Sara Bechkri

Abdulmagid Alabdul Magid

Assia Khalfallah

Laurence Voutquenne-Nazabadioko

Ahmed Kabouche

Charlotte Sayagh

Dominique Harakat

Zahia Kabouche
email: zahiakabouche@umc.edu.dz

Laurence Voutquenne- Nazabadioko

Antioxidant activity-guided isolation of flavonoids from Silene gallica aerial parts

Keywords: Silene gallica L., flavonoid, antioxidant, DPPH, HORAC, CUPRAC

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

The Silene genus (Caryophyllaceae) is one of the largest genera of the world's flora [START_REF] Greuter | Silene (Caryophyllaceae) in Greece: a subgeneric and sectional classification[END_REF]. Silene gallica L., native to central Europe, is an annual growing to 40 cm in height [START_REF] Agrawal | NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides[END_REF]. Previous investigations of S. gallica led to the isolation of eleven triterpene saponins (silenegallisaponin A-K) [START_REF] Bechkri | Triterpene saponins from Silene gallica collected in North-Eastern Algeria[END_REF] from the aerial parts and ten cyclic fatty acyl glycosides (gallicasides A-J) from the glandular trichome exudate [START_REF] Agrawal | NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides[END_REF].

In continuation of our investigation on Caryophyllaceae species [START_REF] Voutquenne-Nazabadioko | Triterpenoid saponins from the roots of Gypsophila trichotoma Wender[END_REF][START_REF] Gevrenova | Gypsophila saponins enhance the cytotoxicity of etoposide in HD-MY-Z lymphoma cells[END_REF][START_REF] Bechkri | Triterpene saponins from Silene gallica collected in North-Eastern Algeria[END_REF], we studied the phenolic constituent of this species, growing in North-Eastern Algeria. This paper focuses on the bio-guided isolation and structure determination of ten compounds (1-10) including four undescribed acylated flavone Cglycosides (1-4) and six known compounds (5-10) from the 80 % aqueous ethanolic extract of the aerial parts of this plant. Their structures were elucidated by spectroscopic methods including 1Dand 2D-NMR experiments ( 1 H, 13 C, HSQC, HMBC, COSY and NOESY) in combination with HR-ESI-MS. The antioxidant capacity of extracts from different solvents (80% EtOH, petroleum ether, CHCl3, EtOAc and n-BuOH), fractions and isolated compounds was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals scavenging (HORAC) and by cupric ion reducing antioxidant capacity assays (CUPRAC).

Results and discussion

The 80% aqueous ethanol extract of the aerial parts (SGE) of S. gallica was partitioned successively between H2O and petroleum ether, chloroforme, ethyl acetate and n-butanol. The free radical scavenging activities of SGE was tested and the resulting extracts were determined by the DPPH, and HORAC assays and the cupric reducing capacity was assessed by the CUPRAC assay. The most active fraction (Sg-n-BuOH) was subjected to vacuum liquid chromatography (VLC) on silica gel, eluting with CH2Cl2-MeOH-H2O (95:5:0 to 0:1:0) to obtain 8 fractions (B1-B8). These fractions were evaluated for their antioxidant activity and the fraction B4 showed the highest antioxidant potential in DPPH (EC50 29.3 ± 1.2 µg/mL), HORAC (EC50 126.3 ± 5.1 µg/mL) and CUPRAC (EC50 170.0 ± 8.2 µg/mL) assays (Table 1). In order to determine which compounds were responsible for these activities, fraction B4 was purified using preparative and semi-preparative HPLC to yield four acylated flavone C-glycosides (1-4) along with six known compounds (Fig. 1).

The known compounds were identified as vitexin 2''-O-β-D-glucopyranoside (5) [START_REF] Pauli | Phenolic glycosides from Adonis aleppica[END_REF], orientin 2''-O-β-D-glucopyranoside (6) [START_REF] Gluchoff-Fiasson | Flavone O-and C-glycosides from Setaria italic[END_REF], vitexin 2''-O-β-D-(6'''acetyl)-glucopyranoside (7) [START_REF] Faiella | Diterpenes, ionol-derived, and flavone glycosides from Podocarpus elongates[END_REF], orientin 2''-O-β-D-xylopyranoside (8) [START_REF] Gluchoff-Fiasson | Flavone O-and C-glycosides from Setaria italic[END_REF], 20-hydroxyecdysone (9) [START_REF] Girault | The complete 1 H-NMR assignment of ecdysone and 20hydroxyecdysone[END_REF], and regaloside A (10) [START_REF] Shimomura | Regaloside A and B, acylated glycerol glucosides from Lilium regale[END_REF], by comparison of physical data with literature values and from spectroscopic evidence.

Structure elucidation of compounds 1-4

Compound 1 was isolated as a yellow amorphous powder. Its UV spectrum exhibited absorption bands characteristic for flavonoids at 334 and 272 nm (Harborne, 1989). Its positive HR-ESI-MS showed an [M+Na] + ion at m/z 701.1702 (calcd for C31H34O17Na, 701.1694) consistent with the molecular formula C31H34O17. The 1 H NMR spectrum (Table 2) showed an AA'BB' spin system at H 6.96 (2H, d, J = 8.4 Hz) and 8.02 (2H, d, J = 8.4 Hz), together with two singlets at H 6.27 and 6.58, a sugar chain [two anomeric protons at H 4.46 (1H, d, J =7.7 Hz) and 5.07 (1H, d, J = 10.0 Hz)]. Analysis of 13 C NMR spectrum (Table 2) combined with COSY, NOESY, HSQC-TOCSY and HSQC experiments suggested that 1 was a vitexin derivative and that a β-glucopyranosyl unit was assigned starting from the anomeric proton at H 4.46. The H-1, H-2, H-3 and H-4 of glcII exhibited large vicinal coupling (J =7.8 Hz) indicating that all the protons are axial [START_REF] Pauli | Phenolic glycosides from Adonis aleppica[END_REF][START_REF] Faiella | Diterpenes, ionol-derived, and flavone glycosides from Podocarpus elongates[END_REF][START_REF] Benmerache | 6"'-O-acetylisospinosin, a new C-glycosylflavone and known compounds from the aerial parts of Cladanthus mixtus[END_REF]. The diaxial coupling constants between the anomeric protons and H-2 in 1 H NMR indicated that all glucosyl units were β-linked [START_REF] Agrawal | NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides[END_REF]. 1 H NMR displayed two acetyl groups at H 1.95 and 1.97 (each 3H, s).The exact locations of the sugars and the acetyl moieties were revealed by the HMBC correlations observed between the anomeric proton signal at H 5.07 (glcI-H-1) and C-8 (C 103.8), C-9 (C 156.9), and C-7 (C 163.0) and between the anomeric proton signal at H 4.46 (glcII-H-1) and glcI-C-2 (C 77.8). NOE connectivities were found between the resonances at H 4,46 of the anomeric proton (glcII-H-1) and at H 4.35 of the proton (glcI-H-2) confirming the interglycosidic linkage from (glcII-H-1) to (glcI-H-2). The β configuration of the two glucose moieties was confirmed by observation of NOE correlations from H-1'' to H-3'' and H-5''and from H-1''' to H-3''' and H-5'''. The downfield shift for glcII-H-4 (H 4.58) and the upfield shift for glcII-C-5 (C 71.2) indicated the presence of acylation at glcII-C-4 and glcII-C-6, respectively. The location of the acetyl groups was confirmed by the HMBC correlations observed between the glcII-H-4 and the carbonyl carbon at C 170.3 and between the glcII-H-6 (H 3.97) and the carbonyl carbon at C 171.3. Therefore, the structure of 1 was determined to be vitexin 2''-O-β-D-(4''',6'''-di-acetyl)-glucopyranoside.

Compound 2 was also isolated as a yellow powder with a molecular formula C31H34O18 as determined by HR-ESI-MS ([M+Na] + at m/z 717.1651]) (calcd for C31H34O18Na, 717.1643). The UV spectra exhibiting absorption bands at 346 and 256 nm were very similar to those of 1, suggesting a flavone glycoside structure. A comparison of its NMR spectroscopic data with those of 1 (Table 2) indicated that the only difference between 2 and 1 was the aglycone moiety. As in 1, two singlets at H 6.26 and 6.50, two anomeric protons at H 4.47 (d, J =7.8 Hz) and 5.07 (d, J = 10.0 Hz)], and two acetyl groups at H 1.94 and 1.97 (each 3H, s). The 1 H and COSY NMR spectra of 2 exhibited in aglycone region an ABX system at δH 7.58 (1H, d, J= 2.0 Hz, H-2'), 7.53 (1H, dd, J= 8.3 and 2.0 Hz, H-6') and 6.93 (1H, d, J= 8.3 Hz, H-5'), due to a 3',4'-disubstituted B-ring. Full assignment of the remaining aglycone resonances in the 13 C NMR spectrum of 2 was achieved by analysis of HSQC and HMBC data, which confirmed the presence of an orientin derivative and a βglucopyranosyl unit [START_REF] Sientzoff | Fast identification of radical scavengers from Securigera varia by combining 13 C NMR-based dereplication to bioactivity-guided fractionation[END_REF]. Each assignment of the aglycone moiety was confirmed by 2D NMR experiments, which also revealed that the saccharide moiety of compound 2 was identical to that of compound 1 (Table 2). Therefore, compound 2 was identified as orientin-2''-O-β-D-(4''',6'''-di-acetyl)-glucopyranoside.

Compound 3 was obtained as a yellow powder and exhibited UV absorptions at 249, 272, 295, and 332 nm. The HR-ESI-MS spectrum displayed a molecular ion peak [M + Na] + at m/z 809.1913809. (calcd for C37H38O19Na, 809.1905)), in agreement with a molecular formula of C37H38O19. The 1 H-NMR and COSY spectra of 3 was very similar to that of 2, except for the absence of acetyl signals and the presence of three aromatic protons of an ABX system at H 7.04 (d, J= 8.0 Hz, H-6''''), 6.86 (d, J= 8.0 Hz, H-5''''), 7.21 (s, H-2''''), a signal at H 3.96 (3H, s), and two olefinic protons of an AX system at H 7.53 (d, J= 15.9 Hz, H-8''''), and 6.85 (d, J= 15.9 Hz, H-7'''') indicative of a feruloyl moiety (Table 2) [START_REF] Gossan | New acylated flavonol glycosides from the aerial parts of Gouania longipetala[END_REF]. The large value of coupling constant J = 15.9 Hz indicates trans geometry of the double bond. The presence of an E-feruloyl moiety was evident from the connectivities observed in the HMBC spectrum between the trans double-bond and the 1,3,4substituted aromatic ring as well as the ester carbonyl (C 167.8), and from the protons of the methoxy group and the C-3'''' (C 147.9) of the feruloyl moiety. The HMBC correlation between glcII-H2-6 (H 3.75, 4.11) and the carbonyl carbon of the feruloyl moiety defined glcII-C-6 as the site of acylation. The complete assignment of the proton and carbon resonances of 3 was achieved by analysis of COSY, HSQC and HMBC spectra (Table 2). Consequently, the structure of 3 was elucidated as orientin-2''-O-β-D-(6'''-feruloyl)-glucopyranoside.

Compound 4, isolated as a yellow powder, displayed a molecular ion peak [M+Na] + at m/z 839.2020 in the positive HR-ESI-MS (calcd for C38H40O20Na, 839.2011), in agreement with the molecular formula of C38H40O20Na and suggesting an additional methoxy group compared to 3. UV spectra exhibiting absorption bands at 245, 275, 295 and 332 nm were very similar to those of 3, suggesting a flavone glycoside structure. Analysis of 2D NMR experiments of 4 and comparison of its 1 H and 13 C NMR data with those of 3 showed that 4 was orientin-2''-O-β-D-glucopyranoside acylated with sinapic acid derivative, and also showed that 4 contained the same disaccharide chain linked to C-8 of luteolin (Table 2). This disaccharide was elucidated as above as β-Dglucopyranosyl-(12)-β-D-glucopyranoside. Thus, the additional methoxy group should be located at the sinapoyl derivative. This latter was represented on the 1 H NMR spectrum by three signals comprising two doublets for coupled trans double-bond protons as in compound 3, one singlet at H 6.93 integrating for two protons, and one singlet at H 3.94 integrating for six protons suggesting the presence of E-sinapoyl moiety. The 1 H and 13 C resonances of this E-sinapoyl unit were assigned using HMBC spectrum showing connectivities from the trans double-bond to the 1,3,4,5substituted aromatic ring as well as to an ester carbonyl, and from the methoxy groups to the C-3'''' and C-5'''' of the sinapoyl residue (Table 1) [START_REF] Bendaikha | Acylated flavonol glycosides from the flower of Elaeagnus angustifolia L[END_REF]. The protons of the methoxy groups were also correlated to the protons 2'''' and 6'''' at H 6.93 in the NOESY spectrum. Full assignment of the spectra was carried out by analyzing the 1D and 2D NMR spectra of compound 4.

In the HMBC spectra of 4, correlations defining the site of acylation, glycosylation, and interglycosidic linkages were similar to those detected for 3. Thus, compound 4 was concluded to be orientin-2''-O-β-D-(6'''-sinapoyl)-glucopyranoside. Spectra of flavonoids 1-4 (see supporting material) exhibited extensive doubling of signals (6/4 ratio). This is due to the existence of obvious rotational isomerism in these spectra, which is a general characteristic of 8-C-hexosyl-flavones. Several studies have observed the rotational isomerism of 8-C-glycosyl flavonoids through NMR experiments [START_REF] Markham | NMR Spectra of Flavone Di-Cglycosides from Apometzgeria pubescens and the Detection of Rotational Isomerism in 8-C-Hexosylflavones[END_REF][START_REF] Zhou | The overlooked rotational isomerism of C-glycosyl flavonoids[END_REF].

Biological assays

A study guided by the antioxidant activity of the 80% aqueous ethanolic extract of S. gallica was conducted to tentatively determine the active constituents. The free radical scavenging activity of SGE and the resulting fractions was determined by the DPPH and hydroxyl radicals (HORAC) assays and the cupric reducing capacity was evaluated by the CUPRAC assay. Liquid-liquid partitioning in petroleum ether, CHCl3, EtOAc, and n-BuOH, combined with biological screening, identified the n-BuOH soluble fractions (Sg-n-BuOH) as the most active parts of the crude methanol extract. The best antioxidant activity was observed for the extracts in this decreasing order: Sg-n-BuOH > Sg-EA > SgE > Sg-CHCl3 >Sg-PE, as shown in Table 1. The SG-n-BuOH fraction, with the most interesting antioxidant activity, was fractionated by VLC on silica gel and the resulting fractions (B1-B8) were tested for their antioxidant properties (Table 1). Fraction B4 showed the best antioxidant activity in DPPH (EC50: 29.3 ± 1.2 µg/mL), HORAC (EC50 126.3 ± 5.1 µg/mL) and CUPRAC (EC50: 170.0 ± 8.2 µg/mL) assays (Table 1). In order to determine the compounds responsible for this activity, fraction B4 was further purified using preparative and semi-preparative HPLC to obtain compounds 1-10. As summarized in Table 1,compounds 2,3,4, 6 and 8 had the highest DPPH radical scavenging activity (EC50 from 15.0 to 23.2 µg/mL), similar to that of ascorbic acid used as reference compound (EC50 18.3 ± 1.3 µg/mL). Compounds 3, 4, and 6 showed the best hydroxyl radical scavenging activity (EC50 from 95.0 to 106.6 µg/mL) compared to quercetin used as reference compound (EC50: 52.2 ± 1.8 µg/mL). Compound 3 showed good cupric ion reducing antioxidant capacity (EC50 18.0 ± 0.7 µg/mL) compared to the reference compound, Trolox (EC50 10.4 ± 0.2 µg/mL), while compound 4 was moderately active (EC50 51.3 ± 1.1 µg/mL) (Table 1). These results showed that compounds 3 and 4 have good antioxidant capacity according to the three methods tested: DPPH, HORAC and CUPRAC assays.

In general, the antioxidant activity of flavonoids is influenced by hindrance effect, the planar skeleton, the B ring OH moiety, and the number of OH groups in the structures [START_REF] Bendaikha | Acylated flavonol glycosides from the flower of Elaeagnus angustifolia L[END_REF]. In most cases, flavonoids with two free ortho OH groups at positions 3' and 4' of the B-ring exhibited higher antioxidant activity than compounds with one OH group [START_REF] Schmitt | Bio-guided isolation of new phenolic compounds from Hippocrepis emerus flowers and investigation of their antioxidant, tyrosinase and elastase inhibitory activities[END_REF]. Our results confirm this relationship since the most active compounds 2, 3, 4, 6 and 8 were luteolin glycosides.

Compounds 2, 3 and 4 and 6 shared the orientin 2''-O-β-D-glucopyranoside, but it can be noted that acylated flavonoids 3 (feruloyl) and 4 (sinapoyl) were more active than 2 (acetylated) or 6 (without acyl group), indicating that acylation with feruloyl (3) or sinapoyl (4) moieties might contribute to antioxidant activity. Compound 3 was slightly more active than 4, suggesting that acylation with the feruloyl moiety (3) was more favorable for the antioxidant activity than acylation with the sinapoyl (4) residue. Unacylated compound 8 was slightly more active than 6, suggesting that xylose (8) was more favorable to antioxidant activity than glucose (6).

Conclusions

In this study, antioxidant activity detection was applied to fractions of an 80% EtOH extract of S. gallica aerial parts containing components of different polarities. Purification of the active fractions by preparative and semi-preparative HPLC afforded four new compounds (1-4) and six known compounds. All flavonoids (1-8) showed antioxidant potential. Two flavonoids were identified as the main antioxidant compounds (3 and 4) which showed a good effect in DPPH, HORAC and CUPRAC assays.

Experimental

General experimental procedures

Optical rotations were measured in MeOH using a Perkin-Elmer 341 Polarimeter. UV-Vis spectra were acquired in MeOH with Shimadzu UV-Vis U-2450 spectrophotometer. NMR spectra were recorded in CD3OD on a Bruker AvanceIII-600 spectrometer (Karlsruhe, Germany) ( 1 H at 600 MHz and 13 C at 150 MHz). HR-ESI-MS spectra were recorded on a Micromass Q-TOF micro instrument (Manchester, UK). Flash chromatography was carried out on a Grace Reveleris system equipped with dual UV and ELSD detection using Grace® cartridges (Silica gel or RP-C18).

Preparative HPLC was performed on Armen Instrument apparatus equipped with an AP 250 pump and a Knauer (Merck) detector UV K-2501. The mobile phase was composed of H2O acidified with TFA (0.025%) and CH3CN with a flow rate of 5 mL/min for the semi-prep HPLC and 75 mL/min for the preparative HPLC and the chromatograms were monitored at 255 nm. Semi-preparative HPLC purification was carried out on a Dionex apparatus equipped with an ASI-100 automated sample injector, a STH 585 column oven, a P580 pump, and a UVD 340S diode array detector, all driven by the Chromeleon ® software version 6.8. A prepacked C18 column (Interchim, 250 × 10 mm, 5 μ) was used for semi-preparative HPLC. Thin-layer chromatography (TLC) was carried out on silica gel 60 F254 pre-coated aluminum plates (0.2 mm, Merck), using CHCl3/MeOH/H2O (14/6/1, v/v/v) as mobile phase. After developing with solvent systems, spots were visualized under UV light (254 and 366 nm) and sprayed with 50% H2SO4 followed by heating. A FLUOstar Omega spectrophotometer (BMG LABTECH) was used for absorbance measurement in antioxidant assays.

Plant material

The aerial parts of Silene gallica L. were collected from Djebel El-Ouahch, Constantine (North-Eastern Algerian (GPS: x=6.671694, y=36.394611, z=888 m) in May 2016. Plant specimen was identified by Mr. Kamel Kabouche. Voucher specimen (LOST Sg.05/16) are on files in the herbarium of LOST Laboratory, University frères Mentouri-Constantine 1, Algeria.

Extraction and isolation

The dried and powdered aerial part of S. gallica (1 kg) was extracted three times with 80% aqueous ethanol (each 5 L, 24 h), and concentrated under vacuum in order to evaporate the ethanol and obtain an aqueous solution. The aqueous solution (700 mL) was extracted successively with petroleum ether, CHCl3, EtOAc and n-ButOH (3 × 300 mL, each), then dried under reduced pressure to yield 4 g of Sg-PE, 0.8 g of Sg-CHCl3, 2.3 g of Sg-EtOAc and 33.2 g of Sg-n-BuOH extracts, respectively. A part of Sg-n-BuOH extract (10.8 g) was subjected to vacuum liquid chromatography (VLC) over silica gel (9 cm  5.5 cm) eluted with the system CH2Cl2-MeOH (95:5, 9:1:0, 8:2, 7:3), CH2Cl2-MeOH-H2O (14:6:1, 6:4:0.7, 1:1:0) and finally by 100% MeOH to obtain 8 fractions B1-B8, respectively. A part of fraction B4 (1.75 g) was subjected to preparative HPLC eluted with CH3CN-H2O (15-20% CH3CN, in 45 min) to yield compounds 10 (tR=22 min, 125 mg), 2 (tR=27 min, 34.2 mg) and 3 (tR=40 min, 16 mg). Fractions 9-13 (111 mg) were purified by flash chromatography over silica gel, eluted with MeOH-CH2Cl2 (20-80%, in 40 min) to afford compound 6 (30 mg). Fractions 14-15 (67 mg) were purified by semi-prep. HPLC eluted with CH3CN-H2O (16:84) to yield compound 9 (tR=9.8 min, 4 mg). Fractions 18-19 (70 mg) were purified by semi-prep. HPLC eluted with CH3CN-H2O (17-20% CH3CN, in 20 min) to yield compounds 8 (tR=8.0 min, 12 mg) and 5 (tR=10.0 min, 15 mg). Fraction 23 (59 mg) was purified in the same manner as fractions 18-19 to yield compounds 7 (tR=15.3 min, 10 mg). Fraction 34 (13.9 mg) was purified by semi-prep. HPLC eluted with CH3CN-H2O (2:8) to afford compound 1 (tR=18.0 min, 3.7 mg). Fraction 37 (32 mg) was purified by semi-prep. HPLC eluted with to give compound 4 (tR=6.0 min, 12 mg). -glucopyranoside (1) Amorphous yellow powder;   20 D  -20.7 (c 0.28, MeOH) ; UV (MeOH) max (logƐ) 272 (1.91), 334 (2.05) nm; 1 H and 13 C NMR data; see Table 2 -glucopyranoside (2) Amorphous yellow powder;   20 D  -15.6 (c 0.85, MeOH) ; UV (MeOH) max (logƐ) 256 (1.02), 346

Spectral data

(1.12) nm; 1 H and 13 C NMR data; see Table 2; HR-ESI-MS m/z 717.1651 [M+Na] + (calcd for C31H34O18Na, 717.1643).

Orientin 2''-O-β-D-(6'''-feruloyl)-glucopyranoside (3)

Amorphous yellow powder;   20 D  -61.1 (c 0.83, MeOH) ; UV (MeOH) max (logƐ) 249 (1.51), 272 (1.41), 295 (1.45), 332 (2.28) nm; 1 H and 13 C NMR data; see Table 2; HR-ESI-MS m/z 809.1913

[M+Na] + (calcd for C37H38O19Na, 809.1905). 4.4.4. Orientin 2''-O-β-D-(6'''-sinapoyl)-glucopyranoside (4) Amorphous yellow powder;   20 D  -59.5 (c 0.86, MeOH) ; UV (MeOH) max (logƐ) 245 (1.9), 272

(1.35), 295 (1.30), 332 (2.13) nm; 1 H and 13 C NMR data; see Table 2; HR-ESI-MS m/z 839.2020

[M+Na] + (calcd for C38H40O20Na, 839.2011).

Acid hydrolysis

An aliquot of the fraction Sg-n-BuOH-4 (100 mg) was treated with 2 N TFA (trifluoroacetic acid, aqueous solution, 15 mL) at 90 °C for 6 h. After extraction with CH2Cl2 (10 mL  3), the watersoluble layer was evaporated to dryness (50.1 mg). The sugars mixture was then purified by preparative silica gel TLC eluted with CH3COOEt:CH3COOH:CH3OH:H2O (65:25:15:15) to afford

Fig. 1 .

 1 Fig. 1. Chemical structure of new compounds 1-4, isolated from S. gallica.

  ; HR-ESI-MS m/z 701.1702 [M+Na] + (calcd for C31H34O17Na, 701.1694).

Table 1 .

 1 Antioxidant activity of extracts, fractions and compounds isolated from S. gallica. a Values expressed are means ± S.D. of three parallel measurements, b used as positive control, nd: EC50 is not achieved.

			DPPH	HORAC	CUPRAC
			EC50 (µg/mL)	EC50 (µg/mL)	EC50 (µg/mL)
		SGE	204.4 ± 9.7	366.6 ± 5.1	650.0 ± 10.2
	extracts	Sg-PE	250.0 ± 6.2	533.3 ± 8.3	nd
		Sg-CHCl3	195.0 ± 2.8	489.0 ± 11.5	nd
		Sg-EA	106.8 ± 3.8	423.3 ± 11.5	nd
		Sg-BuOH	57.7 ± 2.1	126.6 ± 7.3	330.0 ± 7.3
		B1	167.3 ± 1.7	110.0 ± 5.8	nd
		B2	104.3 ± 5.7	823.3 ± 10.1	nd
		B3	62.2 ± 5.2	366.6 ± 8.21	nd
	fractions	B4	29.3 ± 1.2	126.3 ± 5.1	170.0 ± 8.2
		B5	58.3 ± 1.4	320.6 ± 2.8	nd
		B6	109.4 ± 3.6	443.3 ± 15.2	nd
		B7	191.5 ± 7.4	nd	nd
		B8	300.8 ± 10.4	nd	nd
		1	nd	301.6 ± 9.5	nd
		2	16.2 ± 0.9	185.1 ± 5.3	170.0 ±5.6
		3	15.0 ± 0.8	95.0 ± 3.8	18.0 ± 0.7
		4	21.3 ± 1.7	106.6 ± 4.2	51.3 ± 1.1
	compounds	5	78.5 ± 5.3	112.5 ± 5.6	nd
		6	23.2 ± 1.1	96.0 ± 2.7	178.6 ± 2.3
		7	77.5 ± 4.5	210.0 ± 7.5	nd
		8	15.2 ± 0.3	135.0 ± 8.2	168.3 ± 3.4
		9	nd	nd	109.0 ± 3.6
		10	nd	nd	nd
	controls	Ascorbic acid b	18.3 ± 1.3	-	-
		Quercetin b	-	52.2 ± 1.8	-
		Trolox b	-	-	10.4 ± 0.2

a

Table 2 .

 2 1 H NMR and 13 C NMR spectroscopic data of compounds 1-4 in CD3OD. a,b Overlapped signals are reported without designated multiplicity. b δ in ppm, J in parentheses in Hz.

	Position	1		2		3		4
		δH m (J in Hz)	δC	δH m (J in Hz)	δC	δH m (J in Hz)	δC	δH m (J in Hz)	δC
	2	-	164.9 -	164.9 -	164.9 -	164.9
	3	6.58 s	102.2 6.50 s	102.2 6.46 s	102.5 6.46 s	102.5
	4	-	182.8 -	182.7 -	182.6 -	182.6
	5	-	161.1 -	161.0 -	161.0 -	161.0
	6	6.27 s	97.9	6.26 s	97.9	6.25 s	97.8	6.23 s	97.8
	7	-	163.0 -	163.0 -	162.8 -	162.8
	8	-	103.8 -	103.7 -	103.8 -	103.8
	9		156.9		156.9		156.9		156.9
	10		104.3		104.3		104.3		104.3
	1′		122.2		122.7		122.9		122.7
	2′	8.02 d (8.4)	128.7 7.58 d (2.0)	113.6 7.57 d (1.7)	113.6 7.56 d (1.3)	113.6
	3′	6.96 d (8.4)	115.7 -	145.8 -	145.7 -	145.6
	4′	-	161.4 -	149.6 -	149.6 -	149.4
	5′	6.96 d (8.4)	115.7 6.93 d (8.2)	115.4 6.85 d (8.2)	115.4 6.87 d (8.5)	115.4
	6′	8.02 d (8.4)	128.7 7.54 dd (8.2,2.0)	119.5 7.45 dd (8.2, 1.7)	119.5 7.44 dd (8.5, 1.3)	119.5
	glcI
	1''	5.07 d (10.0)	72.3	5.06 d (10.0)	72.4	5.06 d (10.0)	72.4	5.06 d (10.0)	72.4
	2''	4.35 t (9.4)	77.8	4.37 dd (9.6, 9.2)	77.7	4.43 t (9.3)	77.3	4.43 t (9.4)	77.2
	3''	3.77 t (9.3)	79.1	3.77 t (9.3)	79.2	3.79 t (9.2)	79.2	3.78 t (8.9)	79.3
	4''	3.69 t (9.3)	70.7	3.73 t (9.3)	70.7	3.73 t (9.2)	70.7	3.73 t (8.9)	70.7
	5''	3.48 m	81.6	3.49 m	81.6	3.48 m	81.6	3.49 m	81.6
	6''	3.81 dd (12.2, 5.5)	61.5	3.89 dd (12.3, 5.8)	61.7	3.86 dd (12.5, 5.4) 61.8	3.87 dd (11.4, 5.2)	61.8
		3.97 dd (12.2, 1.8)		4.00 dm (12.3)		3.98		3.99 dd (11.4, 1.8)
	glcII
	1'''	4.46 d (7.7)	103.2 4.47 d (7.8)	103.2 4.48 d (7.7)	103.1 4.49 d (7.7)	103.1
	2'''	3.03 dd (9.4, 8.2)	74.1	3.01 dd (9.3, 8.0)	74.1	3.00 dd (8.8, 8.2)	74.3	3.00 dd (8.8, 7.7)	74.2
	3'''	3.37 t (9.4)	73.8	3.37 t (9.3)	73.8	3.23 t (8.8)	76.2	3.23 t (8.8)	76.2
	4'''	4.58 t (9.6)	69.8	4.58 t (9.6)	69.8	3.10 t (9.1)	69.2	3.11 t (9.1)	69.2
	5'''	3.18 dt (10.0, 2.3)	71.2	3.20 dt (9.9, 2.3)	71.2	3.05 m	73.8	3.07 m	73.7
	6'''	3.31	61.4	3.32	61.4	3.75 dm (12.5)	62.6	3.72 dd (11.6, 2.4)	62.6
		3.98 dd (12.2, 3.0)		3.97 dd (12.3, 3.0)		4.10 dd (12.5, 3.0)		4.16 dd (11.6, 2.5)
	Ac at glcII-C-4
	1	1.94 s	19.3	1.94 s	19.3
	2	-	170.3 -	170.4
	Ac at glcII-C-6	acetyl		acetyl		feruloyl		sinapoyl
	1''''	1.98 s	19.4	1.98 s	19.3	-	126.5 -	125.5
	2''''	-	171.3 -	171.3 7.21 s	110.2 6.93 s	105.6
	3''''					-	147.9 -	148.0
	4''''					-	149.1 -	138.0
	5''''					6.86 d (8.0)	115.0 -	148.0
	6''''					7.04 d (8.0)	123.0 6.93 s	105.6
	7''''					6.20 d (15.9)	114.0 6.25 d (15.8)	114.5
	8''''					7.53 d (15.9)	145.5 7.52 d (15.8)	145.5
	9''''					-	167.8 -	167.8
	3''''-O-CH3					3.96 s	55.1	3.94 s	55.5
	5''''-O-CH3							3.94 s	55.5

a

Acknowledgements.

The authors are grateful to MESRS Algeria for the Profas grant to Ms Sara Bechkri, to CNRS, Conseil Regional Champagne Ardenne, Conseil General de la Marne, Ministry of Higher Education and Research (MESR) in France, and to the PlANET CPER project for financial support.

DPPH radical scavenging activity

Extracts, fractions and compounds 1-10 were evaluated for their DPPH radical scavenging activity according to a recently published procedure [START_REF] Schmitt | Bio-guided isolation of new phenolic compounds from Hippocrepis emerus flowers and investigation of their antioxidant, tyrosinase and elastase inhibitory activities[END_REF]. Samples were prepared at concentrations of 400,200,100,50,25,12.5 and 6.25 μg/mL. Ascorbic acid was used as positive controls. All the tests were conducted in triplicate and EC50 were determined by interpolation of concentration versus inhibition curves obtained by a MSExcel calculation sheet.

Hydroxyl radical scavenging activity (HORAC)

Extracts, fractions and compounds 1-9 were evaluated for their hydroxyl radical scavenging activity at various concentrations, ranging from 1330 to 41.56 µg/mL, according to [START_REF] Schmitt | Bio-guided isolation of new phenolic compounds from Hippocrepis emerus flowers and investigation of their antioxidant, tyrosinase and elastase inhibitory activities[END_REF].

Ascorbic acid was used as positive controls. All the tests were conducted in triplicate and EC50 were determined by interpolation of concentration versus inhibition curves obtained by a MSExcel calculation sheet.

Power cupric ion reducing (CUPRAC) assay

Extracts, fractions and compounds 1-9 were tested for their cupric ion reducing ability. The cupric ion reducing activity (CUPRAC) was determined according to [START_REF] Schmitt | Bio-guided isolation of new phenolic compounds from Hippocrepis emerus flowers and investigation of their antioxidant, tyrosinase and elastase inhibitory activities[END_REF]. Samples were prepared at concentrations of 572,286,143,71.5,35.75,17.87 and 8.94 µg/mL. Trolox (Sigmaaldrich) was used as positive controls. All the tests were conducted in triplicate and EC50 were determined by interpolation of concentration versus inhibition curves obtained by a MSExcel calculation sheet.

Statistical analysis

All data on bioassays were the average of triplicate analyses. The data were recorded as mean values ± standard deviation. The IC50 and values were calculated by linear regression analysis (MSExcel calculation sheet).

Conflict of interest:

The authors declare no conflict of interest.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supplementary data: HR-ESI-MS, UV, and NMR data for compounds 1-4


Jeroen Wackers
email: jeroen.wackers@ec-nantes.fr

CL ÉMENCE RAYMOND Ganbo Deng

Emmanuel Guilmineau

Alban Leroyer

Michel Visonneau

TOWARDS AUTOMATED COMPUTATION WITH UNCERTAINTY ESTIMATION FOR INDUSTRIAL SIMULATION OF SHIP FLOW

Keywords: Mesh Adaptation, Automatic Simulation, Mesh Convergence, Ship Resistance

Adaptive grid refinement is tested for routine, automated simulations of ship resistance in calm water. A simulation protocol for these computations is fine-tuned on one test case and then applied unchanged to three different cases. The solutions are numerically accurate and compare well with experiments. Effective numerical uncertainty estimation increases the trustworthiness of the solutions.

INTRODUCTION

Today, mesh generation is a bottleneck for the continuing growth of CFD for realistic simulation in research and industry [START_REF] Slotnick | CFD Vision 2030 Study: A Path to Revolutionary Computational Aerosciences[END_REF]. Meshing requires more expert user knowledge than any other aspect of numerical simulation, while the trend towards more complex simulations implies that even experts cannot determine beforehand what mesh fineness, in what positions, is needed to resolve the main flow features correctly. Thus, computations may need to be repeated several times, leading to unpredictable costs and turnaround times, which is an obstacle for large-scale use in industry.

Adaptive mesh refinement, which locally refines the mesh during the simulation according to the requirements of the flow, is a natural solution to these problems. However, for general use of adaptive refinement, it should simplify computations, rather than only increase their accuracy. Mesh adaptation is only acceptable in an industrial context if it can be performed automatically, with little or no user intervention, and if it is robust enough to produce reliable solutions for a large range of test cases.

In the recent paper [START_REF] Wackers | Adaptive grid refinement for ship resistance computations[END_REF], we show that adaptive refinement for the simulation of free-surface water flows around ships, using the ISIS-CFD flow solver which we develop, is mature enough to envisage industrial application. In [START_REF] Wackers | Adaptive grid refinement for ship resistance computations[END_REF], the reliability of the adaptive simulations is ensured by two means. First, standardised guidelines [START_REF] Wackers | Adaptivity for complex flows[END_REF] are introduced to automatically set the simulation parameters correctly for each simulation, independent of the flow conditions and the geometry. And second, adaptation is used to produce series of coarse to fine meshes, which are combined with classical uncertainty estimations to predict the accuracy of the results [START_REF] Wackers | Can adaptive grid refinement produce grid-independent solutions for incompressible flows?[END_REF]. The grid convergence obtained is remarkably smooth for unstructured meshes, which implies that the estimated uncertainty bounds are sharp and accurate.

This paper provides a summary of [START_REF] Wackers | Adaptive grid refinement for ship resistance computations[END_REF]. After an introduction of the flow solver ISIS-CFD and its mesh adaptation (section 2), the approach for defining universal user guidelines is presented in section 3. These guidelines are then applied to three ships, ranging from a destroyer to a supertanker (section 4). To conclude, the range of applications for the simulation protocol and the wider implications of the tests are discussed in section 5.

ISIS-CFD HYDRODYNAMICS SOLVER

Flow solver

The CFD simulations are performed with the Navier-Stokes solver ISIS-CFD developed at ECN -CNRS, available in the FINE TM /Marine computing suite from Cadence Design Systems. ISIS-CFD is an incompressible unstructured finite-volume solver for multifluid flow. The velocity field is obtained from the momentum conservation equations and the pressure field is extracted from the mass conservation constraint transformed into a pressure equation. These equations are similar to the Rhie and Chow SIMPLE method, but have been adapted for flows with discontinuous density fields. Free-surface flow is simulated with a conservation equation for the volume fraction of water, discretized with specific compressive discretization schemes. The method features sophisticated turbulence models, such as an anisotropic EASM model and hybrid RANS/LES models of the DES family.

The unstructured discretization is face-based. While all unknown state variables are cell-centered, the systems of equations used in the implicit time stepping procedure are constructed face by face. Therefore, cells with an arbitrary number of arbitrarily-shaped constitutive faces are accepted, which enables for example adaptive mesh refinement. The code is fully parallel using the message passing interface (MPI) protocol. A detailed description of the solver is given by [START_REF] Queutey | An interface capturing method for free-surface hydrodynamic flows[END_REF].

Mesh adaptation

Adaptive grid refinement in ISIS-CFD is performed by local division of unstructured hexahedral meshes [START_REF] Wackers | Combined refinement criteria for anisotropic grid refinement in free-surface flow simulation[END_REF][START_REF] Wackers | Can adaptive grid refinement produce grid-independent solutions for incompressible flows?[END_REF]. The decision where to refine is based on a metric refinement criterion [START_REF] George | Creation of internal points in Voronoi's type method, Control and adaptation[END_REF][START_REF] Alauzet | Feature-based and goal-oriented anisotropic mesh adaptation for RANS applications in aeronautics and aerospace[END_REF], a tensor field C (x, y, z) computed from the flow. The tensor is based on the water surface position and on second derivatives of the flow variables, which give a crude indication of the local truncation errors. The grid is refined until the dimensions d i, j ( j = 1, 2, 3) of each hexahedral cell i satisfy:

C i d i, j = T r . (1)
The refinement criterion based on the second derivatives of the flow is not very sensitive to grid refinement [START_REF] Wackers | Can adaptive grid refinement produce grid-independent solutions for incompressible flows?[END_REF], so the cell sizes everywhere are proportional to the constant threshold T r .

The adaptation is included in the flow solver and fully parallelised. Computations are started from a coarse initial grid created with the mesher Hexpress (figure 2) and the refinement procedure is called repeatedly during the simulation, until the flow has converged and the mesh is no longer changed by successive adaptation steps.

Refinement criterion

To create the fine meshes entirely using adaptive refinement, the refinement criterion must react to all the flow features which are relevant for ship resistance. This implies a combination of a free-surface capturing criterion and the Hessian of the pressure, which is a suitable indicator of the orbital flow fields in the waves [START_REF] Wackers | Combined refinement criteria for anisotropic grid refinement in free-surface flow simulation[END_REF]. Also the accurate resolution of the boundary layer and wake is needed to correctly predict both viscous and pressure forces, which suggests refinement based on the velocity Hessians. The combined free-surface and flux-component Hessian criterion of [START_REF] Wackers | Can adaptive grid refinement produce grid-independent solutions for incompressible flows?[END_REF] is therefore adopted.

Figure 1 shows a mesh generated with the combined refinement criterion: combined refinement at and below the surface can be seen in waves, while the mesh around the hull is refined at the bow and in the boundary layer.

MESH REFINEMENT PROTOCOL

For routine simulation, default values for the simulation parameters are needed, which must be straightforward and correct, without a need for trial and error. These guidelines have to be valid over a range of ship lengths, velocities, hull shapes, etc. Our approach to defining guidelines [START_REF] Wackers | Adaptivity for complex flows[END_REF] is to use physical arguments, notably dimensional analysis, to reduce the number of input variables that have to be considered and then to use experience to find sensible guidelines for the remaining parameters. Without repeating the full protocol stated in [START_REF] Wackers | Adaptive grid refinement for ship resistance computations[END_REF], we provide two examples here of how the guidelines are defined.

Thresholds The appropriate mesh density is specified here by the threshold T r of equation [START_REF] Alauzet | Feature-based and goal-oriented anisotropic mesh adaptation for RANS applications in aeronautics and aerospace[END_REF]. Many authors adjust such mesh density parameters to obtain a specified number of cells, for example by normalising the metric in such a way that the threshold directly specifies the target number of cells [START_REF] Alauzet | Feature-based and goal-oriented anisotropic mesh adaptation for RANS applications in aeronautics and aerospace[END_REF].

To capture free-surface waves, we prefer a different approach. For non-adapted meshes, we specify target cell sizes at the free surface in terms of the ship length L. To obtain the same behaviour for adaptive refinement, the free-surface refinement criterion is defined with unit vectors in equation ( 1), so its threshold T rS equals the desired cell size normal to the free surface, which is chosen as L/1000 following established ISIS-CFD guidelines.

To keep the criteria compatible, a similar behaviour is sought for the Hessian refinement criterion: the mesh density on the hull should be proportional to the threshold value, independent of the case parameters. Therefore, the criterion is non-dimensionalized using the reference length and velocity. As a result, the choice for T rH no longer depends on the ship length and velocity; only a (weak) dependence on Fr, Re, and the hull shape remains, which can be ignored for a specific class of ship hulls (such as displacement hulls). The tests in [START_REF] Wackers | Adaptive grid refinement for ship resistance computations[END_REF] suggest the range T rH ∈ [0.2L, 0.025L] for coarse to fine grids.

Refinement cutoff filter and limiting box Adaptive refinement based on the combined refinement criterion captures flow details in the entire domain. However, for the accurate evaluation of ship resistance, not all these details may be required. Thus, the criterion is modified in two ways. The resolution can be controlled by specifying a minimum cell size, below which cells are no longer refined. A large minimum cell size works like a cutoff filter: while the adaptive refinement still captures the main flow features, very fine cells to resolve small details are not created. For the KCS test case of section 4.1, figure 3a shows the dependence of the resistance on the minimum cell size. This parameter (chosen small in earlier tests, around 10 -4 L) has a pronounced influence: for a larger minimum cell size, the number of cells is strongly reduced, for only a modest loss of precision. Also, the solutions for all minimum cell sizes converge to the same value when T rH is reduced. Thus, for resistance simulations, the largest minimum cell size tested, i.e. L/1000 like the free-surface threshold, is preferable. Furthermore, resolving the ship wake and the wave field behind the ship may not be needed for resistance computations. Therefore, limiting boxes were tested which forbid horizontal refinement from a given distance behind the stern. Preventing refinement aft of 0.3L behind the stern removes the entire far wake field, but does not significantly alter the forces while reducing the number of cells by up to 40% (figure 3b). An even shorter box (0.1L behind the stern) changes the converged resistance, so it is not adopted. The box at 0.3L is the best compromise.

TESTS OF THE PROTOCOL

The protocol was developed using the KCS test case. To test its generality, it was then applied blindly to other cases with varying hull forms (figure 5); the results are compared with experiments and simulations on non-adapted meshes. This section presents an extract of these tests which illustrates the main results.

KRISO Container Ship

A modern fast cargo ship, the KRISO Container Ship (KCS) [START_REF] Kim | Measurement of flows around modern commercial ship models[END_REF] is simulated in model-scale towed condition at Re = 1.257 • 10 7 and Fr = 0.260. The kω SST turbulence model with a wall law is used. The full setup for this case is given in [START_REF] Wackers | Adaptive grid refinement for ship resistance computations[END_REF]. Figure 6a shows the convergence of the resistance with the Hessian threshold T rH , as well as the numerical uncertainty evaluated with the grid-convergence based method of Ec ¸a and Hoekstra [START_REF] Ec ¸a | A procedure for the estimation of the numerical uncertainty of CFD calculations based on grid refinement studies[END_REF] (where T rH is used as the measure for the grid size, instead of an averaged cell size). A power-law like convergence is obtained and the uncertainty estimations, which overlap for the finer grids, make sense. Figure 7 shows that the free surface is visually converged at T rH = 0.05, since the difference with the finest threshold is negligible. This shows that varying T rH is an effective way to obtain grid convergence and that the selected range for T rH is appropriate. The results are compared with simulations on Hexpress meshes created with the C-Wizard automatic setup tool, which provides uniform cell sizes over the hull. Also, a combined Hexpress and systematic grid refinement (SGR) approach is shown, where the three coarsest grids are generated by Hexpress, using manual refinement at the bow and stern. The finest grids are created from these coarser grids by refining all cells once, using the adaptive refinement algorithm. Figure 6b compares the three results. The adapted series converges much faster to its final value than the other two. A given numerical accuracy is therefore obtained on coarser grids: the same distance from the converged value is typically obtained with 1.5 to 2 times fewer cells than the C-Wizard series. Furthermore, the MS-FCH and SGR series converge to roughly the same values for the resistance, which underlines the reliability of both series. The meshes of the C-Wizard series are not similar: in Hexpress, the boundary layer meshes become thinner as the outer mesh is refined. Therefore, the series converges to a lower value than the other two.

DTMB 5415

The protocol developed for the KCS is applied unchanged to the DTMB 5415 model in towed condition at Fr = 0.41 and Re = 1.74 • 10 7 following the measurements of [START_REF] Olivieri | Towing tank experiments of resistance, sinkage and trim, boundary layer, wake, and free surface flow around a naval combatant INSEAN 2340 model[END_REF]. This case represents a slender military vessel at full speed, where heavy wave breaking occurs both at the bow and the stern (figure 8b).

The adapted-grid resistance is again compared with C-Wizard uniform grids and meshes with manual refinement and SGR (figure 8a). The adapted series converges quickly and produces good uncertainty estimations; however, the experimental results are underestimated, likely due to the difficulty of modelling the breaking wave behaviour. The non-adapted series seem to agree better with the experiments, but they converge to the same underestimated solution -albeit more slowly. Thus, the faster convergence and smaller uncertainty mean that the adapted-mesh results are the most reliable.

KVLCC2

A slow bulky ship, the KVLCC2 tanker, is simulated at Fr = 0.14232 and Re = 4.6 • 10 6 , following the experiments of [START_REF] Kim | Measurement of flows around modern commercial ship models[END_REF]. To test the influence of the turbulence modelling, the anisotropic EASM model, which is recommended to resolve the KVLCC2 wake [START_REF] Duvigneau | On the role played by turbulence closures in hull shape optimization at model and full scale[END_REF], is used. Furthermore, both wall-law and wallresolved boundary conditions are tested. All other settings are the same as for the KCS.

For the wall-law results (figure 9a), the convergence of the resistance for the adapted meshes is nearly perfect with a very low final uncertainty of 0.2%. This shows that the refinement protocol is applicable to the EASM model without modifications. The C-Wizard series is in better agreement with the experiments, but this is an artefact of the wall-law boundary condition: for the more accurate wall-resolved boundary layers (figure 9b), the adapted series gives the best agreement. The difference between the solutions is caused by the aft-body flow. The experiments show a characteristic 'hook' shape in the axial velocity (figure 10c), due to a strong longitudinal vortex. This vortex is well represented by the adapted solution (figure 10a), but underestimated for the uniform grids (figure 10b). Since the creation of a vortex costs energy, this explains the lower resistance for the C-Wizard series.

KCS in self-propulsion

To test the versatility of the refinement protocol, it is finally applied to a case for which it was never intended: the KCS in self-propulsion condition [START_REF]CFD Workshop Tokyo[END_REF] with a body-force model to represent the propeller. This is a challenge since the mesh refinement has never been tested for a force field before. Also, a ship propeller is tiny with respect to the ship itself. Can something so small be captured by mesh adaptation that is configured for the flow around the ship?

The initial Hexpress mesh is equal to the one from section 4.1; no particular refinement is applied around the actuator disk. Still, the adapted grid (figure 11b) captures the propeller flow well, even at the coarse threshold T rH = 0.1L. Thus, refinement based on the ship length scale is appropriate for a detail like the actuator disk. However, the mesh size throughout the actuator disk region is equal to the minimum cell size. While this mesh is fine enough for the actuator disk, it is possible that the minimum cell size prevents the convergence to a mesh-independent solution. All resistance results (figure 11a) agree well with the experiments; the difference between the series is similar to the KVLCC2 for example. These force results confirm that the mesh adaptation protocol can be used for self-propulsion.

CONCLUSIONS AND DISCUSSION

This paper, based on [START_REF] Wackers | Adaptive grid refinement for ship resistance computations[END_REF], presents a mesh adaptation protocol intended for the evaluation of calmwater resistance for displacement hulls. How general is this protocol? The tests show that it can be applied unmodified to hull shapes ranging from slender to full, and to speeds from slow steaming to military top speeds. Also, different RANS turbulence models and wall models were tested. Furthermore, the protocol is suitable for body-force propeller models. Larger variations of the test case, such as fullscale vessels or fast planing hulls, were not tried. However, [START_REF] Wackers | Adaptivity for complex flows[END_REF] shows that adaptation with the same criterion and similar thresholds is even applicable to wings and hydrofoils. Most calm-water ship flow cases cases are probably close enough to what was tested here, that even if the protocol in section 3 has to be adapted for them, it is likely that a similar protocol exists. Thus, routine mesh adaptation for resistance simulations is realistic today. Does Hessian-based adaptation offer advantages with respect to existing meshing approaches? First, the tests show that adapted meshes provide excellent grid convergence, often obtaining 1% difference with the converged resistance on grids of about 1M cells, which is much faster than on non-adapted meshes. Furthermore, mesh adaptation makes the simulation more reliable, since it automatically ensures that the mesh is suitable for the flow. This was observed for the KVLCC2, where only the adapted meshes capture the hook-shaped wake. Finally, the grid convergence in all cases presented here is smooth, which leads to reliable convergence-based uncertainty estimations; this was also observed by [START_REF] Wackers | Can adaptive grid refinement produce grid-independent solutions for incompressible flows?[END_REF] for mono-fluid flows and by [START_REF] Alauzet | Feature-based and goal-oriented anisotropic mesh adaptation for RANS applications in aeronautics and aerospace[END_REF] for wings and aircraft using compressible flow and tetrahedral mesh adaptation. Thus, the good uncertainty estimations are likely a property of the metric-based refinement. For unstructured meshes, this is an important advantage.

The correspondence of the resistance with experiments is often worse than for non-adapted meshes. However, wherever this was tested, the differences were traced to problems with the non-adapted meshes, such as insufficient resolution of breaking waves and wakes. The reason for this is that industrial-standard turbulence models (like kω SST with wall laws) are approximate, while fixed meshes lead to nonnegligible numerical errors. Therefore, successful existing simulation methods rely to some extent on error compensation to get optimal accuracy: over the years, the turbulence modelling has been tuned to work well on industrial-standard meshes. Adaptive grid refinement however reduces numerical errors by an order of magnitude with respect to fixed grids. This is likely to upset the equilibrium of error compensation, leading to overall larger errors. Maybe the turbulence modelling has to be specifically adjusted for adaptively refined grids? Therefore, open questions remain and the simulation protocol will probably be improved and extended. Still, it has proved its generality and the potential advantages are obvious. The current results show that the technique is mature enough to start gaining experience with large-scale practical application.

Figure 1 :

 1 Figure 1: An example of an adapted mesh following the current protocol. Top to bottom: free surface, details of the Y -symmetry plane, X-cut at the stern. The case is the DTMB 5415 at Fr = 0.41 of section 4.2, with T r,H = 0.07.

Figure 2 :

 2 Figure 2: Initial mesh corresponding to the bottom image of figure 1.

Figure 3 :

 3 Figure 3: KCS, effect of the minimum cell size (a) and limiting box (b) on the resistance convergence with T rH .

Figure 4 :Figure 5 :

 45 Figure 4: KCS, Free-surface elevation with limiting box 0.3L behind the stern (left) and 2L behind the stern (right). Hessian threshold T rH = 0.05L. The dashed line indicates the aft edge of the limiting box.

Figure 6 :

 6 Figure 6: KCS resistance, grid convergence as a function of T rH (a) and of the number of cells (b), with estimated numerical uncertainty. The blue lines show the uncertainty interval of the experimental results [6].

Figure 7 :Figure 8 :

 78 Figure 7: KCS, mesh convergence of free-surface elevation. Top: T rH = 0.2L and 0.1L, bottom: 0.05L and 0.025L.

Figure 9 :Figure 10 :

 910 Figure 9: KVLCC2 resistance, grid convergence for wall law (a) and wall-resolved (b) boundary conditions.

Figure 11 :

 11 Figure 11: KCS self-propulsion: (a) resistance, grid convergence for three meshing strategies and estimated numerical uncertainty; the blue line represents the experimental result from [5]. (b) axial velocity in the y-symmetry plane for T rH = 0.1L and location of the body force (pink box).

Jeroen Wackers et al.

ACKNOWLEDGEMENTS

This work was granted access to HPC resources of CINES under the allocation A0092A01308 made by GENCI (Grand Equipement National de Calcul Intensif), which is gratefully acknowledged.


Guillaume Thébault
email: guillaume.thebault@univ-st-etienne.fr

Sophie Martin

Denis Brouillet

Lionel Brunel

Mickaël Dinomais

Emilie Presles

Joel Fluss

Stéphane Chabrier

Guillaume Th Ebault

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| Lionel Brunel

| Micka

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| Joel Fluss

| St

Ephane Chabrier

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Study Group

Guillaume Th

Manual dexterity, but not cerebral palsy, predicts cognitive functioning after neonatal stroke

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

INTERPRETATION As observed in typically developing children, manual dexterity is related to cognitive functioning in children having suffered a focal brain insult during the neonatal period.

on development, as the lesion and its timing are well circumscribed. [START_REF] Kirton | Modeling developmental plasticity after perinatal stroke: defining central therapeutic targets in cerebral palsy[END_REF][START_REF] Grunt | Incidence and outcomes of symptomatic neonatal arterial ischemic stroke[END_REF] In typically developing children, cognitive development is related to early sensorimotor abilities and experiences. [START_REF] Bremner | Developing body representations in early life: combining somatosensation and vision to perceive the interface between the body and the world[END_REF] At 3 months, the motor repertoire is correlated with global intelligence, attention, and visual-motor integration at school age. [START_REF] Hitzert | Motor development in 3-month-old healthy term-born infants is associated with cognitive and behavioural outcomes at early school age[END_REF] In children with early brain insult, CP and epilepsy segregate with lower cognitive functioning, [START_REF] Anderson | Do children really recover better? Neurobehavioural plasticity after early brain insult[END_REF][START_REF] Straub | Effects of cerebral palsy on neuropsychological function[END_REF] while high socio-economic status (SES) is protective. [START_REF] Hackman | Socioeconomic status and the developing brain[END_REF] Yet despite this generally accepted overview, the intimate relation between motor and cognitive functioning deserves further exploration.

In summary, studying children with NAIS may allow disentanglement of the respective contributions of CP on its own and manual dexterity to childhood cognitive functioning. We hypothesized (1) that a diagnosis of CP and manual dexterity are both associated with cognitive functioning; (2) that these associations are mutually Studies on cognitive and motor development after perinatal stroke provide interesting insights not only into the capacity, but also into the limits of brain plasticity. [START_REF] Anderson | Do children really recover better? Neurobehavioural plasticity after early brain insult[END_REF] However, debate still persists on the impact of early brain focal insult on later cognitive outcome. Some studies have reported a decrease of cognitive functioning, emphasizing brain vulnerability. [START_REF] Westmacott | Late emergence of cognitive deficits after unilateral neonatal stroke[END_REF] Other studies have shown stability in cognitive scores, suggesting that the developing brain can buffer the effect of an early focal insult. [START_REF] Ballantyne | Plasticity in the developing brain: intellectual, language and academic functions in children with ischaemic perinatal stroke[END_REF] In most studies, the determinants of outcome have been primarily focused on presumably deleterious factors (lesion size or diagnosis of epilepsy, or a disorder of motor function like cerebral palsy [CP]), [START_REF] Anderson | Do children really recover better? Neurobehavioural plasticity after early brain insult[END_REF][START_REF] Murias | A review of cognitive outcomes in children following perinatal stroke[END_REF][START_REF] Fuentes | Determinants of cognitive outcomes of perinatal and childhood stroke: a review[END_REF] but potentially protective factors have not been addressed. In addition, several studies have included various perinatal stroke syndromes where the timing, localization, and type of insults are diverse. Neonatal arterial ischaemic stroke (NAIS) represents a unique model to study the impact of an early brain insult independent; and (3) that they are also independent of sex, SES, and lesion side.

METHOD Ethical

The study was conducted in accordance with international ethical standards and the Declaration of Helsinki. This specific research project was approved by the regional ethics committee in May 2010. Informed consent was obtained from each family.

Participants

Put together between November 2003 and October 2006 in 39 French neonatology and paediatric neurology units, the AVCnn cohort provides a unique opportunity to follow a population of 100 children born at term (36 females, 64 males) having suffered an NAIS. [START_REF] Chabrier | Multimodal outcome at 7 years of age after neonatal arterial ischemic stroke[END_REF] The stroke, as well as its location and side, were confirmed by brain imaging performed before day 28 of life. This cohort has enabled us to study distinct developmental areas, particularly motor outcome and handedness, quality of life, functional autonomy, multimodal assessment at school age, and epilepsy. The current study is part of the assessment at 7 years of age. [START_REF] Chabrier | Multimodal outcome at 7 years of age after neonatal arterial ischemic stroke[END_REF] Two children died in the neonatal period and 13 families were lost to follow-up. Of the 85 families contacted, 73 accepted the face-to-face encounter at 7 years that was required for this work. The current sample of 60 children excludes those with incomplete data (n=2) and epilepsy (n=11), with the aim of reducing this confounding factor (see Fig. 1).

Epilepsy was defined as the occurrence of two or more febrile unprovoked seizures after the neonatal period or, in the case of a single seizure, if it was decided to introduce antiepileptic treatment. Seizure-free children (i.e. included in the current study) at 7 years were those without seizure and without treatment for over 1 year. [START_REF] Chabrier | Multimodal outcome at 7 years of age after neonatal arterial ischemic stroke[END_REF] All parents were asked to complete a questionnaire about their personal school achievements and current occupation. The Four-Factor Index of Social Status was adapted for French schooling and used as a surrogate of SES. [START_REF] Hollingshead | Four-Factor Index of Social Status[END_REF] This so-called Hollingshead score was obtained by calculating the average of the scores of both parents (or the unique score for an isolated parent) for the Education Schooling Score and the Occupation Score. When only one parent worked, only her/his score was kept. A high score indicates a high SES.

Motor functioning

All children were examined during the 7-year assessment by an experienced clinician: either a paediatric neurologist or a paediatric rehabilitation specialist.

The definition provided by the Surveillance of CP in Europe network was used: (1) permanent but not unchanging disorder of movement and/or posture; and (2) of motor function; (3) caused by a non-progressive interference/lesion/abnormality of the developing/immature brain; and (4) in a child 4-years-old or older when assessed. [START_REF]Surveillance of Cerebral Palsy in Europe (SCPE). Prevalence and characteristics of children with cerebral palsy in Europe[END_REF] Criteria (3) and (4) were, by definition, present in our population. This also means that a child with minimal neurological abnormality (e.g. tendon reflex asymmetry) but without limitation of motor function (criterion 2) was not diagnosed with CP.

Two neonates died

AVCnn cohort n=100

Assessments at 7y n=73 What this paper adds

• Manual dexterity predicts cognitive functioning after neonatal arterial ischaemic stroke.

• Correlations between manual dexterity and cognitive functioning occur irre- spective of sex, lesion side, presence of cerebral palsy, and socio-economic status.

• Residual motor ability may support cognitive functioning.

The assessment of children with CP was completed by the Gross Motor Function Classification System (GMFCS) and the Bimanual Fine Motor Function (BFMF). The GMFCS provides a measure of gross mobility capacities. According to the scale, individuals at level I walk without limitations, individuals at level II have limitations walking outdoors, individuals at level III need assistive mobility device, individuals at level IV are transported or use power mobility outdoors, and a manual wheelchair is needed by individuals at level V. The BFMF scale indicates ability to grasp, manipulate, and hold objects for each hand separately. As the other hand manipulates without restrictions, scores range in children with unilateral CP from level I (the affected hand manipulates without restrictions or has limitations in more advanced fine motor skills) to level II (the affected hand has only ability to grasp or hold) and level III (the affected hand has no functional ability). [START_REF] Elvrum | Bimanual Fine Motor Function (BFMF) classification in children with cerebral palsy: aspects of construct and content validity[END_REF] Manual dexterity was evaluated with the Nine-Hole Peg Test (NHPT) [START_REF] Smith | Normative and validation studies of the nine-hole peg test with children[END_REF] and the Box and Blocks Test (BBT), [START_REF] Jongbloed-Pereboom | Norm scores of the box and block test for children ages 3-10 years[END_REF] which are commonly used in clinical studies and experimental research to measure finger dexterity and hand dexterity respectively. [START_REF] Immerman | Hand function in children with an upper brachial plexus birth injury: results of the Nine-Hole Peg Test[END_REF] The NHPT consists of inserting nine small pegs on a board and then taking them off, as quickly as possible. The time to finish the NHPT was reported. The BBT consists of a box with two compartments separated in the middle. At the beginning, 100 small blocks are located in one of the compartments, on the same side of the tested hand. Children move as many cubes as they can from one compartment to the other. The result is the number of cubes moved into the second compartment in 1 minute. Both hands were evaluated and, for each test, the score of the best hand was considered.

Cognitive functioning

The Wechsler Intelligence Scale for Children, Fourth Edition (WISC-IV), provides a global assessment of intelligence from 15 subtests, including five optional subtests. [START_REF] Wechsler | WISC-IV technical and interpretative manual[END_REF] The 10 main subtests and the arithmetic subtest were administered during the 7-year assessment by an experienced child neuropsychologist. Based on the results, the four WISC-IV indexes were calculated: Verbal Comprehension Index, Perceptual Reasoning Index, Processing Speed Index (PSI), and Working Memory Index (WMI). The Full-scale IQ (FSIQ) was then computed. The WISC-IV has good psychometric properties. Furthermore, this scale was used in previous studies on cognitive functioning in children with NAIS. Only PSI needs unilateral manual dexterity to write.

Statistical analysis

Descriptive statistics were performed using a univariate test to estimate differences between the sample and the norm of WISC-IV on FSIQ and the four indexes.

To test the first association, the FSIQ and the four indexes of the WISC-IV were the dependent variables. Explanatory variables were manual dexterity and CP. For overall manual dexterity, a score averaging the BBT and the NHPT scores was calculated and called the Manual Dexterity Index (MDI). To define it, we converted each individual raw score obtained from NHPT and BBT into z-scores. The NHPT score is a time delay to accomplish the required task; a negative z-score means a good performance. Conversely, the BBT score is the number of cubes translated during a specified period; a positive z-score means a good performance. To average the z-scores of BBT and NHPT of an individual child, the NHPT positive z-scores were converted to negative z-scores and vice versa. A high MDI therefore indicates a good performance. The advantage of the z-score transformation is to retain the position of each child in the sample. [START_REF] Cahna | Differential contributions of cognitive and motor component processesd to physical and instrumental activities of daily living in Parkinson's disease[END_REF] Linear models were used to establish the respective importance of motor variables (i.e. MDI and CP) on WISC-IV scores. We performed linear regressions for both motor variables on WISC-IV indexes to test the first association (models 1 and 2). From the results of this first analysis, multiple linear regressions were used to test the second and third associations, specifically to measure the effect of CP independently of the effect of the MDI on FSIQ and other indexes (second association; model 3). The variables kept in the modelling had a level of significance of p<0.15. [START_REF] Cox | The choice of variables in observational studies[END_REF] The same design was used by integrating sex, lesion side, and SES (third association; model 4).

To appreciate the results of linear regressions, we used adjusted R 2 . Established from R 2 , adjusted R 2 takes into account the number of variables introduced in the analysis. The adjusted R 2 corresponds to the variance of dependent variable explained by regression model. Moreover, the b designs the standardized regression coefficient. It means, for instance, that with every increase of one SD in FSIQ, the MDI rises by 0.41 SD. We expected to observe an adjusted R 2 explaining about 20% of variance as in previous results. [START_REF] Fjørtoft | Adaptive behavior in 10-11 year old children born preterm with a very low birth weight (VLBW)[END_REF] All statistics were performed using R software environment (R Development Core Team, Vienna, Austria). The significant threshold was p<0.05.

RESULTS

Population, sex, lesion side, and socioeconomic status

Twenty-one females and 39 males with NAIS but not epilepsy were examined at a median age of 7 years and 2 months (range 6y 11mo-7y 8mo). The infarct was located in the following territories: middle cerebral artery (n=53), anterior cerebral artery (n=3), posterior cerebral artery (n=2), multiple territories (n=2). It was superficial in 44 cases, deep in six, and involved both regions in 10. Unilateral infarct was more common on the left (n=37) than on the right (n=19). Four children had bilateral infarction. The Hollingshead score ranged from 13.5 to 66, with a normal distribution (Tables I andII).

Motor and intelligence variables

During the 7-year assessment, 15 of the 60 children (25%) were diagnosed with CP by clinical examination, unilateral in all. BBT and NHPT scores of the best hand, cognitive child had no PSI score. These missing data did not allow us to take into account these participants in corresponding analyses (regression concerning FSIQ and PSI). The Motor and cognitive variables had normal distributions.

Relationship between motor and intelligence variables

The results of simple linear regression models showed a strong relationship between FSIQ and both motor variables (CP and MDI; see models 1 and 2 in Table III). A relationship was found between CP and Verbal Comprehension Index but not with other indexes. MDI was significant on all indexes except for WMI.

In the first step of multiple regressions, motor variables were introduced and WMI removed (p>0.15). Whether a child had CP or not, MDI was associated with intelligence scores (see model 3 in Table IV). A second step integrated Hollingshead score, sex, and lesion side (see model 4 in Table IV). MDI was still significant on all indexes except for WMI. The Hollingshead score effect was significant on FSIQ, Verbal Comprehension Index, and Perceptual Reasoning Index. The sex effect was significant on PSI, with females having better scores. The lesion side effect was not significant.

In order to further investigate the lack of significant data observed for the CP effect, we performed a post hoc analysis. In this analysis, the binary CP variable was substituted by the GMFCS and BFMF scores to test the severity of clinical symptoms of CP as an associated variable of FSIQ scores. In this analysis, GMFCS and BFMF scores were defined at 0 for children without CP. We ran multiple regression models using MDI and severity of CP as the explanatory variables and FSIQ score as the dependent variable. This analysis showed no significant effect of CP severity, but MDI did (data not shown).

DISCUSSION

Our results establish a strong relationship between motor and cognitive functioning in children having suffered an NAIS. While significant links were found simultaneously for CP and manual dexterity with IQ scores, manual dexterity acts on its own as an independent variable to explain higher cognitive functioning. Whatever the CP status, manual dexterity associates with IQ. In accordance with previous studies, SES was also correlated with IQ. However, putting the SES as an independent covariable in the statistical model, our main result remains valid. This is in line with studies on children with typical development, in which motor ability is also closely linked to IQ. [START_REF] Jenni | Correlations between motor and intellectual functions in normally developing children between 7 and 18 years[END_REF][START_REF] Wassenberg | Relation between cognitive and motor performance in 5-to 6-year-old children: results from a large-scale cross-sectional study[END_REF] Furthermore, our results show that this relationship is independent of sex, lesion side, SES, and, finally, CP. This highlights the strength of the relationship between manual dexterity and IQ in comparison with other variables. It also supports the fact that in early braindamaged children, some developmental processes are comparable to those of typically developing children. [START_REF] Hadders-Algra | The Neuronal Group Selection Theory: an attractive framework to explain variation in normal motor development[END_REF][START_REF] Karmiloff-Smith | Development itself is the key to understanding developmental disorders[END_REF] Two main hypotheses can be formulated to explain these findings. Firstly, it might be that some subtle limitations in manual dexterity in children not categorized as CP are not detectable through clinical examination. The second hypothesis considers the role of motor ability in development. [START_REF] Thelen | A Dynamic Systems Approach to the Development of Cognition and Action[END_REF] In our cohort, the preservation of fine motor function could contribute to the development of cognitive hypothesis, favouring residual motor ability may support cognitive functioning and help to counterbalance the negative effect of CP.

Having included only part (60/73 children) of the AVCnn cohort assessed at 7 years of age is a limitation. Nevertheless, our primary goal was to analyse a population without confounding deleterious factors, such as epilepsy. Furthermore, although the original cohort is longitudinal in nature, this assessment is cross-sectional. This means that we have not been able to study developmental trajectories and the potential crucial role of fine motor skills in the first months of life as an early determinant of further cognitive functioning after early brain insult.

In conclusion, having taken into consideration well-studied factors contributing to development, we have shown that manual dexterity of children with NAIS is closely linked to cognitive functioning. functioning. One can eventually speculate that in some situations the negative effects of unilateral CP on development can be blurred by the motor ability of the non-affected hand.

Our results show an association between cognitive functioning and manual dexterity. However, as stated by Fjørtoft et al., [START_REF] Fjørtoft | Assessment of motor behaviour in high-risk-infants at 3 months predicts motor and cognitive outcomes in 10 years old children[END_REF] a causal relationship can neither be established nor the direction of this relationship. In other words: is the cognitive impairment a direct consequence of the poor movement quality or does the quality of movements reflect global brain dysfunctioning? More likely both are coextensive, with the necessary implication of motor activity in cognitive or executive tasks, such as visuo-constructional processes. And, conversely, the execution of any ecological motor tasks involves a complex interplay between motor planning, motor control, visual perception, and intention. Although cognitive and motor domains can be considered distinct, they are necessarily overlapping during initial development and also for later cognitive skills. [START_REF] Thelen | A Dynamic Systems Approach to the Development of Cognition and Action[END_REF] For instance, fine motor function and arithmetical ability are intimately intricated. [START_REF] Thevenot | Numerical abilities in children with congenital hemiplegia: an investigation of the role of finger use in number processing[END_REF] Besides these developmental issues, the perspectives are therapeutic and methodological. Regarding the latter, examining the relationships between motor and cognitive processes requires the inclusion of motor performance values more complex than CP as a binary variable. Our results also suggest that a quantitative evaluation of manual dexterity can be valuable when assessing cognitive functioning in children with early brain lesion. Furthermore, our study is consistent with data that efficient motor interactions with the environment are important for cognitive development. [START_REF] Ferre | Coaction of individual and environmental factors: a review of intensive therapy paradigms for children with unilateral spastic cerebral palsy[END_REF][START_REF] Morgan | Optimising motor learning in infants at high risk of cerebral palsy: a pilot study[END_REF] Future therapeutic trials should take into account the potential positive value of favouring such interactions with the valid hand or through bimanual tasks, as well as providing an enriched environment, notably at home. [START_REF] Fjørtoft | Assessment of motor behaviour in high-risk-infants at 3 months predicts motor and cognitive outcomes in 10 years old children[END_REF][START_REF] Thevenot | Numerical abilities in children with congenital hemiplegia: an investigation of the role of finger use in number processing[END_REF] According to this

Figure 1 :

 1 Figure 1: Follow-up until 7 years of age and exclusion of children.

Table I :

 I Individual children information and lesion/neurological characteristics of the population (n=60) FSIQ score was not calculable in three children and one results of the WISC-IV subtests are specified in TableSI(online supporting information).Three children had an FSIQ score below 1.96 SD and one above 1.96 SD. When comparing FSIQ averages and each index to WISC-IV, FSIQ, Verbal Comprehension Index, Perceptual Reasoning Index, and PSI, they were not different from norms, but WMI was significantly lower (t[59]=À3.63, p=0.001).

		Age at
		assessment	Holligshead	Territory and
	Participant/sex	y:mo	score	side of infarct			CP	GMFCS	BFMF	BBT a	NHPT a	FSIQ
	1/M	7:0	14.5	MCA	R	Superficial	No			27	33	94
	2/M	7:2	53	PCA	R	Deep	No			35	29	94
	3/F	7:1	31.5	MCA	R	Superficial	No			23	23	105
	4/M	7:3	22.5	MCA	R	Superficial	No			31	30	71
	5/M	7:4	27.5	ACA	L	Superficial	No			31	30	68
	6/M	7:1	57	MCA	L	Superficial	No			38	30	136
	7/M	7:2	48	MCA	R	Deep	No			31	26	99
	8/F	7:1	28.5	MCA	R	Superficial	No			37	20	108
	9/M	7:0	28.5	ACA	L	Superficial	Yes	II	I	24	32	101
	10/M	7:5	28	MCA	L	Superficial	Yes	II	I	34	27	74
	11/M	7:2	37.5	ACA	L	Mixed	Yes	II	I	45	20	101
	12/M	7:3	36.5	MCA	R	Deep	No			25	33	93
	13/M	7:2	13.5	MCA	B	Superficial	No			31	34	-
	14/F	7:1	35	MCA	L	Superficial	No			39	26	104
	15/M	7:1	38.5	MCA	L	Superficial	No			31	27	98
	16/M	6:11	25	MCA	L	Superficial	No			26	32	81
	17/F	7:4	29	MCA	R	Superficial	No			32	24	112
	18/F	7:3	25.5	MCA	L	Mixed	No			30	25	117
	19/M	7:1	23	MCA	R	Superficial	Yes	I	II	28	30	82
	20/F	7:1	50.5	MCA	L	Superficial	No			32	22	111
	21/M	6:11	18	MCA	L	Superficial	No			33	25	84
	22/F	7:3	32.5	MCA	L	Superficial	No			33	27	107
	23/M	7:2	55.5	MCA	L	Mixed	Yes	I	I	42	29	103
	24/M	7:0	46	MCA	L	Mixed	Yes	II	II	22	29	69
	25/M	7:0	62	MCA	L	Mixed	No			42	27	107
	26/M	7:0	32.5	MCA	R	Superficial	No			44	22	108
	27/F	7:0	44	MCA	R	Superficial	Yes	I	I	34	24	89
	28/F	7:3	26	MCA	L	Superficial	No			34	30	115
	29/F	7:1	19.5	MCA	L	Superficial	No			25	30	71
	30/M	7:1	31	MCA	R	Superficial	No			39	29	115
	31/M	7:2	34	MCA	L	Superficial	No			31	23	110
	32/M	7:2	52	MCA	L	Superficial	No			28	32	92
	33/M	6:11	66	MCA	L	Superficial	Yes	I	II	36	29	92
	34/M	7:3	46	ACA+MCA	L	Superficial	No			45	29	101
	35/M	7:2	58	MCA	L	Superficial	No			39	33	104
	36/F	7:2	37.5	MCA	L	Superficial	No			41	20	100
	37/F	7:4	27	MCA	R	Superficial	No			39	32	90
	38/M	7:0	21.5	ACA+MCA+PCA	L	Superficial	No			33	26	117
	39/M	7:0	23.5	MCA	L	Superficial	No			44	27	94
	40/F	7:3	42.5	MCA	L	Superficial	No			34	30	94
	41/M	7:2	38	MCA	L	Mixed	Yes	II	II	28	26	107
	42/M	7:0	23	MCA	L	Superficial	No			37	25	79
	43/F	7:7	29	MCA	R	Mixed	Yes	II	I	31	23	91
	44/F	7:2	25.5	MCA	L	Deep	No			43	22	93
	45/M	7:8	58	MCA	R	Superficial	No			33	25	116
	46/F	7:2	58	MCA	R	Superficial	No			31	30	111
	47/F	7:2	44	PCA	B	Superficial	No			44	26	109
	48/M	7:4	33.5	MCA	L	Deep	Yes	II	II	30	25	-
	49/M	7:3	26.5	MCA	B	Superficial	No			26	28	84
	50/F	7:2	54.5	MCA	L	Superficial	No			40	29	111
	51/F	6:11	20.5	MCA	L	Mixed	Yes	II	I	17	28	102
	52/F	9:2	26	MCA	L	Mixed	Yes	I	II	27	30	93
	53/M	7:1	31	MCA	L	Superficial	No			35	23	105
	54/M	7:2	31	MCA	R	Superficial	No			31	28	126
	55/M	6:11	21.5	MCA	R	Superficial	No			34	25	94
	56/F	7:1	36.5	MCA	L	Superficial	No			35	25	106
	57/M	6:11	47	MCA	R	Mixed	Yes	I	III	23	46	69
	58/M	7:4	21	MCA	B	Superficial	No			42	31	101
	59/M	7:3	16	MCA	L	Deep	No			32	24	-
	60/M	7:3	21.5	MCA	L	Superficial	Yes	I	I	34	24	93

a Results of the best hand. CP, cerebral palsy; GMFCS, Gross Motor Function Classification System; BFMF, Bimanual Fine Motor Function; BBT, Box and Blocks Test; NHPT, Nine-Hole Peg Test; FSIQ, Full-scale IQ (assessed by the Weschler Intelligence Scale for Children); M, male; MCA, middle cerebral artery; R, right; PCA, posterior cerebral artery; F, female; ACA, anterior cerebral artery; L, left; B, bilateral. assessment in the whole population, and GMFCS and BFMF scores in children with CP are reported in Table

I

.

Table II :

 II Statistical analysis of motor, cognitive, and socio-economic variables

		Mean (SD)	Median	95% CI
	BBT a	33.51 (6.47)	33	29.82-37.21
	NHPT a	27.4 (4.34)	27	23.71-31.09
	CP (yes/no)	15/45
	FSIQ	98.26 (14.59)	101	94.57-101.95
	Verbal	99.63 (13.91)	101	95.93-103.33
	Comprehension Index
	Perceptual	96.27 (15.72)	102	92.57-99.96
	Reasoning Index
	Processing Speed Index	101.43 (16.39)	100	97.74-105.12
	Working Memory Index	92.07 (16.91)	92.5	88.37-95.76
	Hollingshead score	34.83 (13.3)	31.25	31.14-38.53
	a Results of best hand. CI, confidence interval; BBT, Box and Blocks
	Test; NHPT, Nine-Hole Peg Test; CP, cerebral palsy; FSIQ, Full-scale
	IQ.

Table III :

 III Regression analyses from Manual Dexterity Index (MDI), cerebral palsy (CP), and other covariables on Wechsler Intelligence Scale for Children, Fourth Edition

		FSIQ		VCI		PRI		PSI		WMI
	Variables and models	b	p	b	p	b	p	b	p	b	p
	Model 1
	MDI	0.41	0.002	0.39	0.002	0.36	0.005	0.42	0.001	À0.02	0.872
	Adjusted R 2		0.152		0.136		0.116		0.164		À0.017
	Model 2
	Absence of CP	0.31	0.019	À0.33	0.009	À0.09	0.488	À0.25	0.06	À0.05	0.702
	Adjusted R 2		0.079		0.096		À0.009		0.045		À0.015

FSIQ, Full-scale IQ; VCI, Verbal Comprehension Index; PRI, Perceptual Reasoning Index; PSI, Processing Speed Index; WMI, Working Memory Index.

  Male was designated as the reference category. b Left lesion was designated as the reference category. FSIQ, Full-scale IQ; VCI, Verbal Comprehension Index; PRI, Perceptual Reasoning Index; PSI, Processing Speed Index; MDI, Manual Dexterity Index; CP, cerebral palsy.

		b	p	b	p	b	p	b	p
	Model 3
	MDI	0.35	0.007	0.33	0.009	0.36	0.006	0.39	0.003
	Absence of CP	0.22	0.083	À0.26	0.038	À0.01	0.951	À0.02	0.232
	Adjusted R 2		0.183		0.185		0.100		0.171
	Model 4
	MDI	0.31	0.017	0.29	0.013	0.33	0.013	0.34	0.006
	Absence of CP	0.23	0.067	À0.27	0.021	À0.04	0.774	À0.15	0.194
	Sex a	À0.06	0.229	0.08	0.468	0	0.994	0.35	0.004
	Hollingshead score	0.27	0.026	0.4	0.0003	0.3	0.01	0.005	0.966
	Lesion side b
	Bilateral	À0.03	0.789	0.12	0.287	0.002	0.989	À0.19	0.097
	Right	À0.06	0.637	0.11	0.627	À0.001	0.993	À0.08	0.489
	Adjusted R 2		0.221		0.324		0.152		0.283

a

ACKNOWLEDGEMENTS

This study was funded by the Minist ere des Affaires Sociales et de la Sant e (PHRC r egional No. 0308052 and PHRC interr egional No. 1008026), Centre Hospitalier Universitaire de Saint-Etienne (AOL no. 085), and the following supporting organizations: Fondation Paralysie C er ebrale/Fondation Motrice, Fondation Garches, Association des Paralys es de France, and Fondation de l'Avenir. EudraCT number 2010-A00329-30; Clinical trial NCT02511249. We warmly thank Mr Jean-Fabrice Vernet and Mr Sev Fluss for their editing assistance. The authors have stated that they had no interests that might be perceived as posing a conflict or bias.

SUPPORTING INFORMATION

The following additional material may be found online:

Appendix S1: AVCnn Study Group.