acid C15:0, C15:1 and C17:1) were analysed by UHPLC-UV-MS using an Acquity UPLC H-Class Waters system equipped with a diode array detector (DAD) and an Acquity QDa ESI-Quadrupole Mass Spectrometer (software Empower 3). The stationary phase was a Waters Acquity BEH C18 column (2.1 × 50 mm, 1.7 μm) and the mobile phase consisted of H2O/ACN (10:90 with 0.1% formic acid) at 0.3 mL/min. The ionization was performed in a negative mode with a cone voltage at 10 V.

Identification of Salicylic Acids Derivatives

Spectrometric Analysis

Compounds isolated from S. malaccense were analysed by High Resolution Mass Spectrometry (HR-MS) using a Thermo Fisher Scientific Orbitrap Mass Spectrometer with an electrospray ion source (negative mode) and Xcalibur software.

El-MS spectra of thiomethylated derivatives were realised on a GC-MS Trace DSQ (ThermoFisher in CHCl3, 70 eV, positive mode).

NMR spectra were recorded on a Bruker AVANCE 500 spectrometer (1H at 500 MHz-and 13C-NMR) in CDCl3 using standard pulse sequences.

Thiomethylation

To a solution of alkenyl-salicylic acid (1 eq., 0.01 mmol) in diethyl ether (2 mL) was added iodine (9.5 eq., 24 mg, 0.095 mmol) and dimethyl disulfide (450 eq., 0.4 mL, 4.5 mmol) in a sealed tube. The reaction mixture was stirred at 50 °C for 5 h, then cooled to room temperature, poured into 5% aqueous sodium thiosulfate solution (5 mL) and extracted with diethyl ether (3 mL) three times. The combined organic layer was concentrated in vacuo to give a crude dimethyldisulfide (DMDS) adduct, which was addressed to EI MS analysis without further purification. Compound 4: 6-(10′Z-Heptadecenyl)-salicylic acid: ginkgolic acid C17:1 ω5: white wax; IR λmax : 2916, 2848, 1649, 1606, 1445, 1297, 1218, 720, 698 cm -1 ; UV (MeOH) λmax: 215.3, 243.7, 310.3 nm; 1 H NMR (500 MHz, CDCl3, in ppm): δH: 7.39 (1H, dd, J = 7.8, 8.4 Hz), 6.89 (1H, d, J = 8.4 Hz), 6.80 (1H, d, J = 7.8 Hz), 5.36 (2H, m), 3.00 (2H, t, J = 6.4 Hz), 2.09 (4H, m), 1.60 (2H, m), 1.20 to 1.40 (20H, m), 0.90 (3H, t, J = 6.9 Hz). 13 C NMR (500 MHz, CDCl3): δC: 175.7, 163. 5, 147.5, 135.4, 129.9, 129.9, 122.8, 115.7, 110.7, 36.5, 32.3, 31.9 4, 128.3, 127.6, 127.2, 122.8, 115.7, 110.7, 36.5, 32.3, 29.7, 29.6, 29.4, 29.3, 27.3, 25.5, 25.6, 20.

Figure 1 .

 1 Figure 1. Aliphatic-salicylic acid derivatives isolated from the leaves of Syzygium malaccense.

2. 4 . 2 . Compound 2 :

 422 Ginkgolic Acid (C15:1, ω5)The molecular formula C22H34O3 was established by HR EI-MS of the molecular ion [M -H] -at m/z 345.24248. The 1 H-NMR spectrum showed signals characteristic of the same salicylic moiety previously described for compound 1, and NMR data of the aliphatic chain also exhibited the presence of 15 carbons but suggested one unsaturation.

2. 4 . 5 . Compound 5 :

 455 Ginkgolic Acid (C17:3; ω3, ω6, ω9) The molecular formula C24H34O3 was established by HR EI-MS of the molecular ion [M -H] -at m/z: 369.24254. The 1 H-NMR spectrum showed signals characteristic of the salicylic acid moiety previously described with 23 aliphatic and 6 olefinic protons. The presence of 24 carbons was demonstrated by the 13C NMR spectrum, and the heteronuclear 2D NMR (HSQC, HMBC) indicated the presence of 4 non-protonated carbons, 9 methine, 10 methylene, and 1 methyl. The salicylic acid accounted for five of the eight degrees of unsaturation from the molecular formula; the remaining three were attributed to three olefinic double bonds with their methine protons (δH: 5.38 ppm, 6H). The 2D NMR correlations (HSQC and HMBC) experiments allowed assignment of the 13 C-NMR spectrum signals, attesting to correlations of the carbons 7′, 10′ and 13′ at δC: 25.5, 25.6 and 27.3 ppm for the allylic methylene carbons of the 3 cis unsaturations previously mentioned (Figure 2).

Figure 2 .

 2 Figure 2. Heteronuclear 2D NMR correlations (HMBC in bold) and homonuclear selective TOCSY correlations (dotted line) of the aliphatic chain from compound 5.

4. 5 . 3 .

 53 Compound Spectroscopic Data Compound 1: 6-(pentadecyl)-salicylic acid (anacardic acid or ginkgolic acid C15:0): white wax; IR λmax : 2915, 2846, 1652, 1604, 1446, 1110, 1216, 706, 548 cm -1 ; UV (MeOH) λmax: 207.1, 243.7, 311.5 nm; 1 H NMR (500 MHz, CDCl3, in ppm): δH: 7.38 (1H, dd, J 8.4, 7.3 Hz), 6.89 (1H, d, J = 8.4 Hz), 6.80 (1H, d, J = 7.3 Hz), 3.00 (2H, t, J = 6.4 Hz), 1.60 (2H, m), 1.20 to 1.40 (24H, m), 0.90 (3H, t, J = 6.9); 13 C NMR (500 MHz, CDCl3): δC: 175.0, 163.8, 148.0, 135.4, 122.8, 116.4, 110.5, 36.2, 31.9, 32.2, 29.0 à 29.7, 22.7 and 14.4; HR El-MS (negative) m/z: 347.25840 ([M -H] -calcd for C22H36O3). Compound 2: 6-(10′Z-Pentadecenyl)-salicylic acid: ginkgolic acid C15:1 ω5 : white wax, IR λmax: 2918, 2847, 1648, 1607, 1450, 1297, 1218, 720, 697 cm -1 ; UV (MeOH) λmax: 207.1, 243.7, 310.3 nm; 1 H NMR (500 MHz, CDCl3, in ppm): δH: 7.38 (1H, dd, J = 7.3, 8.4 Hz), 6.89 (1H, d, J = 8.4 Hz), 6.80 (1H, d, J = 7.3 Hz), 5.38 (2H, m), 3.00 (2H, t, J = 6.4 Hz), 2.03 (4H, m), 1.60 (2H, m), 1.20 to 1.40 (16H, m), 0.91 (3H, t, J = 7.6 Hz); 13 C NMR (500 MHz, CDCl3): 175.7, 163.8, 148.0, 135.4, 130.3, 122.8, 116.4, 110.5, 36.2, 32.2, 31.9, 29.0 to 29.7, 27.3, 27.2, 22.7 and 14.4 ppm; HR El-MS: m/z : 345,24254 [M -H] -calcd for C22H34O3. Compound 3: 6-(Heptadecyl)-salicylic acid: ginkgolic acid C17:0: white wax; IR λmax: 2910, 2842, 1653, 1601, 1448, 1109, 1212, 704, 547 cm -1 ; UV (MeOH) λmax: 215.3, 243.7, 310.3; 1 H NMR (500 MHz, CDCl3, in ppm): δH: 7.38 (1H, dd, J = 7.5, 8.2 Hz), 6.89 (1H, d, J = 8.2 Hz), 6.80 (1H, d, J = 7.5 Hz), 3.00 (2H, 6.7), 1.60 (2H, m), 1.20 to 1.40 (28H, m), 0.90 (3H, t, J = 6.9 Hz); 13 C NMR (500 MHz, CDCl3): δC: 175.7, 163.5, 147.5, 135.4, 122.8, 116.4, 110.5, 36.2, 32.2, 31.9, 29.7 to 29.0, 22.7 and 14.1 ppm; HR El-MS: m/z: 375.28930 ([M -H] -calcd for C24H40O3).

  , 29.8 to 29.0, 27.2, 26.9, 22.6 and 14.0 ppm; HR El-MS: m/z: 373.27316 ([M -H] -calcd for C24H38O3). Compound 5: 6-(8′Z,11′Z,14′Z-Heptadecatrienyl)-salicylic acid: ginkgolic acid (C17:3 ω3; ω6; ω9 or penladjuaic acid: colorless oil; IR λmax: 2932, 2856, 1647, 1608, 1448, 1297, 1218, 720, 698 cm -1 , UV (MeOH) λmax 215.3, 243.7, 310.3 nm; 1 H NMR (500 MHz, CDCl3, in ppm): δH: 7.38 (1H, dd, J = 7.8, 8.4 Hz), 6.88 (1H, d, J = 8.4 Hz), 6.79 (1H, d, J = 7.8 Hz), 5.38 (6H, m), 3.00 (2H, t, J = 6.4 Hz), 2.82 (4H, m), 2.09 (4H, m), 1.60 (2H, m), 1.20 à 1.40 (8H, m) and 0.99 (3H, t, J = 7.6 Hz); 13 C NMR (500 MHz, CDCl3): δC: 175.7, 163.5, 147.5, 135.0, 132.5, 132.0, 130.

  6 and 14.7 ppm; HR El-MS: m/z: 369.24254 ([M -H] -calcd for C24H34O3). Compound 6: 6-(8′Z,11′Z-Heptadedienyl)-salicylic acid:ginkgolic acid or penladjuaic acid (C17:2 ω6; ω9): white wax; IR λmax : 2920, 2860, 1650, 1609, 1450, 1299, 1216, 718, 695 cm -1 ; UV (MeOH) λmax: 198.4, 243.7, 311.5 nm; 1 H NMR (500 MHz, CDCl3, in ppm): δH: 7.38 (1H, dd, J = 7.8, 8.2 Hz), 6.88 (1H, d, J = 8.2 Hz), 6.79 (1H, d, J = 7.8 Hz), 5.38 (4H, m), 3.00 (2H, t, J = 7.8 Hz), 2.82 (2H, t, J = 6.7 Hz), 2.07 (4H, m), 1.60 (2H, m), 1.31, 1.20 to 1.40 (14H, m), 0.89 (3H, t, J = 6.7 Hz); 13 C NMR (500 MHz, CDCl3): δC: 175.7, 163.5, 147.5, 135.0,

Table 2 .

 2 MIC (mg/mL) of methanolic plant extracts.

	Malvaceae	Myrtaceae	Calophyllaceae	Boraginaceae	Anacardiaceae	Parmeliaceae	Euphorbiaceae	Pandanaceae	Polypodiaceae	Linderniaceae

Hibiscus tiliaceus Thespesia populnea Syzygium cumini Psidium guajava Calophyllum inophyllum Cordia subcordata Mangifera indica Usnea spp. Aleurites moluccanus Pandanus tectorius var. tectorius Microsorum grossum Torenia crustacea

		Staphylococcus aureus	ATCC 6538	0.6	0.1 5	0.6 0.3 0.6	0.0 7	0.6 0.6 1.2 0.6	-	1.2		1.2	-
		Staphylococcus aureus	T28-1	-	0.3 0.6 0.3 0.3	0.0 7	0.3 0.6 1.2 0.6	-	-		1.2	-
		Staphylococcus aureus	T17-4	-	0.3 1,2 0.3 0,6	0.0 7	0.6 0.6 1,2 0.6	-	1.2		1,2	-
		Staphylococcus epidermidis	T46A1	0.6	0.1 5	0,6 0.3 0,3	0.1 5	0,6 0.3 1,2 0.6 0.6	-		1,2	0.6
		Staphylococcus epidermidis	T19A1	0.6	0.1 5	0,6 0.3 0,3	0.1 5	0,6	0.1 5	0.6 0.6 0.6	-		1,2	0.6
		Staphylococcus epidermidis	T21A5	1.2 0.3 0.6 0.3 0.3	0.1 5	0.6	0.1 5	0.6 0.6 1.2	-		1.2	0.6
		Staphylococcus warneri	T12A12	1.2 0.6	-	0.3 0.6 0.3	-	0.3 1.2 1.2	-	-		1.2	-
		Staphylococcus warneri	T26A1	1.2 1.2	-	0.3 0.6 0.3	-	0.3 0.6	-	1.2	-		1.2	-
		Staphylococcus pettenkoferi	T47.A6	1.2 0.3	-	0.3 0.6 0.6	-	0.6 0.6 1.2 1.2	-		1.2	-
		Streptococcus agalactiae	T38.2	-	0.1 5	1.2	-	0.6	0.1 5	0.6	-	1.2 0.6	-	1.2		-	0.3
		Streptococcus agalactiae	T53C9	-	0.1 5	1.2	-	0.6	0.1 5	0.6 0.6	-	0.6	-	1.2		-	0.6
		Corynebacterium striatum	T40A3	1.2 0.6 0.6	0.1 5	0.3	0.1 5	0.3 0.6 1.2 0.6 1.2	-		0.6	-
	Gram -	Citrobacter freundii	11041	-	-	-	-	-	-	-	1.2	-	-	-	-		-	-
		Citrobacter freundii	10268	1.2 1.2	-	0.6	-	-	-	1.2	-	-	-	-		-	-
		Proteus mirabilis	11060	-	-	-	0.6	-	-	-	1.2	-	-	-	-		-	-
		Proteus mirabilis	T28-3	-	-	-	0.6	-	-	-	1.2	-	-	-	-		-	-
		Pseudomonas aeruginosa	8131	-	-	-	0.6 0.6	-	-	1.2	-	1.2 1.2	-		-	-
		Pseudomonas aeruginosa	ATCC 27583	0.6 1.2	-	0.1 5	0.6	-	-	0.3 1.2 0.6 0.6	-		-	0.6
		Pseudomonas aeruginosa	8129	0.6 1.2 1.2 0.3 0.6 1.2 1.2 0.3 1.2 1.2 1.2	1.2		-	0.6
		Microorganisms Tested		Bark	Bark	Leaf	Bark	Leaf	Bark	Leaf	Bark	Leaf	Lichen	Bark	Aerial	Roots	Rhizome	Aerial	Part
	Yeast	Candida albicans	10286	1.2 0.6 1.2	0.1 5	0.6 0.6 1.2 0.6 1.2 1.2	-	1.2		-	0.15
	Gram +	Enterococcus faecalis	C159-6	-	0.3	-	-	0.6 0.6	-	1.2	-	0.6	-	-		-	-
		Enterococcus sp.	8153	-	0.3	-	-	-	1.2	-	-	-	0.6	-	-		-	-
		Staphylococcus aureus	8146	-	0.3 1.2 0.6 0.6	0.1 5	0.6 0.6 1.2 0.6	-	-		-	-
		Staphylococcus aureus	8241	-	0.3 1.2 0.6 1.2	0.0 7	0.6 0.6 1.2 0.6	-	-		1.2	-

Table 3 .

 3 MIC (mg/mL) of methanolic extract from leaves of S. malaccense and rhizome of C. longa.

		Tested Microorganisms		Syzygium malaccense, MIC (mg/mL)	Curcuma longa, MIC (mg/mL)	FIC Index	Effect
	Yeast	Candida albicans	10286	0.15	0.15	1.3	Indifferent
	Gram +	Enterococcus faecalis	C159-6	1.2	0.15	0.9	Additive
		Staphylococcus aureus	8146	1.2	0.3	0.8	Additive
		Staphylococcus aureus	8241	0.6	0.3	0.8	Additive
		Staphylococcus aureus	ATCC 6538	-	0.3	0.7	Additive
		Staphylococcus aureus	T28-1	0.6	0.3	0.7	Additive
		Staphylococcus aureus	T17-4	0.6	0.3	0.9	Additive
		Staphylococcus epidermidis	T46A1	0.15	0.15	1.1	Indifferent
		Staphylococcus epidermidis	T19A1	0.15	0.07	1.3	Indifferent
		Staphylococcus epidermidis	T21A5	0.15	0.07	1.2	Indifferent
		Staphylococcus warneri T12A12	0.6	0.15	1.9	Indifferent
		Staphylococcus warneri T26A1	0.6	0.6	1.5	Indifferent
		Staphylococcus pettenkoferi	T47.A6	0.6	0.6	1.5	Indifferent
		Streptococcus agalactiae T53C9	1.2	0.15	1.3	Indifferent
		Corynebacterium striatum T40A3	0.6	0.15	0.9	Additive
		Streptococcus pyogenes 16138	0.3	-	-	-
		Streptococcus pyogenes 19140	0.15	-	-	-
	Gram -	Proteus mirabilis	11060	1.2	-	-	-
		Proteus mirabilis	T28-3	0.6	-	-	-
		Pseudomonas aeruginosa 8131	1.2	-	-	-
		Pseudomonas aeruginosa	ATCC 27583	0.15	-	-	-
		Pseudomonas aeruginosa 8129	0.15	0.6	0.5	Synergic

MIC: 0.6 mg/mL, MIC: 0.6 mg/mL ≥ MIC > 0.3: mg/mL, MIC ≤ 0.3 mg/mL; (-

Table 4 .

 4 1 H and13 C NMR chemical shifts in ppm of compounds 1-7 (500 MHz in CDCl3).

	Comp
	C (H)	1	2	3	4	5	6	7
	.
	1	110.5	110.7	110.5	110.7	110.7	110.7	110.5
	2	163.8	163.8	163.8	163.5	163.5	163.5	163.5
		116.4	116.4	116.4	115.7	115.7	115.7	115.8
	3	(6.89, d:8.4	(6.89, d:8.4	(6.89, d:8.2	(6.89, d:8.4	(6.88, d:8.4	(6.88, d:8.2	(6.90, d:7.7
		Hz)	Hz)	Hz)	Hz)	Hz)	Hz)	Hz)
		135.4	135.4	135.4	135.4	135.0	135.0	135.4
	4	(7.38, d:7.3	(7.38, dd:7.3,	(7.38, dd:7.5,	(7.38, dd:7.8,	(7.38, dd:7.8,	(7.38, dd:7.8,	(7.39, dd:7.7,
		Hz)	8.4 Hz)	8.2 Hz)	8.4 Hz)	8.4 Hz)	8.2 Hz)	8.0 Hz)

Table 5 .

 5 MIC (µg/mL) of compounds isolated from S. malaccense and standard molecules (n = 3); (NA: Not Active).

	MIC (µg/mL)

Acknowledgments:

We are grateful to the association "Te Rau Ora No Tahaa" and its President Joel Hahe, the communities of Tahaa and Raiatea, Aline Kahn, Poema Moutame, Miza Ohiu and the participants of this study by sharing with us their knowledge about Polynesian traditional medicine. The authors also gratefully acknowledge the Lille ComUE (Communauté d'Université et d'Etablissements) and the project BioScreen-Smartbiocontrol (Interreg V and CPER Alibiotech). We also thank the DIREN and the Minister for Health and Social Services of French Polynesia for their kind attention. The authors are grateful to Amandine Descat and Laurence Goosens from « Plateau de Spectrométrie de Masse »: PSM (ULR 7365 GRITA and Vincent Ultré, Alexandre Rech and Nathalie Azaroual from « Laboratoire de Physique et d'Application RMN » (Faculté de Pharmacie de Lille).

Data Availability Statement: