Jonathan Sorres

Abir Sabri

Orianne Brel

Didier Stien

Véronique Eparvier
email: veronique.eparvier@cnrs.fr

Ilicicolinic acids and ilicicolinal derivatives from the fungus Neonectria discophora SNB-CN63 isolated from the nest of the termite Nasutitermes corniger found in French Guiana show antimicrobial activity

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The search for antimicrobial molecules represents a global challenge in public health. Then, the emergence of multiresistant pathogenic microbial strains and the global increase of the incidence of bacterial and fungal infections encourage the search for new antibiotic compounds [START_REF] Martinez-Rossi | Antifungal resistance mechanisms in dermatophytes[END_REF][START_REF] Davies | Opinion: the pharmacometrics of infectious disease[END_REF]. Novel lead-like antibiotic molecules, in particular those drugs belonging to new structural classes, are urgently needed [START_REF] Borysowski | Is phage therapy acceptable in the immunocompromised host?[END_REF].

Recently, the exploration of the chemical basis and well-described bacteria-host or fungal-host interactions have been shown to be a powerful strategy to identify novel chemical entities, in particular, in natural products from microbes like fungi associated with insects [START_REF] Beemelmanns | Natural products from microbes associated with insects[END_REF]Rhamadar et al., 2014;[START_REF] Nirma | Antifungal agents from Pseudallescheria boydii SNB-CN73 isolated from a Nasutitermes sp. termite[END_REF][START_REF] Adnani | Symbiosis-inspired approaches to antibiotic discovery[END_REF]. In fact, insects are the most diverse animal groups on earth [START_REF] Purvis | Getting the measure of biodiversity[END_REF]. Social insects, particularly ants, are well studied for their symbiosis with microbes to protect colonies [START_REF] Berenbaum | Ecology. Bugs' bugs[END_REF][START_REF] Stow | Antimicrobial defences increase with sociality in bees[END_REF][START_REF] Stow | Chemical and genetic defenses against disease in insect societies[END_REF][START_REF] Currie | Coevolved crypts and exocrine glands support mutualistic bacteria in fungus-growing ants[END_REF]. These mechanisms are also described with termites [START_REF] Matsui | Antibiotics production by an actinomycete isolated from the termite gut[END_REF][START_REF] Nirma | Antifungal agents from Pseudallescheria boydii SNB-CN73 isolated from a Nasutitermes sp. termite[END_REF], Nirma et al., 2015). Therefore, the symbiotic nature of some fungi favours metabolic interactions with their host and environment, thereby increasing the production of bioactive compounds [START_REF] Beemelmanns | Natural products from microbes associated with insects[END_REF][START_REF] Adnani | Symbiosis-inspired approaches to antibiotic discovery[END_REF].

In a preceding study, we isolated Neonectria discophora fungus from a termite nest. This strain was chemically studied, and we isolated the new antibacterial ilicicolinic acids (C and D) and the original structure named ilicicolonal (Nirma et al., 2015a). These molecules, with 5-chloro-2,4dihydroxy-6-methylbenzoic acid or 5-chloro-2,4-dihydroxy-6-methylbenzaldehyde moieties, such as aschochlorins, ascofurons, colletochlorins, ilicicolinic acids and ilicicolins, are less represented in the literature and are isolated from diverse macroscopic and microscopic fungi genuses in association with plants, sponges or insects [START_REF] Beemelmanns | Natural products from microbes associated with insects[END_REF]Takamatsu et al., 1994;[START_REF] Gutiérrez | Bioactive Metabolites from the Fungus Nectria galligena, the Main Apple Canker Agent in Chile[END_REF][START_REF] Zhang | Anti-inflammatory sesquiterpenoids from a sponge-derived fungus Acremonium sp[END_REF]. These compounds show inter alia cytotoxicity on cancer cells lines and antimicrobial activities [START_REF] Hashimoto | Isolation, synthesis and biological activity of grifolic acid derivatives from the inedible mushroom Albatrellus dispansus[END_REF][START_REF] Beemelmanns | Natural products from microbes associated with insects[END_REF].

Results and Discussion

During our first work on Neonectria discophora, many potentially new compounds were detected in the active fractions by LC-MS (Nirma et al., 2015a). The amount of extract available was, however, too low to isolate these molecules, establish their structures and evaluate their biological activities. We then decided to reinvestigate this strain chemically. The bioguided fractionation allowed us to isolate the known ilicicolinal (1) and ilicicolin A, also named LL-Z1272 (2), which were identified by comparison with literature data, (Mori and Fujioka, 1984;[START_REF] Ellestad | Some new terpenoid metabolites from an unidentified Fusarium species[END_REF]Kawagachi et al., 2013) along with 12 new derivatives of ilicicolin and ilicicolinic acids 3-14 (Fig. 1).

Compound 3 was obtained as a dark purple oil. Its molecular formula (C 23 H 31 ClO 4 ) was determined by HRESIMS ( [M+H] + peak at m/z 407.1991 calcd at m/z 407.1989), suggesting eight degrees of unsaturation. The 1 H and 13 C NMR spectra revealed similarities to 1 (Table 1 and2), with only some minor differences observed on the benzoyl group. Therefore, the 1 H-13 C HMBC and 1 H-1 H COSY correlations of 3 were similar to 1 (Fig. 2). However, we observed for compound 3 a hydroxyl proton at δ H 8.59 contrary to a hydroxyl at 12.50 ppm for 1. This fact, permitted to determinate that compound 3 is cyclized on benzoyl group on C-2 contrary to compound 3, which was cyclized on C-4. This compound was named ilicicolinal B.

Compound 4 was obtained as a yellow oil. Its molecular formula (C 23 H 31 ClO 4 ) was determined by HRESIMS ([M-H] -peak at m/z 405.1821, calcd 405.1833), suggesting eight degrees of unsaturation. The 1 H and 13 C NMR spectrums were similar to that of compound 2 (Table 1 and2).

The main difference was its localization on the side chain since there was only one methylene carbon at C-9 at δ C 27.0 ppm. COSY correlations between H-9 and the protons H-8 and H-10, in addition to the HMBC correlations between H-9 and the carbons C-7, C-8 and C-10, led to a diunsaturated hydrocarbon chain on C-8' and C-10' (Fig. 2). To access the cyclopentane structure, we first noticed the COSY correlations between H-6 at δ H 2.49 ppm and the protons H-2 and H-5 at δ H 1.90 and 1.83 ppm, respectively. The methylene 3-CH 3 at δ H 1.03 ppm had HMBC correlations with the carbons C-2, C-3 and C-4, which allowed the cyclization into C-6'. This was well supported by the H-5 HMBC correlations with the carbons C-2 and C-6. The presence of the carbinol at position C-3' was suggested by the deshielded chemical shift at δ C 80.6 ppm.

The nOe correlation observed between the CH 3 -3 and the H-2, as well as the coupling constant between H-6 and H-2 as well as H-5 at J = 11.0 and 10.3 Hz, respectively, confirm the pseudoaxial position of H-6 (Fig. 3), and allow us to deduce the relative configuration of the compound

4.

Compound 5 and 6 were obtained as a dark purple powder. Their molecular formula (C 23 H 31 ClO 4 ) was identical to molecule 4 ([M-H] -peak at m/z 405.1828 for 5 and 405.1842 for 6, respectively, calcd 405.1833). The 1 H and 13 C NMR spectra of 5 and 6 were very similar to those of 4 (Table 1 and 2), with only one difference on the carbon side chain. For compound 5, the 1 H NMR spectra revealed the presence of an exomethylene group at δ H 4.93 ppm. The HMBC correlations between the proton at 2.15 and 2.83 ppm with the carbon at 150.9 and 110.6 ppm and, 1 H-1 H coupling between δ H at 2.83 ppm with a proton at 2.12, 2.23 and 5.17 ppm unambiguously positioned this exomethylene in position C-7 (Fig. 2). In molecule 6, the difference was localized on the double bond in C-7. In fact, the clear and intense nOe correlation between the proton of methyl 7'-CH 3 at 1.77 ppm and the olefinic proton H-8' at 5.30 ppm allowed us to deduce the Z configuration for the C-7'-C-8' double bond. The relative configuration was based on the nOe correlations analysis and by comparison with molecule 4 (Fig. 3). These compounds 4, 5 and 6 were named ilicicolinal C, D and E. 1993, calcd 407.1989) corresponding to eight degrees of unsaturation. We first noticed that compound 7 and 2 presented similar 1 H and 13 C NMR spectra (Table 2 and3). The geranyl side chain was easily deduced from the COSY correlations H-4/H-5, H-5/H-6 and H-9/H-10, together with the intense HMBC correlations observed for the protons of methyl 7'-CH 3 with C-6/C-7/C-8 and the protons of methyl 12-CH 3 /13-CH 3 with C-10/C-11 (Fig. 2). The chromane moiety was determined with the COSY correlation H-1/H-2 and with the HMBC correlations given by the protons H-1 with the aromatic carbons C-2/C-3/C-4, the aldehydic proton at 10.14 ppm with C-4/C-5 and the methyl 6-CH 3 with C-1/C-5/C-6. The connection of the geranyl chain was deduced from the HMBC correlations H-4/H-5 with the oxygenated quaternary carbon C-3 and 3-CH 3 with C-2/C-3/C-4.

Compound 8 was obtained as a yellow oil. Its molecular formula (C 23 H 32 O 4 ) was determined by HRESIMS ( [M+H] + peak at m/z 373.2374 calcd at m/z 373.2379) suggesting eight degrees of unsaturation. Compared to 7, the molecular formula of 8 indicated the presence of additional hydrogen and the lack of the chlorine. It was confirmed by comparison of the 1 H NMR spectra between 7 and 8, which showed a unique difference: a supplementary aromatic proton at δ H 6.23 ppm (Table 2 and3) for 8.

Compound 9 was obtained as a dark purple powder and has the same molecular formula as 7 407.1994, calcd 407.1989). The 1 H and 13 C NMR spectra revealed similarities to 7 (Table 2 and3), with only some minor differences observed on the benzoyl group.

([M+H] + peak at m/z
However, the presence of the hydroxyl proton at δ H 8.59, contrary to 12.73 ppm in molecule 7, permitted the determination that compound 9 was cyclized on C-2 (as compound 3) but not on C-4 as molecules 7 and 1. 2 and3). Like compound 8, molecule 10 had no chlorine on the aromatic and presented a proton at  H 6.40 ppm. Compared to compound 8, 13 C NMR data revealed the presence of one additional carbinol at  C 69.2 ppm, and the lack of one olefinic carbon. The HMBC correlations of H12'/H13' with the carbinol mentioned above suggested that compound 10 resulted from the hydration of a C-10-C-11 double bond according to the Markovnikov's rule (Fig. 2). The chemical shift of the hydroxyl proton at δ H 8.19 ppm permitted to determinate that compound 10 was cyclized on C-2.

The relative configurations of compounds 7 to 10 were determined by NMR analyses of the nOe correlations between 3-CH 3 with H-1b and H-2 (Fig. 3.). Indeed, on the NOESY spectrum nOe correlations are observed between 3'-CH 3 and only one of the methylene protons H-1', and this methylene proton does not correlate with the H-2' proton. These compounds with the chromane core were named ilicicolinal F, G, H and I. [START_REF]D -24 (c 0.1, MeOH); UV (λ max CHCl 3 ) (log ) 213 nm (3.4); NMR spectral data, see supporting information and Tables 2 and 3; HRESIMS [M+H] + m/z 407[END_REF]Nirma et al. 2015a). And, for compound 13 and 14, with the HMBC correlations of the aromatic proton observed at  H 6.05 and 6.46 respectively (Fig. 2). Compounds 11-13 were named ilicicolinic acid E, F and G, and compound 14 was named ilicinolinol.

From the 17 metabolites produced by Neonectria discophora SNB-CN63, the possible biosynthetic correlations among them are summarized in Fig. 4. In the literature, it was reported that ilicicolin A

(2) was biosynthesized through the mevalonate and polyketide pathways [START_REF] Hunter | Detection of deuteride shifts in the biosynthesis of the fungal triprenylphenol, ascochlorin, by 13C nuclear magnetic resonance spectroscopy following incorporation of [3-13C, 4-2H2]mevalonic acid[END_REF][START_REF] Kawaguchi | A new ascochlorin derivative from Cylindrocarpon sp. FKI-4602[END_REF]. In this study, we proposed for groups of key intermediates between ilicicolin A (2).

First, an oxidation of 2 can conduct ilicicolinic acid A (Fig. 4, track 1), and a hydration of its farnesyl chain can conduct ilicicolinic acids, such as compounds 11, 12 and 13. Epoxide formation in the alkyl chain of compound 2, and epoxide cyclisation with the acidic phenolic hydroxyl group at C-2 or C-4 by an S N 2 opening of the epoxide ring (6 endo-trig cyclisation) results in the formation of the compounds with a chromane core, molecules 7, 8, 9 and 10 (Fig. 4, track 2a).

From the same epoxidate 2, a 5-exo-trig cyclization with C-2 or C-4 can produce benzofuran cores:

1 and 3 (Fig. 4, track 2a). Finally, a 5-exo-trig cyclisation by epoxide cyclisation with a double bond of the alkyl chain gives compounds 4 to 6 with a resorcinol core (Fig. 4, track 2b). This biosynthetic pathway supported the relative configuration of compounds 1 and 3 and the configurations of molecules with a resorcinol and a chromane core. The C-3 configuration of 1 has never been described in the literature. To determine absolute configuration of this molecule, (S)-and (R)-MTPA esters of this compound were synthetized (respectively 1a and 1b), The methyl CH 3 -C-3' is at δ H 1.28 and δ C 22.7 for the (R)-Mosher ester of 1, and at δ H 1.27 and δ C 22.5 for the (S)-Mosher ester. Based on HMBC correlations of this methyl, it was possible to find protons H-2' (R: δ H 4.776 and δ C 91.5; S: δ H 4.801 and δ C 91.6) and H-4' (R: δ H 1.43-1.51 and δ C 37.8; S: δ H 1.43-1.51 and δ C 37.9). The analysis of protons H-2' is quite straightforward as they are well separated in the proton spectra. However, H-4' protons are a large multiplet in both case, and it was not possible to measure differences in chemical shifts even with HSQC (see supporting information). In the (S)-ester of compound 1, H-2' appears more downfield than in the (R)-ester, and is therefore deshielded by the phenyl group. It can be concluded that C-3' is (R), and so is C-2' (if we assume that this compound is generated by a SN 2 on an epoxide) (Fig. 4). Finally, if we take into account the absolute configuration determined for compound 1, and if we consider that all the compounds come from the formation of the same epoxide, we can deduce the absolute configurations of compounds 1-10, as being 2'R, 3'R for 3; 2'S, 3'R for compounds with a chromane core (7-10), and 2'R, 3'R and 6'R for compounds 4, 5 and 6. 4). Overall, the antimicrobial activities of compounds 1 to 14 were moderate to good, depending on the strain tested. We demonstrate that compounds 3, 4, 5, 10, 11, 12 and 14 have antimicrobial activities less than 32 µg/mL. These molecules show significant activities against S. aureus and/or MRSA (Methicillin-resistant Staphylococcus aureus), with vancomycin as a positive control, with the best MIC for 4 at 8 µg/mL. Contrary to other compounds, molecules 11 and 12 showed growth inhibition on T. rubrum with a MIC at 8 µg/mL too. If we compare ilicicolinic acids 11 to 14, it seems clear that the presence of the chlorine element is necessary to have activity against T. rubrum (by comparison between 11 and 13), and carboxylic acid is necessary for activity against MRSA (by comparison between 11 and 14). This structure-activity relationship against S. aureus and MRSA between compounds 1 to 14 is not clear, but we observed that the presence of a C-11 hydroxyl group (as in molecules 10, 11, 12 and 14) or the cyclisation between C-2 and C-3 (as compounds 9 and 10) allow us to determine that the hydroxyl in position C-4 and/or the C-11 groups are important for these antimicrobial activities. Finally, if we compare compounds 3 to 5, it seems that the proton configuration of H-8 is important for having activity against MRSA. We also tested the cytotoxicity on studied MRC5 and KB cell strain extracts and fractions, on which we detected inhibition growth 20% at 1 µg/mL (See supporting information).

Conclusion

In conclusion, this article provides the first description and isolation of 4 new ilicicolonic acids (11-14) and 8 original ilicicolinal derivatives (compounds 3-10). Contrary to compound 1, ilicicolin A (2) was isolated from other natural products: Cylindrocarpon sp. and Fusarium sp.

(filamentous fungi), which were isolated from soil samples [START_REF] Ellestad | Some new terpenoid metabolites from an unidentified Fusarium species[END_REF]Mori and Fujioka, 1984;[START_REF] Kawaguchi | A new ascochlorin derivative from Cylindrocarpon sp. FKI-4602[END_REF]. Prenylated phenols are known for their antibiotic properties, and compound 2 was previously synthetized. Compound 2 is also described in the literature as anti-protozoan, antineoplastic and as a dihydroorotic acid dehydrogenase inhibitor [START_REF] Saimoto | Novel dihydroxybenzene derivatives and antiprotozoal agent comprising same as active ingredient[END_REF][START_REF] Saimoto | Novel dihydroxybenzene derivatives and antiprotozoal agent comprising same as active ingredient[END_REF]. In our study, we detail for the first time complete NMR 1 H and 13 C and 2D data on this molecule. From ilicicolin A, we have been able to propose a biosynthetic pathway for all the new isolated molecules from Neonectria discophora extract. This biosynthetic pathway allowed us to deduce the configuration of all the isolated compounds. Moreover, the absolute configuration of the compound 1 was determined for the first time. Finally, six compounds had antibacterial activities against MRSA, and two compounds had antifungal activity against T. rubrum with MIC  16 g/mL: ilicicolinal C, D, E and I and ilicicolinic acid E and F. This finding may inspire the discovery of new and selective antibacterial agents. These compounds were obtained from a Neonectria discophora strain isolated from an aerial nest of Nasutitermes corniger. It is reasonable to hypothesize that this microbe provided its host with effective antimicrobial protection. The biosynthetic pathway it utilized by this microbe and whether this strain is shared by other colonies is a question that remains to be addressed.

Experimental

General experimental procedures

Optical rotations were measured on an Anton Paar MCP 300 polarimeter in a 100 mm-long 350 µL cell. Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker 500 MHz spectrometer or a Bruker 600 MHz spectrometer equipped with a 1-mm inverse detection probe.

Chemical shifts (δ) are reported as ppm based on the TMS signal, and the coupling constants (J) are reported in Hertz. HR-ESI-MS measurements were performed using a Waters Acquity UPLC system with a column bypass coupled to a Waters Micromass LCT Premier Time-of-Flight mass spectrometer (Milford, MA, USA) equipped with an electrospray interface (ESI). Flash chromatography was performed on a Grace Reveleris system with dual UV and ELSD detection equipped with an 80 g silica column. For UV-based experiments, effluents were monitored at 254 and 280 nm. Analytical and preparative HPLCs were conducted with a Gilson system equipped with a 322 pumping device, a GX-271 fraction collector, a 171 diode array detector and a prepELSII detector electrospray nebulizer. Columns used for these experiments included a Phenomenex Luna C 18 5 µm 4.6 x 250 mm, Phenomenex PFP 5 µm 4.6 x 250 mm analytical column, Phenomenex Luna C 18 5 µm 21.2 x 250 mm and PFP 5 µm 21.5 x 250 mm preparative column. The flow rate was set to 1 or 17 mL/min using a linear gradient of water mixed with an increasing proportion of acetonitrile. All solvents were HPLC grade. UV spectra were recorded using a Perkin-Elmer Lamba 5 spectrophotometer. The CD spectra were measured at 25°C on a JASCO J-810 spectropolarimeter. Potato Dextrose Agar (PDA) was purchased from Fluka Analytical.

Collection and identification of Neonectria discophora SNB-CN63.

N. discophora was collected, identified and preserved as described in the literature (Nirma et al., 2015a). The strain was isolated from an N. corniger termite aerial nest, sampled in Rémire-Montjoly, French Guiana, in 2011. A piece of the nest matter was placed in supsension in sterile water, and this solution was spread on PDA medium placed in Petri dishes. After 7 days of incubation at 25°C, the first fungus hyphae were removed and transferred to another Petri dishes.

The colony is maintained in triplicate at -80°C in glycerol-Water (50/50). A sample was submitted for amplification and nuclear ribosomal internal transcribed spacer ITS4 sequencing allowed for strain identification by NCBI sequence comparison. The ITS4 sequence has been registered in the NCBI GenBank database (http://www.ncbi.nlm.nih.gov) under registry number KJ023733.

Molecular analyses were performed externally by BACTUP, France.

Culture, extraction and isolation

The N. discophora strain was cultivated on PDA at 26°C for 15 days, initially on a small scale and then on 150 Petri dishes (of 14-cm diameter). The fungus and culture medium were then transferred into a large container and macerated with ethyl acetate for 24 h. The organic solvent was then collected by filtration, washed with water in a separating funnel and evaporated, yielding 0.92 g of crude extract. A portion of the crude extract (803 mg) was purified by flash chromatography with a linear gradient of hexane-AcOEt followed by another gradient of AcOEt-MeOH. Six fractions were gathered based on their TLC profiles. Fractionation of fraction IV on the C 18 prep-HPLC with ACN/Water gradient (87/13 to 100/0) in 15 min permitted to obtain compound 1 (91.5 mg, rt 14.1 min), 2 (8.6 mg, rt 17.3 min) and 7 (8.1 mg, rt 16.6 min). Two subfractions were submitted to PFP HPLC-prep in other conditions (ACN/Water 75/25 to 100 % in 24 min) to give compounds 9 (1.8 mg, rt 6.6 min) and 4 (1.2 mg, rt 11.0 min) from the first fraction and compound 6 (0.8 mg, rt 8.2 min) from the second. Purification of fraction II with C 18 prep-HPLC with isocratic ACN/water for 4 min followed by a gradient of 87/13 to 100/0 for 15 min permitted to isolate 14 (3.1 mg, rt 8.7), 10 (0.9 mg, rt 9.6 min), 13 (1.5 mg, rt 12.5), 11 (2.6 mg, rt 12.9) and 12 (3.3 mg, rt 15.2). Fractionation of fraction VI on a C 18 prep-HPLC with an ACN/Water gradient (80/20 to 83/17) in 30 min gave compounds 5 (0.5 mg, rt 10.4 min), 3 (0.9 mg, rt 11.0 min), and 8 (0.7 mg, rt 15.2).

Ilicicolinal (1)

Dark red oil; [α] 25 D +99 (CHCl 3 ; c 0.1); UV (λ max CHCl 3 ) (log ) 240 nm (3.2), 302 (3.6); 1 H NMR: 3.22 m (H-1), 4.87 dd (8.5, 9.8) (H-2), 1.55 t (8.4) (H-4), 2.14 m (H-5a), 2.18 m (H-5b), 5.14 t (6.8) (H-6), 1.98 m (H-8), 2.09 t (6.3) (H-9a), 1.97 m (H-9b), 5.08 t (6.8) (H-10), 1.77 s (H-12), 1.60 s (H-13), 10.14 s (1-CHO), 12.48 s (2-OH), 2.59 s (6-CH 3 ), 1.35 s (3-CH 3 ), 1. 1 and2; HRESIMS [M+H] + m/z 391.2034 (calcd for C 23 H 32 ClO 3 m/z 391.2040).

Ilicicolinal B (3)

Dark purple oil; [α] 25 D +21 (c 0.1, CHCl 3 ); UV (λ max CHCL 3 ) (log ) 240 nm (1.7), 301 nm (2.6); NMR spectral data, see supporting information and Tables 1 and2; HRESIMS [M+H] + m/z 407.1991 (calcd for C 23 H 32 ClO 4 m/z 407.1989).

Ilicicolinal C (4)

Yellow oil; [α] 25 D +67 (c 0.1, MeOH); UV (λ max CHCl 3 ) (log ) 240 nm (3.1), 300 nm (3.4); NMR spectral data, see supporting information and Tables 1 and2; HRESIMS [M-H] -m/z 405.1821 (calcd for C 23 H 30 ClO 4 405.1834).

Ilicicolinal D (5)

Beige powder; [α] 25 D -80 (c 0.02, MeOH); UV (λ max MeOH) (log ) 222 nm (3.8), 299 (3.2); NMR spectral data, see supporting information and Tables 2 and3 2 and3; HRESIMS [M-H] -m/z 397.2146 (calcd for C 22 H 34 ClO 4 m/z 397.2146).

For compounds 3, 6 and 8-10 13 C chemical shifts were deduced from proton-carbon correlation experiments.

MTPA derivatives of 1

In an NMR sample tube (3 mm diameter) 1 mg of dimethylamynopyridine (DMAP) was added to 1.0 mg of compound 1. Nine microliter of pyridine-d 5 and 600 l of CDCl 3 were added to this mixture. Then, 1.35 l of (R)-MTPA chloride was added. The resultant reaction mixture was allowed to stand at RT for 120 min, to afford (S)-MTPA ester 1a. The reaction was monitored directly by NMR and upon completion, the chemical shifts of the relevant protons were obtained directly from 1 H and 2D NMR experiments. The identical procedure was carried out to obtain the (R)-MTPA 1b ester from (S)-MTPA chloride.

Antimicrobial assays.

The ATCC strains were purchased, and the clinical isolate was provided by Prof. P. Loiseau, at the University Paris Sud (ITS sequence deposited in the NCBI GenBank database under the registry number KC692746 corresponding to SNB-TR1 strain). Crude extracts, fractions and pure compounds were tested according to the reference protocol of the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2003(EUCAST, , 2008)). The MIC value was obtained after 18 h for yeasts, 5 days for T. rubrum and 24 h for S. aureus.

Cytotoxicity assays.

The cytotoxicity assays were conducted with the KB (nasopharyngeal epidermoid carcinoma) and MRC5 (Human Fetal Lung Fibroblast) cell lines according to the procedure described by Tempête et al. with the positive control Taxotère (with IC 50 = 0.2 and 0.5 nM respectively) [START_REF] Tempête | In vitro cytostatic activity of 9demethoxyporothramycin B[END_REF].

Acknowledgment

This work has benefited from an "Investissement d'Avenir" grant managed by Agence Nationale de la Recherche CEBA, ref. ANR-10-LABX-0025.

  Ilicicolinic acids and ilicicolinal derivatives from the fungus Neonectria discophora SNB-CN63 isolated from the nest of the termite Nasutitermes corniger found in French Guiana show antimicrobial activity Jonathan Sorres a , Abir Sabri a , Orianne Brel a , Didier Stien b , Véronique Eparvier a,* a CNRS, Institut de Chimie des Substances Naturelles, UPR 2301, Université Paris-Saclay, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France b Sorbonne Universités, UPMC Univ. Paris 06, CNRS, Laboratoire de Biodiversité et Biotechnologies Microbiennes (LBBM), Observatoire Océanologique, 66650 Banyuls/Mer, France

Fig. 1 .

 1 Fig. 1. Structures of isolated compounds 1 to 14.

Fig. 2 .Fig. 3 .

 23 Fig. 2. Key 1 H-1 H COSY (bold line) and 1 H-13 C HMBC (dashed arrows) of compounds 3 to 14.

  Compounds 11 and 12 were obtained as a light beige powder. A molecular formula of C 23 H 35 ClO 6 was determined via HRESIMS, which showed a pseudomolecular ion at m/z 441.2053 and 441.2047 [M-H] -(calcd 441.2044). Compound 13 had a molecular formula of C 23 H 36 O 6 and was determined via HRESIMS analysis, which showed a pseudomolecular ion at m/z 407.2441 [M-H] - (calcd 407.2434) and the compound 14 molecular formula of C 22 H 35 O 4 Cl was deduced from HRESIMS analysis, which showed a pseudomolecular ion at m/z 397.2146 [M-H] -(calcd at m/z 397.2146). Compounds 11-14 shared the same di-hydroxylated farnesyl side chain. Structural differences occurred on the resorcinol part with variations in the nature and position of the substituents. The structure of the side chain was deduced from the analysis of COSY correlationsand the clear and intense HMBC correlations observed for the methyl groups. The methyl group of these compounds linked to 7' had  H around 1.06-1.11 ppm instead of 1.58 ppm for compounds 2, and the C-7' quaternary carbon exhibited a signal around  c 70.9-72.0 ppm, which indicated that it carried out an oxygen. Farnesyl chain of 11-14 was hydrated across C-6-C-7'. Therefore, the COSY correlations between the methyl group at  H 1.14-1.16 ppm (H-12' and H13') and a carbon at  c 69.2-70.1 ppm (C-11') placed a tertiary alcohol in C-11'. The position and the nature of the substituents on the aromatic moiety were determined with the 13 C NMR data and HMBC correlations observed with 6-CH 3 as described in the literature for ilicicolinic acids A-D (Kuroda

Fig. 4 .

 4 Fig. 4. Possible biosynthetic pathway to produce the compounds isolated from Neonectria discophora SNB-CN63.

Fig. 5 .

 5 Fig. 5. R-MTPA (1a) and S-MTPA (1b) esters of 1.

  63 s (7-CH 3 ), 1.35 (3-CH 3 ) see supporting information; 13 C NMR: 114.6 (C-1), 159.4 (C-2), 112.2 (C-3), 163.4 (C-4), 108.3 (C-5), 141.3 (C-6), 27.4 (C-1), 91.7 (C-2), 73.6 (C-3), 36.9 (C-4), 21.8 (C-5), 123.7 (C-6), 136.0 (C-7), 39.6 (C-8), 26.6 (C-9), 124.1 (C-10), 131.5 (C-11), 25.7 (C-12), 17.7 (C-13), 14.0 (6-CH 3 ), 22.7 (3-CH 3 ), 16.0 (7-CH 3 ); HRESIMS [M+H] + m/z 407.1992 (calcd for C 23 H 32 ClO 4 m/z 407.1989). 4.5. Ilicicolin A (2) White pink powder; [α] 25 D 0 (c 0.1, MeOH); UV (λ max CHCl 3 ) (log ) 210 nm (3.0) 240 nm (3.3), 293 nm (3.5); NMR spectral data, see supporting information and Tables

  ; HRESIMS [M-H] -m/z 407.2441 (calcd for C 23 H 35 O 6 m/z 407.2434). 4.17. Ilicicolinol (14) Beige powder; [α] 25 D -8 (c 0.03, MeOH); UV (λ max MeOH) (log ) 205 nm (3.1); NMR spectral data, see supporting information and Tables

Appendix A. Supplementary data

Experimental methods, full spectroscopic data and NMR spectra of compounds 1-14 can be found at http://dx.doi.org/.


Ana T F Batista

Dorothea Wisser

Thomas Pigeon

David Gajan

Fabrice Diehl

Mickael Rivallan

Leonor Catita

Anne-Sophie Gay

Anne Lesage

Céline Chizallet

Pascal Raybaud
email: pascal.raybaud@ifpen.fr

Beyond γ-Al 2 O 3 Crystallite Surfaces: the Hidden Features of Edges Revealed by Solid-State 1 H NMR and DFT Calculations

Keywords: alumina, density functional theory, edge, hydroxyls, NMR spectroscopy

) and (100) alumina facets. The sharp 1 H NMR peak at 0 ppm corresponds to µ 1 -OH groups which are located on this edge and are free from hydrogen bonding. Moreover, we show that these edge sites are the most reactive with respect to chlorine exchange.

Introduction

The gamma polymorph of Al 2 O 3 is used in numerous industrial applications thanks to its remarkable catalytic and adsorptive properties [START_REF] Euzen | Handbook of Porous Solids[END_REF] which have been extensively studied by experimental and theoretical approaches. [START_REF] Knözinger | [END_REF][3][4][5][6][7][8][9] In particular, surface hydroxyl groups, responsible for Brønsted acidity, have been characterized by infra-red (IR) spectroscopy [START_REF] Knözinger | [END_REF]3,[10][11][12] and by 1 H solid-state nuclear magnetic resonance (NMR) spectroscopy. [12][13][14][15][16] Moreover, DFT calculations [5] enabled the refinement of the empirical assignment of the main IR bands thanks to the determination of the hydration of the three main exposed γ-Al 2 O 3 surfaces: [17] (110), (100) and (111). In 1 H NMR work, the use of high fields and fast magic angle spinning (MAS) provided improved spectral resolution, revealing various, partially resolved, 1 H signals. Three main spectral regions were identified at around 0 ppm, 1-3 ppm and 3-5 ppm, respectively assigned to non H-bonded µ 1 -OH; µ 2 -OH and µ 3 -OH. These hydroxyls are connected to one, two or three Al atoms exhibiting different coordination (Al IV , A lV , Al VI ). [15,16] Also, broad signals above 5 ppm were associated with hydrogen bond donor species. [16] However, to go beyond the current knowledge of high surface aluminas, atomic scale insights into the nature and location of the hydroxyls that originate each signal are required. In particular, considering extended surfaces only to interpret NMR data overlooks that γ-Al 2 O 3 crystallites are finite nano-objects exhibiting various morphologies. Like metallic nanoparticles with stepped surfaces and edges that provide low coordination sites active in catalysis, [18][19][20][21] edge architectures present on alumina crystallites should harbour original hydroxyl and Al sites distinct from those on such surfaces. In a recent review, Busca points to the likely role of edges and corners on alumina's reactivity, which are suspected to be the location of the strongest Lewis acid sites bearing hydroxyl groups more resistant to dehydration. [8,22] Here, the interpretation of the proton NMR spectra of γ-Al 2 O 3 is revisited, providing new insights into the nature and location of the hydroxyls in order to refine our current structural knowledge of high surface area aluminas. 1 H NMR spectra was recorded on two Al 2 O 3 samples of different morphologies, and it is shown by chemical shift DFT calculations that considering alumina facets only leads to an incomplete description of the spectra. This challenge is solved by establishing edge models highlighting the selective chlorination of the edge sites.

Materials and methods

Two relevant commercial high surface alumina samples are considered, PuralSB3 and TH100 (Sasol), labelled P-egg and T-flat respectively, exhibiting different high BET surfaces (S.I.S1). Their characterization by high-resolution transmission electron microscopy (HR-TEM) suggests that T-flat crystallites are larger and have a better defined and more parallelepipedic platelet-like morphology than P-egg's, which appear to have a round shape (S.I.S1). Chlorinated alumina samples were prepared by exposing alumina to a HCl solution (3.5% w Cl/g dry alumina ) for 45 minutes, followed by drying and calcination at 520°C. After thermal treatment under H 2 for 2h at 500°C (ramp 5°C/min) and rotor packing under inert atmosphere, quantitative solid-state 1 H NMR spectra were acquired. H SS MAS NMR spectra were obtained on a SB Bruker Avance III 800 (800 MHz 1 H resonance frequency, 18.8 T) spectrometer using a zirconia 2.5 mm rotor at 30 kHz MAS. Quantitative 1 H spectra were recorded using a DEPTH [23][24][25] sequence for probe background suppression. Pre-scan delays were set to five times the 1 H longitudinal relaxation time (T 1 ). Chemical shifts were referenced relative to adamantane. Spectral deconvolution was done using DMFit [26]. These spectra recorded at high magnetic field and relatively fast MAS reveal the surface OH signals and subtle changes in chemical shifts between the two different types of alumina (Figure 1 a).

To help for the interpretation of NMR experiments, chemical shifts (S.I.S2.3.) have been calculated by using the linear response approach [27,28] within the PBE-dDsC exchange correlation functional [29,30] and PAW pseudopotentials [31] with an energy cut off of 400 eV as implemented in the VASP code. [32,33] The average of the isotropic chemical shielding (σ) of each proton on a TMS (tetramethylsilane) model (a single molecule surrounded by vacuum) was used as reference to calculate the isotropic chemical shift (δ) of the protons of the various hydroxyls of the alumina surfaces or at the edges : δ iso =σ iso -σ ref [START_REF] Euzen | Handbook of Porous Solids[END_REF] For that purpose, relevant periodic models of alumina ( 110), ( 100) and (111) surfaces and ( 110)-(100) edges have been constructed and optimized for various thermodynamically relevant hydration coverages depending on the experimental (T, P) analytical conditions (S.I.S2.). Geometry optimizations were performed using a conjugate-gradient algorithm and convergence criterion on forces of 0.01 eV Å -1 .

Results and Discussion

In order to rationalize these experimental results, chemical shifts were calculated (Figure 1 b and S.I.S2.3) for hydrated surface models of the three main exposed γ-Al 2 O 3 surfaces [17] (110), ( 100) and (111) as defined in previous DFT works [4,5,7] For each surface, several hydration degrees were considered (S.I.S2.1.) in order to well represent the alumina samples after thermal treatment, (Figure S8). In the experimental conditions, 10 First, it can be noted that most of the hydroxyls on these surfaces are involved in hydrogen bonding (Tables S8, S9, S10). Almost half of them are H-bond donors, resulting in high chemical shifts: several µ 2 -OH appearing at >5 ppm. In this case, the chemical shift correlates with hydrogen bond length (Figures S11 andS12), [16,[34][35][36] leading to large variations in .

Experimentally, H-bond donors are not expected to provide well defined signals such as the ones observed up to 3 ppm. [36] Thus, we observe a poor correspondence between the previously proposed range and the simulated 1 H chemical shifts which do not explain the below 0 ppm signal, expected to correspond to µ 1 -OH. [14,15] Contributions calculated at 0. These rows of atoms on both edges will be referenced to as edge sites. The systematic study of the hydration state of the nano-rod (and the corresponding edges)

shows that water is the most strongly stabilized at the edge sites, rather than on the facet sites.

For the adsorption of the first water molecule per unit cell of simulation, this leads to the formation of one µ 2 -OH, the H + bonding to the O II atom on the O-edge, and of one µ 1 -OH, the OH -bonding to the Al III on the lower row on the Al-edge that relaxes into a tetragonal geometry (Table S7). Both these hydroxyls are not involved in hydrogen bonding. The corresponding adsorption energy is -436 kJ.mol -1 , which is far greater than usual adsorption energies reported on the alumina surfaces, [4] and is consistent with the chemical intuition that the reactivity of Lewis Al edge sites should be greater. [8] Such a configuration is a priori striking when thinking in terms of water dissociation, because the hydroxyl and the corresponding proton are not lying on the same kind of edge. However, one shall consider that experimentally, such low hydration states are obtained upon dehydration of the surface sites, making it possible to leave at the surface distant OH and H groups in the end, after recombination of other OH and H pairs. When more than two water molecules are adsorbed per unit cell of simulation, all the "edge sites" are saturated and the near edge sites on the (110) top surface of the nano-rod start being occupied (Figure 2 b), while the (100) facet of the rod remains dehydrated. These "near edge" sites exhibit a H-bond network identical to that of the surface models. In the conditions of thermal treatment (10 -4 < P(H 2 O) <10 -6 bar and 700K< T <800K), among the multiple hydration degrees equally stable (S.I. Figure S10), we choose two relevant cases: one and six adsorbed water molecules per pair of edges.

The calculated 1 H chemical shifts for the (110)-(100) edge with one and six adsorbed water molecules are represented in Figure 1 c). µ 1 and µ 2 hydroxyls on edge sites appear in the expected  range: ≈0 ppm and ≈2 ppm respectively. These OH are not only free from hydrogen bonding, but also isolated from other neighbouring hydroxyls. For 6 H 2 O molecules, hydroxyls with  >3 ppm are located on near edge sites and are H-bond donors, as the red outlining indicates.

With this edge model a significantly improved correlation between experimental and calculated chemical shifts is achieved, especially for the sharp peak at ≈0 ppm. Thus, this peak corresponds mostly to isolated µ 1 -OH located on the edges of alumina crystallites, which are free from hydrogen bonding. While the contribution of some free and H-bond acceptor species on the (111) surface cannot be ruled out, their contribution to the signal is minor, as discussed ahead. The 1-3 ppm region is expected to result from non-isolated µ 1 -OH and µ 2 -OH that are free from H-bonds or H-bond acceptors located on the edges and on the surfaces of the crystallites. The fact that in average, the µ 2 -OH sites are much more represented in this region with respect to the 0-1 ppm region, is in agreement with 1 H- 27 Al RESPDOR experiments by Taoufik et al.. [15] While not much insight was gained into the 3-5 ppm broad signal, its empirical assignment to µ 3 -OH is strongly questioned. Indeed, for the hydration degree of our samples, only two µ 3 -OH were found in the models of interest (one for (110)

12.0 OH/nm² and other for edge 6H 2 O) and both are hydrogen bond donors with  > 5 ppm (7.9 and 14.2 ppm, respectively). Moreover, µ 1 -OH and µ 2 -OH species acting as hydrogen bond donors are also impacting the 3-5 ppm broad signal. Lastly, broad signals with  > 5 ppm are thought to correspond to hydroxyls involved in the hydrogen bond network of the surfaces as hydrogen bond donors.

With this improved assignment, it is now possible to rationalize the impact the alumina nanocrystallite morphology on the 1 H NMR spectra. As mentioned above, P-egg crystallites are rounded and smaller than those of T-flat, which are more parallelepipedic. Thus, P-egg presents a higher edge to surface ratio than T-flat, which explains the relative intensities of the ≈0 ppm peaks of edge µ 1 -OH: 12% and 4% of 1 H signal intensity for P-egg and T-flat respectively (Table S2). In the 1-3 ppm region, the  value of the most intense peak depends on the sample which is a strong indication of different proportions of exposed surface types for each alumina. Indeed, the electron diffraction analysis (Figure S4) suggests that the (111)

termination is more exposed in T-flat crystallites than in P-egg's. Finally, the large signal for  > 3 ppm is more intense for T-flat, which is explained by a H-bond network between hydroxyls that is more developed on the extended surface planes of T-flat than on P-egg.

To get further insights into the surface structure of P-egg and T-flat, the reactivity of the alumina hydroxyls were probed with chlorine. Chlorinated aluminas are used in many catalytic processes [START_REF] Marcilly | Acido-basic catalysis: Application to refining and petrochemistry[END_REF] but Cl can also be used as a probe for µ 1 -OH. [12,14,[START_REF] Digne | [END_REF] Chlorinated samples were prepared so as to have 0.5 and 1.4% w/w Cl deposited on each alumina (referred as P-egg-x%Cl and T-flat-x%Cl with x=0.5, 1.4). The effect of chlorine on the 1 H NMR spectra is clearly different for both aluminas (Figure 1 d and Figure S6). For P-egg, the 0 ppm signal disappears completely, as previously reported, [12,14] while the rest of the spectrum remains unchanged. For T-flat, not only does the 0 ppm signal not disappear completely, but it is also observed an intensity increase in the 3-5 ppm region, associated to an intensity decrease for  > 5 ppm (Table S2).

According to the DFT OH/Cl exchange energies (S.I. S2.5, Figure 2 c), the edge µ 1 -OH sites are predominantly exchanged with chlorine out of all the considered hydroxyls. The subsequent exchanged hydroxyls would be the µ 1 -OH of the (110) surface and after that only the µ 1 -OH and µ 2 -OH sites of the (111) surface. Since the ≈0 ppm peak completely disappears for P-egg, and no impact is observed on the remaining parts of the spectrum, it is believed that only the µ 1 -OH located on the edges are exchanged with chlorine at 1.4%Cl and not those of the surfaces. For T-flat, since the peak at ≈0 ppm does not fully disappear while the 3-5 ppm and  > 5 ppm regions are perturbed, not only the edge µ 1 -OH are exchanged but also surface hydroxyls (most likely on the (110)), disturbing the H-bond network. The signal remaining at ≈0 ppm after chlorination should correspond to the (111) surface µ 1 and µ 2 hydroxyls. Just as the electron diffraction analysis, the NMR results also suggest that the (111) surface is relatively more exposed in T-flat crystallites than in P-egg's. This implies that the number of edge µ 1 -OH of T-flat is not sufficient to exchange 1.4% Cl while it is the case on P-egg.

Conclusion

The

  -4 < P(H 2 O) <10-6 bar and 700K< T <800K, the (110) surface exhibits 3.0 OH/nm² and 9.0 OH/nm², the (111) surface 12.3 OH/nm², while the (100) surface is dehydrated.

Figure 1 .

 1 Figure 1. a) 1 H MAS NMR spectra of two aluminas with different morphologies, P-egg and

  4 and 0.8 ppm from μ 1 -OH and μ 2 -OH (respectively) of the (111) surface are found, while the classical assignment does not expect μ 2 -OH in this range. In addition, no µ 3 -OH are predicted in the 3-5 ppm region.Overall, these results show that the resonances observed in the experimental 1 H NMR spectra of these γ-Al 2 O 3 samples cannot be fully interpreted by considering only their crystalline surfaces. This trend holds true for any hydration degrees (S2.1 and S2.3).To go beyond, another site architecture must be conceived. Apart from the empirical model proposed by Busca,[8] no atomistic model of alumina edge was previously reported in the literature. Thus, a novel model for the (110)-(100) edge was determined (Figure2 a) based on a nano-rod structure (S.I.S2.2) resulting from the cleavage of the alumina bulk[37] in the two directions perpendicular to the (110) and (100) surfaces. This induces two edge-terminations, one exposing Al atoms (here called Al-edge) and the other exposing O atoms (O-edge). The Al-edge is constituted of one upper row of Al III atoms (also three-fold coordinated on the (110) surface) and one lower row of Al III atoms that correspond to Al IV on the (110) surface.The O-edge exhibits a row of alternating O II and O III , both formally O III on the (110) surface.

Figure 2 .

 2 Figure 2. a) Alumina platelet scheme[5] and dehydrated edge model corresponding to two possible edge terminations (blue) between the (110) (green, top) and the (100) (orange, sides) surfaces. Edge sites are depicted by bigger balls. b) hydrated edge model with 6H 2 O and c) chlorinated edge model constructed from the 6H 2 O hydrated model by exchanging Al-side µ 1 -OH. Blue traced lines indicate hydrogen-bonds (bond length's threshold of 2.5 Å).

  construction of the first DFT model of γ-Al 2 O 3 crystallite (110)-(100) edge has allowed a refined1 H NMR peak assignment. It was found that the sharp peak at 0 ppm corresponds mostly to µ 1 -OH located on the edges of the crystallites that are isolated and free from hydrogen bonding. These hydroxyls are also the most favourably exchanged with chlorine, which can be considered as a selective probe of alumina edges. Overcoming the simple empirical assignment, the 1-3 ppm region corresponds to signals from not only µ 2 -OH but also from µ 1 -OH located either on the surfaces or on the edge, and that are either H-bond acceptors or free hydroxyls. Moreover, hydroxyls that are hydrogen bond donors are abundant on the surfaces and contribute to the high chemical shift broad signals. Hopefully, this novel alumina edge model and the improved assignment open new perspectives to further explore the potential of the edge sites present in industrially relevant high surface alumina crystallites.

H NMR spectra of P-egg and of T-flat (Figure 1 a) present the main features expected for γ-

O

Al 2 O 3 : a resolved signal at around 0 ppm; intense and well-defined peaks in the 1-3 ppm range and broad components ranging from 3-7 ppm. Moreover, the spectra of the two aluminas are clearly distinguished. The most remarkable difference is that the higher-field signal (at respectively -0.1 and -0.4 ppm) is much more intense for P-egg than for T-flat (

12% vs 4% of total[START_REF] Euzen | Handbook of Porous Solids[END_REF] H signal from spectral deconvolution, TableS2). The 1-3 ppm region is also distinguishable: for P-egg two main signals (1.6 and 2.2 ppm) are observed while for T-flat only one is (1.2 ppm), albeit some shoulders indicating other contributions. Lastly, the contribution of the broad signals ranging from 3-7 ppm is significantly more intense in the Tflat spectrum.

Acknowledgements A.T.F.B. thanks A.-L. Taleb for her collaboration in HR-TEM analysis and E. Rosati, C. Guegan and C. Mancia for their contribution to sample preparation. M. Marsman (University of Vienna) is acknowledged for advice with respect to chemical shift calculations, and K. Larmier for preliminary computational investigations. Calculations were performed using HPC resources from GENCI-CINES (Grant A0020806134) and from IFP Energies nouvelles. TGIR RMN THC (FR3050 CNRS) is acknowledged for the NMR characterizations.

This work was funded by IFPEN and financial support from Equipex contract ANR-10-EQPX-47-01 is acknowledged. This work was supported by the LCR "CARactérisation des Matériaux pour l'Energie" (CARMEN), IFPEN/CNRS/UCBL/ENS Lyon/UNISTRA/Sorbonne Université, and is part of the "RatiOnAl Design for CATalysis" (ROAD4CAT) industrial chair, project IDEXLYON funded by the French National Research Agency (ANR-16-IDEX-0005).


Abdellatif Dahi

Mohamed-Lemine Abdellahi

Mohamed Fadel Deida

Nicolas Hucher
email: nicolas.hucher@univ-lehavre.fr

Catherine Malhiac

Frédéric Renou

Chemical and physicochemical characterizations of the water-soluble fraction of the Commiphora Africana exudate

Keywords: Commiphora Africana exudate, water-soluble fraction, chemical composition, Arabinogalactan-Protein-like polymer, Surface activity, compact structure

The present study focuses on the chemical and physical characterization of the watersoluble fraction of the Commiphora Africana exudate. The chemical analysis proved that this fraction is mainly composed of carbohydrates and hydrophobic amino acids. Size exclusion chromatography (SEC) revealed the presence of three distinct polysaccharide populations that are all bonded to protein. In addition, both SEC and rheology demonstrated that the gum owns highly compact structure that suggests an AGP-like polymer.

commiphora Africana gum exhibits excellent surface activity at concentration 2 orders of magnitude lower than the reference, acacia gum and was attributed to the fact that the three populations contribute to the surface tension lowering. This remarkable property has been confirmed by preliminary experiments on model emulsions and suggests a very promising application in formulation.

Introduction

Hydrocolloids own many industrial applications in pharmaceutic, cosmetic or food products. Their applications are based on their functional properties, for example, their ability to form viscous solutions at low concentration, or their ability to stabilize emulsions. The possible surface activity of several food gums has been studied in order to evaluate their potential emulsifying properties [START_REF] De Paula | Composition and rheological properties of cashew tree gum, the exudate polysaccharide from Anacardium occidentale L[END_REF][START_REF] Garti | Hydrocolloids as food emulsifiers and stabilizers[END_REF][START_REF] Phillips | Gums and Stabilisers for the Food Industry 12[END_REF][START_REF] Phillips | Handbook of Hydrocolloids[END_REF].

Among hydrocolloids, naturally produced ones show multiple benefits: they are biodegradable, less toxic and have activity over wide temperature, pH and ionic strength ranges. This may explain the intensive search of new natural gums and the effort to characterize their functional properties [START_REF] Grein | Structural characterization and emulsifying properties of polysaccharides of Acacia mearnsii de Wild gum[END_REF]; [START_REF] Ozaki | Structural Characterization and Hypoglycemic Effects of Arabinogalactan-Protein from the Tuberous Cortex of the White-Skinned Sweet Potato (Ipomoea batatas L.)[END_REF]; [START_REF] Steinhorn | Isolation and characterisation of arabinogalactan-proteins from New Zealand kanuka honey[END_REF]; [START_REF] Vinod | Morphological, physico-chemical and structural characterization of gum kondagogu (Cochlospermum gossypium): A tree gum from India[END_REF].

Commiphora Africana [A. rich] Engl. is a tropical and subtropical plant of the genus Commiphora (Burseracea) and widespread over Africa. Hence, Commiphora Africana are found across the south part of Sahel, from West (Mauritania) to East (Ethiopia). The Commiphora genus is known to produce resinous exudates largely studied because of their historical medicinal use [START_REF] Shen | The genus Commiphora: A review of its traditional uses, phytochemistry and pharmacology[END_REF]. Hence, there is a rich literature concerning the chemistry of Myrrh, from Commiphora Myrrha, opopanax from Commiphora Molmol or Commiphora Mukul [START_REF] Hanus | Myrrh -commiphora chemistry[END_REF] which are known to contain large amount of various hydrocolloids. In many case, these exudates are used by traditional medicine, including their oral medications [START_REF] Lemenih | Gum and resin resources from some Acacia, Boswellia and Commiphora species and their economic contributions in Liban, south-east Ethiopia[END_REF]; [START_REF] Abdellahi | Caractérisation chimique et physicochimique de la résine du c mmiph ra africana d' rigine mauritanienne en vue de sa val risati n[END_REF]. Concerning Commiphora Africana although, to our knowledge, only an old study by Jessenne [START_REF] Jessenne | Gums of two gum resins, bdellium and galbanum[END_REF], took interest in the chemical composition of its exudate. Unfortunately, the authors could not certify the origin of their studied gum (Bdellium) because, as stated in the PhD thesis of Jessenne [START_REF] Jessenne | Les Gommes de deux gommes-résines (Bdellium, Galbanum)[END_REF], it came from a local traditional market. Recently, Gundidza et al. [START_REF] Gundidza | Rheological, moisture and ash content analyses of a gum resin from Commiphora Africana[END_REF] conducted a viscosimetric analysis of a gum resin from Commiphora Africana, unfortunately without a previous purification of the hydrocolloids parts.

In this paper we will present the first chemical and physicochemical analyses of the water-soluble part of the Commiphora Africana exudates after intensive purification. The aim of this study is to elucidate the fine structure of this fraction and to determine the relationship between the structure and the functional properties, which, to the best of our knowledge, has never been done so far.

Materials and methods

Materials

Commiphora Africana (CA) gum was extracted from air-dried exudates of the CA tree in Africa. The studied trees are located in the province of Rosso (Wilaya Trarza) in the south of Mauritania, on the right bank of the Senegal River. The exudates (nodules), were collected manually, two weeks after wounding, during the month of April. Nodules were air dried and stored in polyethylene bags. They present a dark reddish brown colour.

Monosaccharides and L-amino-acids, used as standards, were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France) (purity ≥99%). All other chemicals used were of analytical grade and ultrapure water (18.2 mΩ resistivity) was used in all experiments.

CA gum Extraction and purification

CA exudates (nodules) were frozen with liquid nitrogen and powdered in a high-speed mechanical blender. 10 g of the obtained powder was poured in water (1000 cm 3 ) and stirred for 30h at 70°C. The suspension was clarified by centrifugation at 2000 g for 10 min to remove insoluble part and the resultant solution was freeze-dried. The resulting powder was again dissolved in water at room temperature and precipitated with absolute ethanol. The clear supernatant was sucked out after centrifugation at 20°C for 5min at 2000 g, and the insoluble fraction (gum) was dried under vacuum to remove traces of solvents. The dried solid was again dissolved in water, and finally freeze dried. This gave a cotton-like and slightly white CA gum. The overall yield was 38% ± 2%.

Preparation of CA gum solutions

CA gum was dispersed in water at the target concentration (weight %). Dispersions were gently stirred at 35°C for 4 h, and left at 3 ±1°C overnight for complete hydration. Then, filtration using syringe filter (0.45 µm, cellulose acetate membrane) gave clear solutions.

Chemical composition analysis

Moisture and ash content of the CA gum were measured by Thermal Gravimetric Analysis (Setaram Setsys 12) realized between 30 and 900°C at a heating rate of 10°C/min under nitrogen.

The total protein content was estimated by the Bradford protein assay taking bovine serum albumin (BSA) as standard.

Specific optical rotation

The specific optical rotation (

C  25 589 ] [
) of a 2.5 g/L gum solution was measured at 25°C with a Perkin Elmer polarimeter (model 214 MC) at wavelength 589 nm, using a 1 dm length cell and a Na lamp.

Monosaccharides composition

Hydrolysis of the CA gum was conduct trough acidic condition by dissolving the solid in 200 µL 4 M trifluoroacetic acid into a sealed tube flushed with N 2 and heated at 100°C for 4 h. The solution was then dried under vacuum, dissolved in methanol and dried again twice before precolumn derivatization.

For the precolumn derivatization adapted from Tapie et al. [START_REF] Tapie | Determination of galactose and mannose residues in natural galactomannans using a fast and efficient high-performance liquid chromatography/UV detection[END_REF], a 4-(3-methyl-5-oxo-2-pyrazolin-1-yl) benzoic acid (PMPA) solution was prepared immediately before use by the following procedure: PMPA crystals were dissolved in 0.25 M NaOH methanol-water (1:1) up to the point at which the pH stabilized at 7.8-8 and then complete with methanol-water (1:1) to obtain a final concentration of PMPA of 0.15 M.

The monosaccharides, either pure references or from the hydrolysis of the CA gum, were incubated with 1 mL of PMPA solution (0.15 M) at 70°C for 2 h. After incubations samples were acidified by 150 µL of an aqueous 2M HCl solution and shaked up to precipitated most of the excess of derivatization agent. After few minutes samples were centrifuged for 10 min at 5 000 g. Supernatant was sucked out and filtered through 0.2 µm nylon membrane. Mass spectrometry detection was performed in positive mode with optimal conditions determined by direct infusion.

Amino acid composition of the protein part

The overall amino acid composition of the protein part was determined by GC/MS as described by [START_REF] Sassaki | Application of acetate derivatives for gas chromatography-mass spectrometry: Novel approaches on carbohydrates, lipids and amino acids analysis[END_REF]. Freeze dried sample of CA gum (1mg) was hydrolysed with HCl 6M at 100°C for 20h. A 0.6N HCl solution in methanol was added to the liberated amino acids or pure amino acids used as reference and these solutions were heated at 100°C during 15 min. After drying under N 2 stream, the residue containing amino acids methyl esters were treated with pyridine/MeOH/Ac 2 O (300 µL 1:1:4), then heated at 100°C

during 1 hour. The solutions were directly analysed by GC/MS. GC/MS analyses were performed on a Shimadzu GCMS-QP2010SE equipped with a split injector (1/50) heated at 250°C. The carrier gas was set up at 1 mL min -1 . The amino acid acetate methyl esters were separated on a capillary column ZB-5MS-Phenomenex (L=30 m; id = 0.25 mm; film thickness = 0.25 µm). The oven temperature was programmed at 50°C (hold 2 min) to 90°C (20°C min -1 , 1 min),then to 280°C (5°C min -1 ) then held for 2 min.

Electron Impact (EI) spectra were obtained at 70 eV at 200°C. The amino acid acetate methyl esters were identified using NIST database and by comparison with pure reference of amino acid acetate methyl esters (MS spectra and retention time).

Size exclusion chromatography

SEC analysis were performed using a shimadzu HPLC system (Shimadzu, Japan) coupled to four detectors: multi-angle laser light scattering (MALLS) operating at eighteen angles (Dawn Heleos II, Wyatt, CA, USA), differential refractometer (Optilab T-rEX, Wyatt, CA, USA), on-line viscosimeter (VISCOSTAR II, Wyatt, CA, USA) and UV-VIS detector activated at 280 nm (SPD-20A, Shimadzu, Japan). The system was composed of one Shodex OHPAK SB-G pre-column followed by three columns in series (Shodex OHPAK SB 806 HQ, OHPAK SB 805 HQ and OHPAK SB 804 HQ).

Aqueous solution of CA gum (1mg.ml -1 ) was dissolved overnight in milliQ water, then 50 mL were injected and eluted through the system with 100 mM LiNO 3 solution containing 0.02% NaN 3 at a constant flow rate of 1 mL.min -1 at room temperature.

2.9 FT-IR spectroscopy IR spectra of the CA gum were measured by a Perkin Elmer Frontier FT-IR spectrometer using the ATR mode ( Diamond/ZnSe crystal). 8 scans were collected for each measurement over the spectral range 4000-650 cm -1 with a resolution of 8 cm -1 .

Viscosity measurements

Gum solutions at different concentrations (0.01 to 165 g/L) were prepared in water. Their dynamic viscosity was measured using a rolling Ball Viscometer (Anton PaarAMVn) allowing measurements at different angles (15 to 85°). The rolling Ball Viscometer uses the simple Höppler principle to measure viscosity. The basic concept is to measure the elapsed time required for the ball to roll under gravity through a sample-filled tube inclined at an angle. First, the tube (1.6 mm diameter) was filled with a gum solution. Then, it was placed in the thermostatic measurement chamber and equilibrated to a constant temperature maintained within ± 0.1°C for about 15 min. Thereafter, rolling times of solution were measured at different temperature (10, 20, 30, 40 and 50°C). At each temperature, the angle of tube from the horizontal was varied progressively from 30, 40, 50, 60, 70 to 80 degrees in order to study the flow behaviour of CA gum in solution. At each angle, the measurement was carried out 4 times to determine an average. Viscosities were directly given by the viscometer according to the rolling times.

The intrinsic viscosity ]

[ was determined at 20°C using the Huggins equation ( 1) [START_REF] Huggins | The Viscosity of Dilute Solutions of Long-Chain Molecules. IV. Dependence on Concentration[END_REF] by plotting

C sp / 
against the polymer concentration, and subsequently extrapolating to zero polymer concentration (2):

C C k C s s H sp           2 ] [ ] [
(1)

C sp C   0 lim ] [   (2)
where  and  s are the viscosity of the solution and the solvent respectively,  sp is the specific viscosity and C and k H are the polymer concentration and the Huggins coefficient, respectively.

Surface tension measurements

Surface tension measurements were made using Du Noüy ring tensiometer (Krüss k11, Germany) with platinum ring at room temperature (25°C). Gum solutions at concentrations ranging from 0.01 to 35 g/L were used.

Evaluation of emulsifying properties

Oil-in-water emulsions were prepared by mixing 0.35 g of orange essential oil, provided by Laboratoire Rosier Davenne (Montfavet, France) and 4.65 g of CA gum solutions at 10 and 50 g/L. For comparison, commercial Acacia Senegal gum (food grade), provided by Alland & Robert (Port Mort, France) was also tested in the same conditions.

First, gum solutions were prepared and kept at rest for 24h. The oil phase was then dispersed in the gum solutions under strong shear using a T10 basic ULTRA-TURRAX® (IKA, Germany) at 14450 rpm for 5 min at 20°C. All emulsion samples were kept at 20°C after preparation and sheltered from the light after emulsification.

Characterization of size and size distribution of emulsions during aging were conducted using static laser diffraction apparatus SALD-7500nano (Shimadzu, japan).

Results and discussion

CA gum main characteristics

The main composition of CA gum is summarized in Table 1. Two major groups of exudates can be distinguished, on the one hand the resin which are soluble in organic solvents (e.g. pine resin) and on the other hand the gum which are soluble in water (e.g. acacia gum).

This first shows that when compared to other well characterized exudates , in the exudate from Commiphora Africana trees the water-soluble gum is a minor fraction, representing only about 40% of the total content. This is in accordance with the characteristics of exudates from the other Commiphora species where 30-60% of this exudates are water-soluble gum, and 25-40% are ethanol soluble resin [START_REF] Hanus | Myrrh -commiphora chemistry[END_REF]. So the Commiphora Africa exudates can be considered as a resin-gum exudate. This low water-soluble fraction points out the importance of purification prior to any measurement and thus data obtained without proper purification have to be taken with extreme caution. CA gum showed to be mostly composed of carbohydrates (80%) with a low content of inorganic matter but a relatively high content of proteins (Table 1). Hence, when compared to many others gums from trees (acacia gums: 1.4-2.2% [START_REF] Idris | Characterisation of gum from Acacia senegal trees of different age and location using multidetection gel permeation chromatography[END_REF], Tragacanth Gums: 2-4 % [START_REF] Balaghi | Physicochemical and Rheological Characterization of Gum Tragacanth Exudates from Six Species of Iranian Astragalus[END_REF] or Barijeh gum: 4.3% [START_REF] Mohammadzadeh Milani | Physicochemical and emulsifying properties of Barijeh (Ferula gumosa) gum[END_REF], the 10.2% measured as protein content in CA gum could have been related to impurities through unwashed free proteins. But SEC measurement(Figure 1), and especially from the UV-Vis detector, showed that the UV signal, generally the signature of the presence of amino acid, is very similar to the RI signal on the whole range of elution time. Hence, this suggests that the carbohydrates own covalently bonded protein with similar ratio, which will be discussed in more detail later.

FT-IR

The FTIR spectrum of CA gum is given in Figure 2. Two characteristic peaks of polysaccharides were observed at 3286 and 2934 cm -1 , which were related to stretching vibration modes of OH bound to carbons and C-H stretching vibration, respectively. The bands at 1637 cm -1 and 1539 cm -1 are attributed to the amide I and amide II bands of the polypeptidic backbone of glycoproteins and proteoglycans [START_REF] Barth | Infrared spectroscopy of proteins[END_REF]. However, polysaccharides containing uronic acids will also give bands close to 1637 cm -1 and to 1413 cm -1 that were attributed to the vibration of C=O and the deprotonated carboxylic function respectively [START_REF] Boulet | A Fourier transform infrared spectroscopy study of wine polysaccharides[END_REF]. The region between 1200 and 600 cm -1 represents the so-called carbohydrate fingerprints. This region is typical of the skeletal stretching vibrations of the backbone and side chains of polysaccharides, which is dominated by ring vibrations overlapped with stretching vibrations of (C-OH) side groups and (C-O-C) glycosidic bond vibrations ( ac ur v ape asin v ellner ringer v 2 ).

All these observations are in accordance with the presence of both polysaccharides and proteins in CA gum. (Lopez-Torrez, Nigen, Williams, Doco, & Sanchez, 2015)

Monosaccharides composition by HPLC

The composition of the monosaccharides obtained by hydrolysis of CA gum is given in Table 2. The analytic procedure allowed quantifying the derivated monosaccharides and, if any, the di or three-saccharides not fully hydrolysed. The two major monosaccharides found were arabinose and galactose (47% and 28%, respectively). Also these two monosaccharides are usually present in exudates of Commiphora species [START_REF] Hanus | Myrrh -commiphora chemistry[END_REF]. Then galacturonic acid represent about 7% of the total monosaccharide. Finally, mannose, xylose, glucosamine and fucose are also present at relatively high percentage (between 4 and 5%)

which is quite unusual for plant gums, especially for glucosamine. Indeed, for instance, Wiendl et. al. [START_REF] Wiendl | Proteoglycans from the gum exudate of myrrh[END_REF] reported small amount of mannose (less than 3%) in Myrrh from unprecise botanic origin among with larger amount of 4-O-Methylglucuronic acid (up to 43.9%). Fucose was also identified by TLC in gum resin of Commiphora Mukul [START_REF] Bose | Structure of C. mukul -Part II. Structure of the Degraded Gum[END_REF]. If Jessenne et. al. [START_REF] Jessenne | Gums of two gum resins, bdellium and galbanum[END_REF] reported galacturonic acid from gum of Bdellium, there were no evidence that this gum samples came fr m identified tanic rigin even if stated "pr a ly fr m Commiphora Africana". This is the only paper, to-date, that discussed the chemical composition of exudate from Commiphora Africana. Glucosamine is far less common in plant exudates, but already reported in botanic species from Acacia, Araucaria and some others [START_REF] Anderson | The amino acid and amino sugar composition of some plant gums[END_REF] and is closely related to the linkage of proteins to carbohydrate in glycoproteins and proteoglycans.

Amino acids composition

To our knowledge, the amino acid composition of the proteins from Commiphora Africana gum had never been determined. The main amino acids of Commiphora Africana are given in Table 3. The major amino acids therefore are Hydroxyproline (Hyp 22.7%), Proline (18.1%), Alanine (14.9%), PhenylAlanine (8.7%), Leucine (7.1%), Serine (6.2%), Glutamic and aspartic acids (6.1% and 5.3%). Hyp is also described to be the main amino acid in Myrrh (Commiphora molmol) exudate. [START_REF] Wiendl | Proteoglycans from the gum exudate of myrrh[END_REF]. Hyp is also the main amino acid in case of Arabinogalactan-Protein (AGP) structure such as AGP fraction of acacia gum.

Furthermore, this amino acid is often described as the link between the protein fraction and the polysaccharide fraction [START_REF] Mahendran | New Insights int the tructural haracteristics f the ra in galactan-Pr tein ( GP) racti n of Gum Arabic[END_REF].

One can notice a high percentage of phenylalanine which can explain the strong absorbance at 280nm (Figure 1). Furthermore, the major identified amino acids were hydrophobic, which is usually related to compact globular proteins.

Viscosity measurements

The viscosity of many liquids depends exponentially on temperature, and the most appropriate model that best explains the temperature dependence of the viscosity of macromolecules is the Arrhenius equation which is expressed as:

       RT E a ref exp   (3)
where  (Pa.s) is viscosity, R is the gas constant (8.31 J.K -1 .mol -1 ), T (K) is temperature, The values of activation energy evolved slightly from 17 to 23 KJ.mol -1 for CA gum solutions with concentrations from 20 to 165 g/L. It is well known that the activation energy reflects the flexibility and interactions of macromolecules. For instance, acacia and cashew gums branched polysaccharide with few interactions, have comparable Ea values of respectively 15 kJ.mol -1 [START_REF] Varfolomeeva | On the possibility of estimating weak interactions of macromolecules in solutions from the experimental viscous flow activation energies data[END_REF] and 15.6 kJ.mol -1 (de Paula & Rodrigues, 1995) for a 20 g/L solution. On the contrary, linear polymers strongly bonded by intra and intermolecular interactions involving hydrogen bonding show high activation energies. For example, xanthan gum exhibits Ea value about 70-80 kJ.mol -1 for concentration ranging from 1 to 10 g/L [START_REF] Renou | Effect of xanthan structure on its interaction with locust bean gum: Toward prediction of rheological properties[END_REF]). It appears clearly that the viscosity behaviour of CA gum is closer to branched polysaccharides, than to linear ones. Accordingly, the intermolecular interactions between polysaccharide chains in CA gum are weak.

On the other hand, CA gum solutions from 20 to 110 g/L exhibits at 20°C viscosities ranging from 1.06 to 10.3 mPa.s. These very low (for this range of concentration) values are similar to that of acacia gum in solutions up to 100 g/L [START_REF] Glicksman | Gum Technology in the Food Industry[END_REF]. The low and newtonian (as will be seen below) viscosity at higher concentrations is a second sign of weak intermolecular interactions between polymer chains. All these results indicate probably a polymer structure in CA gum of high compactness.

Intrinsic viscosity

The intrinsic viscosity is a measure of the hydrodynamic volume occupied by a macromolecule, which is closely related to both the conformation and size (weight) of the macromolecular chain in a specific solvent [START_REF] Lai | Rheology of decolorized hsian-tsao leaf gum in the dilute domain[END_REF]. Intrinsic viscosity of CA gum was determined according to the Huggins equation (3), as explained before. To this end, dilute solutions was used, ranging from 20 to 80 g/L. The obtained ]

[ value for CA gum was 20.3 ml.g -1 . Again, this low value was close to that found for acacia gum (Al-Assaf, [START_REF] Al-Assaf | Studies on acacia exudate gums. Part I: the molecular weight of Acacia Senegal gum exudate[END_REF][START_REF] Lopez-Torrez | Acacia senegal vs. Acacia seyal gums -Part 1: Composition and structure of hyperbranched plant exudates[END_REF].

Flow behaviour of CA gum

From viscosity measurements, it was observed that the flow times for all solutions (20 to 165 g/L) decrease proportionally with the tube angle from 30 to 80°, proving that solutions remain Newtonian even at concentrations as high as 165 g/L.

The power law equation below (4) relating specific viscosity and concentration can be used as an indicator for estimating the conformation of polymer chains [START_REF] Lai | Solution properties of hsian-tsao (Mesona procumbens Hemsl) leaf gum[END_REF]:

b sp aC   (4)
where a and b are constants. Figure 3 shows this power-law plot for CA gum solutions at different temperature.. From this plot, it can be seen that both the slope (b value) and intercept (a value) remain stable, irrespective to the temperature. This means that the polymer chains adopt the same conformation over the studied temperature range. It has been established that b value greater than one is associated with a spherical or compact conformation, while a value of b less than one is associated with rod like conformation [START_REF] Lai | Rheology of decolorized hsian-tsao leaf gum in the dilute domain[END_REF]. From plots, the b value was around 1.75 ±0.05 (an average of all the slopes). Therefore, the molecular conformation of CA gum is more spherical like than rod like. Again, this result is in good accordance with polymer structure in CA gum of high compactness.

On the other hand, shape factor value was determined to be 2.54, from equation 5:

E V ] [   (5)
where V E , the voluminosity, was determined following Antoniou et al. methodology [START_REF] Antoniou | Structure and dynamics of dextran in binary mixtures of a good and a bad solvent[END_REF] A shape factor value of 2.5 suggests that the polymer coils are spherical in nature [START_REF] Joseph | Viscosity behaviour of acrylonitrile-acrylate copolymer solutions in dimethyl formamide[END_REF]. This indicates that the effective molecular shape of CA gum is spherical. To verify this, the Krieger & Dougherty model [START_REF] Krieger | A Mechanism for Non-Newtonian Flow in Suspensions of Rigid Spheres[END_REF] was also applied to the data. This model was developed to characterize the volume fraction dependence of the relative viscosity of hard spheres and is frequently used for food systems, using equation ( 6 respectively. Thus, this equation was applied to the experimental data of CA gum (Figure 4).

For reasons of clarity and ease of understanding, our results were compared to a linear and spherical star-like polymer (32 arms, considered as relatively dense system) [START_REF] Vlassopoulos | Colloidal star polymers: Models for studying dynamically arrested states in soft matter[END_REF]. A deviation from the Krieger & Dougherty model (hard spheres) was found for the CA gum. This result indicates that CA gum doesn't exhi it a hard-sphere behaviour. CA gum is also not linear because its behaviour diverges for linear one and is in between linear and the star-like polymer conformation. To conclude, the polymer chains in the CA gum adopts a relatively compact structure. This explains both the low viscosifying properties of CA gum, and the weak intermolecular interactions between chains as evidenced previously by the low Ea values. To get deeper into the polymer structure of CA gum, size-exclusion chromatography was used and results are discussed in the next section.

Polymer conformation in solution

Size exclusion chromatography measurements have been carried out with using UV, RI, LS and viscosimetric detectors and the chromatograms are displayed in Figure 1. One can clearly see that the system is composed of three main populations which are quite well defined at 21, 24.5 and 27.4 min elution times, which correspond to 8 000 000, 950 000 and 150 000 g/mol, respectively as determined by the LS detector. The main population in number is eluted at 27.4 min, thus corresponding to the latter molecular weight. All the average parameters (on the whole elution time range) are summarized in Table 4. Mw is about 1 300 000 g.mol -1 while Mn is 260 000 g.mol -1 , proving that the system is highly polydisperse as it is often observed for natural polysaccharide from plants [START_REF] Eagles | Molecular structures of gum exudates from Hakea species[END_REF]; [START_REF] Phillips | Molecular association and function of arabinogalactan protein complexes from tree exudates[END_REF]. In addition, thanks to viscosimetric detector, the intrinsic viscosity was determined and is very close to the one measured by the rolling ball viscometer. The hydrodynamic radius is 14.1 nm, which is quite small for molecule of such a high molecular weight, showed again that the molecules are highly compact. This observation is consistent with the viscosimetric analysis that shows that CA gum behaves as a highly branched polymer.

The UV signal measured at 280 nm showed a maximum absorbance that correlate with the three main populations at 21, 24.5 and 27.4 min elution times. This suggests that the three population own covalently bonded protein with similar ratio, proving that bonded proteins are homogeneously distributed between the three populations. This is an unusual property, very different to what is observed for gums containing glycoproteins such as acacia gum for instance [START_REF] Phillips | Molecular association and function of arabinogalactan protein complexes from tree exudates[END_REF] but that were already found for closely related Myrrh gum (Commiphora molmol) [START_REF] Wiendl | Proteoglycans from the gum exudate of myrrh[END_REF].

Surface tension

The surface activity of CA gum was characterized by measuring the tension at air/water interface acc rding t the Du N üy's ring meth d at 25°C. Figure 5 shows the variation of the surface tension of water as a function of CA gum concentration. Surface tension of ultrapure water at 25 °C was 71.90 mNm -1 . One can observe a rapid lowering of the surface tension from 71.90 to 50.43 mN.m -1 for concentration ranging from 0 to 7 g/L. For higher CA gum concentrations, surface tension remained constant. Critical aggregation concentration (CAC), determine as the concentration at which the surface tension starts to decrease, was determine at 0.2 g/L of CA gum. Hence, CA gum reduced the surface tension more efficiently than classic commercial pectin (63 mN.m -1 in apple pectin) [START_REF] Lutz | Structure and physical properties of pectins with block-wise distribution of carboxylic acid groups[END_REF] or Acacia tortuosa (53 mN.m -1 ) [START_REF] Muñoz | Rheological properties and surface tension of Acacia tortuosa gum exudate aqueous dispersions[END_REF]. In addition, this very low CAC is about 135 times lower than the reference gum (Acacia Senegal), for which CAC is close to 27 g/L in the same conditions of measurement [START_REF] Grein | Structural characterization and emulsifying properties of polysaccharides of Acacia mearnsii de Wild gum[END_REF]. This may open a promising application of CA gums as efficient surface agent.

Emulsifying properties

Preliminary experiments on simple emulsions (orange essential oil/water) were conducted on CA gum and Acacia Senegal gum for comparison. Figure 6 shows the volume droplet size distribution for o/w emulsions containing 10 and 50 g/L of gum (CA and Acacia Senegal), and their monitoring over time at 20°C. At 50 g/L, the droplet size distribution of the freshly prepared emulsions is very close for both gums. The main population can be seen with a volume mean diameter (D4,3) of 12 µm. However, for the CA gum, a small population with a D4,3 of 4 µm slowly disappeared after few days, probably through Ostwald ripening. In general, and for both gums, no changes in the droplet size distribution nor in the D4,3 were observed during the experiment (up to 10 days).

At 10 g/L, the droplet size distribution of the freshly prepared emulsions is also very close for both gums. The main population can be seen with a D4,3 of 19µm. In case of CA gum, both the droplet size distribution and the mean droplet size remain stable over time, evidencing a powerful emulsifying capacity of CA gum at 10 g/L. Indeed, this moderate concentration is already at 50 times the CAC of CA gum. In the case of Acacia Senegal gum, a demixing was observed within one hour after the emulsion preparation (as seen in the picture caption figure 6) and thus no monitoring was possible afterwards. At 10 g/L, Acacia Senegal gum could not create a stable emulsion probably because the used concentration (10 g/L) is below the CAC of this gum (close to 27 g/L according to [START_REF] Grein | Structural characterization and emulsifying properties of polysaccharides of Acacia mearnsii de Wild gum[END_REF]). These preliminary results demonstrate the excellent emulsifying properties of CA gum, which is efficient at much lower concentrations than acacia.

Discussion

All of the chemical analyses of CA Gum lead to a proposed structure for this hydrocolloid that is clearly related to arabinogalactan-like glycoprotein (AGP) structures. First, we showed that the two major monosaccharides were arabinose and galactose, and that, minor ones, galacturonic acid and glucosamine, are usually related to glycoproteins and proteoglycans [START_REF] Nguema-Ona | Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function[END_REF]. Secondly, The CA gum protein fraction is rich in Hyp and its overall composition is also compatible with the AGP nature of the polysaccharides, as stated for acacia gum [START_REF] Idris | Characterisation of gum from Acacia senegal trees of different age and location using multidetection gel permeation chromatography[END_REF].

Besides, the amino acids are mainly hydrophobic, with a large amount of proline, which is consistent with a globular structure of the resulting proteins [START_REF] Lesser | Hydrophobicity of amino acid subgroups in proteins[END_REF]; [START_REF] Schulz | Structural rules for globular proteins[END_REF]. This will most likely lead to a compact AGP-like structure, which is consistent with the high compactness that we determined for CA gum.

The present study shows that CA gum lowers the surface tension of water down to 50.43mN/m with less than 10 g/L of CA gum. Therefore CA gum possesses excellent capacities to lower the surface tension using concentrations at least 100 times lower than acacia gum for the same performance, which would open up promising opportunities in formulation. In general, the capacity of food biopolymers to lower the surface tension is related to their chemical structure, concentration, and molecular conformation at the interface [START_REF] Dickinson | Mixed biopolymers at interfaces: Competitive adsorption and multilayer structures[END_REF][START_REF] López-Franco | Classification and physicochemical characterization of mesquite gum (Prosopis spp.)[END_REF]. More precisely, both the accessibility of the protein moieties to the interface and the net surface charge of adsorbing molecules control the interfacial activity of polymers [START_REF] Vasile | Physicochemical, interfacial and emulsifying properties of a non-conventional exudate gum (Prosopis alba) in comparison with gum arabic[END_REF]. Some reports have shown that a relatively high protein content is required to provide good interfacial properties. Hence, the interfacial properties of acacia gum were, for example, attributed to the arabino-galactan protein fraction and its high molecular weight in the gum sample [START_REF] Dickinson | Mixed biopolymers at interfaces: Competitive adsorption and multilayer structures[END_REF]; [START_REF] Phillips | Molecular association and function of arabinogalactan protein complexes from tree exudates[END_REF]. In addition, contrary to acacia gum, for which protein are mainly located in one fraction (AGP), for CA gum, the 3 fractions possess covalently bonded protein. Thus, all fractions contribute to the surface tension lowering which may explain the much better surface activity of CA gum.

The preliminary experiments on the emulsifying properties of CA gum showed that 1) CA gum presents excellent stabilizing properties as good as Acacia Senegal gum then 2) CA gum is even more efficient than Acacia Senegal gum at low concentration, thanks to it much lower CAC. These preliminary results prove the effective ability of CA gum to stabilize interfaces and thus it potential interest in formulation.

Conclusion

The chemical composition of the water-soluble fraction of the Commiphora Africana exudate has been determined for the first time without ambiguity. This gum exudate is manly composed of glycoproteins, which is composed of 79.5 % carbohydrates and 10.2 % proteins.

The major monosaccharides found were arabinose (47 %) and galactose (28%) together with galacturonic acid (7%) and minors one. The major amino acids found were hydroxyproline (22.7 %), proline (18.1 %) and alanine (14.9 %). Rheological measurement among with size exclusion chromatography lead to a proposed structure of an highly compact AGP-like polymer. Whereas the Commiphora Africana gum is composed of three main populations (ranging from 8 000 000 to 150 000 g.mol -1 ), they all possess covalently bounded protein.

Hence, in accordance with what is expected for AGP-like polysaccharides, the gum showed a great capacity to lower the surface tension in water (down to 50.43 mN.m -1 for a 7 g/L solution). In addition, preliminary experiments on simple emulsions gave encouraging results.

In further works we will conduct a complete characterization of stabilization capacities of the Commiphora Africana gum, in order to confirm its promising efficiency as surface agent. 79.5 ± 0.6 pH (solution 5 w%) 4.9 ± 0.1 Optical rotation (°.dm -1 .g -1 .cm 3 ) -36.6 ± 3.4

Ea

  (J/mol) is the Arrhenius activation energy and  ref (Pa.s) is the viscosity of the solution at the reference temperature (Tref=20°C, in this study).

  equation for mono-dispersed spherical systems are 0.64 and 2.5,

Figure 1 .

 1 Figure 1. Chromatogram from SEC measurement. Detectors signals from differential refractometer (RI), UV-VIS detector activated at 280nm (UV) and multi-angle laser light scattering (LS).

Figure2.

  Figure2. ATR FTIR spectrum of CA gum.

Figure 3 .

 3 Figure 3. Specific viscosity as a function of CA gum concentration (g/L) at different temperatures from 10°C to 50°C.

Figure 4 .

 4 Figure 4. Relative viscosity as a function of concentration of the CA gum solutions (filled symbols). Comparison with Hard Sphere model (line) linear polymer (open circles) and 32 arms starlike polymer (open triangles).

Figure 5 .

 5 Figure 5. Water surface tension as a function of CA gum concentration. Line represents fit using a single exponential decay.

Figure 6 :

 6 Figure 6: Size and size distribution of droplets in o/w emulsions (orange essential oil/water) prepared from 50 g/L of Acacia Senegal (A) or CA gum (B) and 10 g/L of Acacia Senegal (C) and CA gum (D). Captions: pictures of emulsion samples 1 hour after emulsification.

Table 1 :

 1 CA gum main characteristics Extraction yield (from 10g of nodules) (w%)

	38 ± 2

Table 2 :

 2 Monosaccharide composition of the CA gum polysaccharides from HPLC-ESI-MS

	measurement
	Monosaccharide	Proportion % (n=5)
	Arabinose	46.90 ± 0.45
	Galactose	28.10 ± 0.45
	Galacturonic acid	6.87 ± 0.47
	Mannose	4.96 ± 0.28
	Xylose	4.61 ± 0.21
	Glucosamine	4.33 ± 0.19
	Fucose	4.24 ± 0.05

Table 3 :

 3 Amino acids composition of the CA gum proteins from GC-MS measurement

	Amino acids	Proportions % (n=3)
	HydroxyProline	22.7 ± 4.0
	Proline	18.1 ± 1.4
	Alanine	14.9 ± 2.0
	Phenylalanine	8.7 ± 0.8
	Leucine	7.1 ± 0.8
	Serine	6.2 ± 1.1
	Glutamic Acid	6.1 ± 1.5
	Aspartic Acid	5.3 ± 1.8
	Threonine	4.9 ± 0.9
	Glycine	4.3 ± 0.7
	Isoleucine	1.2 ±0.6
	Valine	0.9 ± 0.2

Table 4 :

 4 CA gum characteristics from SEC measurement

	Elution time (min)	19.1 -28.5
	Mw (g/mol)	1 303 000
	Mn (g/mol)	258 600
	Polydispersity	5
	Rh (nm)	14.1
	 (ml/g)	22.8
	Rg (nm) (form 19.1 to 24.3 min)	57.7

ACKNOWLEDGMENT

This project was supported by the Ministère des affaires étrangères Française (French Foreign Policy). Bah M-L Abdellahi was recipient of a Doctoral fellowship from the Coopération Française.