Mohammad Kazem Medlej

Batoul Cherry

Ghassan Nasser

François Zaviska

Akram Hijazi

Suming Li

Céline Pochat-Bohatier

Optimization of polysaccharides extraction from a wild species of Ornithogalum combining ultrasound and maceration and their antioxidant properties

Keywords: Ultrasonic-assisted extraction, optimization, polysaccharides, anti-oxidant activity

Polysaccharides were extracted from a wild species of Ornithogalum by using three methods: maceration, ultrasound-assisted extraction, and combination of maceration and ultrasound.

Extraction conditions were optimized by using response surface method (RSM) with a central composite design (CCD). Four parameters were considered in the optimization method, i.e. total extraction time, extraction temperature, ratio of water volume to raw material mass, and time percentage of ultrasound treatment in the extraction process. The optimal extraction yield was 81.7 %, 82.5 % and 85.7%, and the optimal polysaccharides yield was 74.7 %, 75.7 %, and 82.8% under the optimum conditions of maceration, ultrasound-assisted extraction and combined extraction, respectively. These results indicate that the combination method significantly improves the extraction and polysaccharides yields compared to traditional extraction methods. The combination method also allows reducing the time of ultrasound treatment and thus its adverse effects on polysaccharides. In addition, these results well corroborate with the theoretically predicted values. The NMR ( 1 H, 13 C, HSQC, HMBC, and COSY) analysis shows that the extract is composed of fructo-polysaccharides with a backbone of (2→ 6)-linked β-d-fructofuranosyl (Fruf)and (2→1)-linked β-d-Fruf branched chains, and terminated with glucose and Fructose residues. The antioxidant activities of the extract were evaluated from ABTS radical scavenging activity, total antioxidant capacity, metal-chelating power and βcarotene bleaching test. Data show that the extract presents outstanding antioxidant activities.

Introduction

Synthetic antioxidant compounds such as butylated hydroxyanisole (BHA) are extensively employed as preservatives in pharmaceuticals, cosmetic and food industry. However, some studies have shown that BHA causes adverse effects on cells [START_REF] Labrador | Cytotoxicity of butylated hydroxyanisole in Vero cells[END_REF], and thus could have negative impact on human health. Therefore, naturally occurring antioxidants have been attracting more and more interest as they actually represent an alternative to synthetic ones.

More than 90% of the carbohydrate mass in nature is in the form of polysaccharides. Many studies reported that various polysaccharides present remarkable antioxidant activities, and could be used as healthy food additives [START_REF] Yan | Ultrasonic effects on the degradation kinetics, preliminary characterization and antioxidant activities of polysaccharides from Phellinus linteus mycelia[END_REF]. Ornithogalum is a genus of perennial plants with various species, including Ornithogalum arabicum, saundersiae, caudatum Ait, dubium, nutans, pyrenaicum, thyrsoides and umbellatum. Polysaccharides extracted from Ornithogalum caudatum Ait present outstanding antioxidant, anticancer, antimicrobial and antiinflammatory activities [START_REF] Chen | Antitumor activities of different fractions of polysaccharide purified from Ornithogalum caudatum Ait[END_REF]. They have been used for the treatment of hepatitis, parotitis, and cancers [START_REF] Chen | Antitumor activities of different fractions of polysaccharide purified from Ornithogalum caudatum Ait[END_REF]. Ornithogalum billardieri is a non-edible wild plant widespread in Lebanon which could be a potential source of valuable polysaccharides with promising bioactive properties. To the best of our knowledge, this plant has never been studied, so far.

The extraction of polysaccharides represents an important issue for their applications in pharmaceutical and agri-food industries [START_REF] Persin | Challenges and opportunities in polysaccharides research and technology: The EPNOE views for the next decade in the areas of materials, food and health care[END_REF]. Maceration is the most commonly used method to extract polysaccharides due to its simplicity. Nevertheless, the major inconvenience of maceration is the low extraction yield [START_REF] Gray | Natural Products Isolation[END_REF], long extraction time, and large energy consumption. Ultrasound (US) treatment is a time and energy saving method which is widely used for extraction of various polysaccharides [START_REF] Hrom | Ultrasonic extraction of plant materials --investigation of hemicellulose release from buckwheat hulls[END_REF]. It allows to reduce the particle size, to disrupt the cell wall, and to improve the mass transfer from solid to liquid phase due to the intense shear forces, thus leading to higher extraction yield [START_REF] Ying | Ultrasound-assisted extraction of polysaccharides from mulberry leaves[END_REF]. Compared to maceration, US treatment presents many advantages such as reduced extraction time, lower temperature, and in some cases less water consumption [START_REF] Wang | Effects of Ultrasound Treatment on Extraction and Rheological Properties of Polysaccharides from[END_REF]. Importantly, US extraction allows to improve the purity of extracted polysaccharides, and their antioxidant activity [START_REF] Ebringerová | An overview on the application of ultrasound in extraction , separation and purification of plant polysaccharides[END_REF]. However, US treatment can provoke chain cleavage and compositional changes of polysaccharides due to the cavitation effects [START_REF] Ebringerová | An overview on the application of ultrasound in extraction , separation and purification of plant polysaccharides[END_REF]. Side reactions could also happen and lead to the formation of carbonyl and hydroxyl radical groups.

The aim of this work was to implement and optimize a new extraction process combining both US and maceration in order to improve the extraction yield while reducing the adverse effect of US treatment. Different combinations of maceration and US treatment were tested to extract polysaccharides from Ornithogallum billardieri using the Surface Response Methodology (RSM) applied to an experimental model such as Box-Behnken design (BBD) and central composite design (CCD) [START_REF] Wang | Optimization of enzyme-assisted extraction of polysaccharides from alfalfa and its antioxidant activity[END_REF]. The studied operating parameters include the total duration of the extraction process (time), the extraction temperature, the ratio of added water volume to raw material mass, and the time ratio of US treatment in the total process (% US).

The extracted polysaccharides were characterized by using 1 H, 13 C, HSQC, HMBC, and COSY NMR analysis.

The antioxidant properties of the polysaccharides extracted under optimal conditions were investigated, including ABTS radical scavenging activity, total antioxidant capacity (TCA), metalchelating power, and β-carotene bleaching test so as to evaluate their potential for applications in the agro-food industry as alternative to synthetic antioxidants.

Experimental

Materials and reagents

The onion plant Ornithogalum was collected in the region of Bekaa in Lebanon. The plant was carefully washed, cut into small pieces, and freeze dried. The dried plant was manually crushed, and sieved at 0.6 mm to obtain a homogeneous powder. Sulfuric acid, phenol, 2,2-azino-bis (3ethylbenzothiazoline-6-sulfonic acid) (ABTS), ascorbic acid, potassium peroxydisulfate, sodium phosphate, ammonium molybdate, α-tocopherol, iron dichloride, ferrozine, diethylenediamine (EDTA), phenol, anhydrous ethanol, butanol, and chloroform of analytical grade were purchased from Sigma Aldrich, and used as received.

Extraction of polysaccharides

Before extraction, the powder was purified at 70°C using a Soxhlet for 2 days with ethanol as solvent. This preliminary step allowed to eliminate all pigments, polyphenols, oligosaccharides, simple oses and amino acids, and also to inactivate enzymes. The powder recovered from the Soxhlet cartridge was vacuum dried overnight at 40°C, and then crushed and sieved at 0.6 mm.

The powder was added in ultrapure water, and homogenized for 30 sec at 3500 rpm in a Speed mixer (Hauschild DAC 150.1 FVZ-K). Extraction then proceeded at a given temperature (25-65°C) for predetermined time periods (10-60 min). The extraction temperature was controlled (± 0.2°C) using a thermo-cryostat (Vacuo-Temp P, Selecta) and a thermostat cell. The ratio of the water volume to the powder mass varied in the range of 10 to 40 mL/g. Ultrasound treatment was performed at fixed frequency (35 KHz) and power (120 W). The time ratio of ultrasound treatment in the total extraction process (%US) varied between 0% and 100%. After extraction, the aqueous solution was immediately centrifuged at 6000 rpm for 15 min three times. The supernatant was lyophilized using a freeze-dryer (Freezone 4.5 Labconco). Finally, the obtained powder was washed with ethanol, and vacuum dried up to constant weight.

The extraction yield (%) was calculated using Eq. 1.

Extraction yield (%) = [(Weight of dried product / Weight of crude powder)] × 100(Eq. 1)

Total carbohydrates content

The total carbohydrate content (TCC), also known as the polysaccharides yield, was determined according to Dubois method [START_REF] Dubois | Colorimetric Method for Determination of Sugars and Related Substances[END_REF]. The method allows to quantify all carbohydrate species, including mono-, di-, oligosaccharides, their methyl derivatives and polysaccharides. A calibration curve was first established from standard glucose solutions at concentrations from 0 to 100 μg/mL. 1 mL of standard solution was added to 5 mL of concentrated sulfuric acid, followed by addition of 1 mL of 5% (v/v) phenol solution. After 10 min stirring at 100 °C, the solution was cooled down to room temperature away from light. Finally, the absorbance was measured using a spectrophotometer at 490 nm (λ max). The absorbance was then plotted against glucose concentration to obtain a calibration curve. The samples are analyzed using the same procedure to obtain the concentration of polysaccharides in samples. The total carbohydrate content (%) of samples is determined using the following equation:

Total carbohydrate content (%) = [(Weight of polysaccharides / Weight of sample)] ×100 (Eq. 2)

Extraction optimization

Single-factor experiments

A series of preliminary experiments were first performed in order to determine the intervals of the 4 parameters (time, temperature, volume to mass ratio, and US%), and the range of extraction yield and polysaccharides yield. For each experiment, a single parameter was varied while keeping the others constant: the time was varied from 10 to 60 min, the temperature from 20°C to 80°C, the volume to mass ratio from 10 to 50 mL/g, and the US% from 0 to 100%.

The results obtained from single factor analysis were used to build a model based on RSM to identify the interactions between parameters.

Experimental design

The Surface Response Methodology (RSM) is applied to the central composite design (CCD) for the adaptation of a second order polynomial by the least square technique. Eq. 3 is used to determine the effects of test variables to the searched responses (extraction yield and polysaccharides yield) and the correlation between variables.

Y = βκ 0 ∑ βκ 𝑖 4 𝑖=1 X 𝑖 + ∑ βκ 𝑖𝑖 4 𝑖=1 𝑋 𝑖 2 + ∑ βκ 𝑖𝑗 X 𝑖 4 𝑖<𝑗=2 X 𝑗 (Eq. 3)
Where Y is the predicted response, βκ0 the intercept, and βκi, βκii, and βκij the coefficients of linearity, quadratic and interaction, respectively. And Xi and Xj are the coded independent variables. The response function was also related to the coded variables (Xi, i = 1, 2, 3) and i≠j, by a second-degree polynomial using the method of least squares.

The The regression coefficients of individual linear, quadratic and interaction terms were determined using Design-Expert (Version 11) software. They were then used to make statistical calculations, which generate dimensional and contour maps from the regression models.

Structural characterization

Fourier transform infrared (FT-IR)

FT-IR spectra were recorded using an IR-TF Nicolet IS50 Fourier counter exchange absorption infrared spectrometer (Bruker, Germany) over a range of 400-4000 cm -1 . The samples were analyzed as KBr pellets.

Size-exclusion chromatography (SEC)

Size-exclusion chromatography (SEC) was carried out using HPLC (DW-LC1620A) equipped with TSK gel PW5000 + PW3000 columns and refraction index and ultraviolet detectors. The temperature of the columns and detectors was 20 and 35 °C, respectively. A pH 6 phosphate buffer at 10 mg/mL was used as eluent at a flow rate of 1 mL/min. Calibration was realized using pullulan standards with molar masses from 500 to 25000 Da. The results were processed using OmniSEC software.

NMR Spectroscopy

1 H, 13 C, HSQC, HMBC, and COSY NMR spectra were recorded using a Bruker Avance III spectrometer operating at 600 MHz and 150 MHz, respectively. D2O was used as solvent, and the chemical shifts were expressed in ppm. The spectra were treated using Mestre Nova 12.0.0. software package.

Antioxidant activities

ABTS radical scavenging activity

2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS .+ ) stock solution was prepared by dissolving 7 mM ABTS with 2.45 mM K2S2O8 in ultra-pure water, followed by stirring in the dark for 12 h. The solution was diluted with ethanol to obtain an absorbance of 0.7 (± 0.02) at 734 nm at 30 °C. 0.1 mL of sample at concentrations from 0.02 to 10 mg/mL was mixed with 3.9 mL of the diluted solution of ABTS .+ . After 20 min incubation at 30 °C, the absorbance was measured at 734 nm. Ultrapure water was used as blank sample [START_REF] Tang | Food Hydrocolloids Characterization of tea catechins-loaded nanoparticles prepared from chitosan and an edible polypeptide[END_REF], and ascorbic acid as positive control. The ABTS radical scavenging activity was calculated from the following equation:

Scavenging rate (%) = [A0 -(Ae -Aie)]/A0 × 100 (Eq. 4)

Where A0, Aie and Ae are the absorbance of the blank sample, the positive control, and the samples, respectively.

Total antioxidant capacity (TCA)

100 μL of samples at concentrations ranging from 0.025 to 10 mg/mL were mixed in a tube with 1 mL of reagent (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The mixture was incubated at 95°C for 90 min. A phosphomolybdenum complex was formed after cooling down to room temperature. Absorbance of the solution was measured at 820 nm. Ultrapure water was used as control [START_REF] Mohamed | Antioxidant capacity of chewing stick miswak Salvadora persica[END_REF]. The antioxidant activity is presented in terms of absorption.

Metal-chelating power

FeCl2 (2 mM), and vigorously stirred for 5 min. Then 100 μL of Ferrozine (5 mM) were added together with 2.75 mL of ultrapure water. After 10 min at room temperature, the absorbance at 562 nm was measured.

The blank sample was prepared without addition of ferrozine, while the negative control was prepared without addition of test sample. Ethylenediaminetetraacetic acid (EDTA) was used as positive control [START_REF] Sutharson | Free radical scavenging activity of leaves of BISCHOFIA JAVANICA BLUME AND FRAXINUS FLORIBUNDA WALLICH[END_REF].

The antioxidant activity was calculated from the following equation:

Antioxidant activity % =[(Acontrol-Asample)/ Acontrol] x 100 (Eq. 5)

Where Acontrol and Asample are the absorbance of the negative control and the sample, respectively.

β-carotene bleaching test

The inhibition of β-carotene bleaching was evaluated according to the method described by Koleva et al. [START_REF] Koleva | Screening of Plant Extracts for Antioxidant Activity : a Comparative Study on Three Testing Methods[END_REF]. 0.5 mg of β-carotene was dissolved in a mixture of 1 mL of chloroform, 25 μL of linoleic acid and 200 μL of Tween-80. The solvent was evaporated in a rotary evaporator at 45°C. 100 mL of ultrapure water was then added to the suspension under stirring. 2.5 mL of freshly prepared suspension were transferred to a tube containing 0.5 mL of test samples at concentrations from 0.05 to 5 mg/mL. The mixture is incubated for 2 h at 50 °C after homogenization. Ultrapure water was used as control. Absorbance was then measured at a wavelength of 470 nm. The antioxidant activity was calculated from the following equation:

Antioxidant activity (%) = [1-(A0-A120)test / (A0-A120)control] x 100 (Eq. 6)
Where A0 and A120 are the absorbance of the sample or the control before and after 120 min incubation.

Statistical analysis

The data processing is based on the statistical analysis performed by using the ANOVA Test (Analysis of Variance). A value of p <0.05 is considered statistically significant. The data are expressed as mean ± standard deviation (SD) for three replicates. The lack-of-fit test, F value, determination of coefficient (R 2 ), adjusted determination coefficient (Radj 2 ), coefficient of variation (C.V.%) calculated from Design Expert were used to evaluate the adequacy of the models.

Results and discussion

The challenge of this work is to find a compromise between the positive and negative effects of US extraction and temperature on the extraction and polysaccharides yields, in order to maximize the mass transfer without degrading the quality of the product of interest.

Optimization of the extraction

Single-factor experiments

Single-factor experiments were first performed to determine the effect of the 4 parameters, i.e. extraction time, temperature, volume to mass ratio, and US %, on both of the total extraction yield and polysaccharides yield. The results are presented in Fig. S1 (Supporting information). The same trend is observed in all cases, i.e. an increase of the yields to reach a maximum followed by a decrease.

Based on the results of single factor experiments, it appears that both the extraction and polysaccharides yields are largely dependent on the extraction conditions, and the 4 parameters are inter-dependent. Opposite effects of the parameters are clearly evidenced. In fact, hydrolysis of polysaccharides occurs under harsh conditions. It is thus essential to understand the effect of each parameter on the extraction and polysaccharides yields and the relationship between the parameters by applying the RSM, an efficient statistical technique for optimizing complex processes. The following intervals are selected for the optimization study: time from 22.5 to 47.5 min, temperature from 35 to 55°C, volume to mass ratio from 20 to 40 mL/g, and %US from 25 to 75%.

RSM statistical analysis and models validity

Based on the results obtained from single factor analysis, optimization was carried out by using RSM to study the effect of four independent variables [X1: time, X2: temperature, X3: volume (ratio of volume to mass), X4: %US]. The preliminary results pave the way to determine the interactions between parameters and their influence on the investigated responses. Table 2 shows the matrix of variables in real values and the obtained responses. The predicted response Y can be correlated to the variables by applying multiple regressions analysis method. shown in Table 3. 286

The ANOVA results show that both quadratic models developed for Y1 and Y2 are highly 287 significant with F values (Fisher coefficient) of 25.1 and 121.3, respectively. Considering also 288 the p-values well below 0.05 (P<0.0001) and the negligible pure errors towards the quadratic 289 model, the lack of fit of these models is insignificant. The correlation coefficient R 2 , adjusted R 2 290 and predicted R 2 are 0.950, 0.912, and 0.780 for the extraction yield, and are 0.989, 0.981, and 291 0.959 for the polysaccharides yield, respectively. These values are reasonably close to 1, showing a high degree of correlation. On the other hand, the Adeq. Precision is used to measure the signal to noise ratio, and a ratio above 4 is considered as acceptable. The obtained Adeq Precision is 15.5 and 32.3 for the extraction and polysaccharides yields, respectively, indicating a very high degree of precision and a good reliability of the experimental data. Further analysis of the statistical indicators in Table 3 also shows that the model developed for the polysaccharide yield presents slightly better precision than the one for the extraction yield. The coefficients associated to the parameters or the interactions give a precise idea of how they affect the corresponding responses (positively or negatively). Firstly, it appears that the effect of the parameters and interactions follow the same trend for both Y1 and Y2. Regarding the coefficients, the ratio of volume to mass (X3) is the most important factor affecting the extraction process, followed by the US % (X4) and the extraction time (X1). The extraction temperature (X2) is the less significant parameter. It is well known that temperature generally enhances the polysaccharide solubility and mass transfer, but it is also a destructive factor as the US treatment. Raza et al. reported similar adverse effect of temperature [START_REF] Raza | Optimization of ultrasonic-assisted extraction of antioxidant polysaccharides from the stem of Trapa quadrispinosa using response surface methodology[END_REF]. Thus, opposite effects could happen, minimizing the overall effect on the response. Regarding the interactions, the most important one is referred to the synergistic (positive) effect between the treatment time and the volume to mass ratio (X1X3) which enhances the polysaccharide extraction, in agreement with literature [START_REF] Raza | Optimization of ultrasonic-assisted extraction of antioxidant polysaccharides from the stem of Trapa quadrispinosa using response surface methodology[END_REF]. Indeed, both factors (X1 and X3) are nondestructive parameters. When the ratio of volume to mass is higher, the overall driving force is higher, thus accentuating the effect of the extraction time.

It is also noticed that US has systematically a negative effect when combined with another factor. This means that US has a positive effect under mild extraction conditions, i.e. all other parameters at low level. But as soon as the extraction conditions become harsher, the destructive effect becomes predominant. This phenomenon is more accentuated with the treatment time (X1X4), which makes sense as US (percentage of X1) is directly dependent on the treatment time.

Response surface analysis and optimization

The two responses Y1 and Y2 follow the same trend as indicated above. Therefore, the same effects and interactions of the parameters are expected for both responses. Only the polysaccharides yield was investigated by using the Design-Expert to construct a threedimensional surface according to Eq. 8. The RSM was used to determine the relationship between extraction parameters and responses, as shown in Fig. 2. Two variables are continuously varied while keeping the two others constant.

Fig. 2A presents the effect of interaction between volume to mass ratio and extraction time on

the polysaccharides yield, while the extraction temperature is fixed at 35°C and US% at 25%.

The highest polysaccharides yield of 78.2% is observed at 40 min and 36 mL/g. In fact, the increase of volume to mass ratio results in increase of the polysaccharides yield. But in the meantime, the extraction time required to obtain the maximum of polysaccharides increases due to the dilution effect [START_REF] Jean | Extraction solide-liquide[END_REF]. Fig. 2C presents the effect of volume to mass ratio and US% on polysaccharide yield at fixed time (47.5 min) and temperature (55°C). Under these harsh conditions, the US treatment above 42% leads to a decrease by 8.2% of the maximum of polysaccharides yield.

Fig. 2D presents the effect of time and US% on polysaccharides yield while keeping the volume to mass ratio at 30 mL/g and the temperature at 45°C (level 0). A remarkable increase of the polysaccharides yield is detected with extraction time. However, an extraction time longer than 37.1 min leads to decrease of the polysaccharides yield. In fact, prolonged extraction time could lead to cleavage of polysaccharide chains, resulting in decrease of the polysaccharides yield [START_REF] Chen | Optimization of ultrasonic extraction process of polysaccharides from Ornithogalum Caudatum Ait and evaluation of its biological activities[END_REF]. The polysaccharides yield increases with the increase of US% up to a maximum at 51.7%.

Beyond, a decrease of the polysaccharides yield is observed. Additionally, the extraction time required to obtain the maximum of polysaccharides yield becomes shorter with increase of US%. These results evidence the effect of the main parameters and their interactions, which allows to better understand the extraction process. In particular, it is clearly shown that the US is definitely promoter of extraction because of improved mass transfer. But US also has a destructive effect in combination with other parameters such as high extraction temperature. temperature and US% at (-1, -1), (C) volume to mass ratio and US % at (+1, +1), and (D) time and US % at (0, 0).

Validation of predictive models

The optimal extraction conditions to obtain the highest extraction and polysaccharides yields are given below: time (38.5, 37.1 min), temperature (43.5, 44.0°C), volume to mass ratio (35.4, 33.8 ml/g), and US% (52.8, 51.7%). Under these conditions, the theoretical extraction and polysaccharides yields are 85.9% and 82.7% respectively.

Extraction was repeated three times under the optimal conditions in order to validate the two models for the extraction and polysaccharides yields. The obtained extraction yield is 85.7 ± 0.2 %, and the polysaccharides yield is 82.8 ± 0.1%. These values are very close to the theoretical ones, thus validating the models for evaluation of the effects of the 4 parameters on the extraction and polysaccharides yields.

In order to evidence the ambivalent effects of the US treatment, additional experiments were conducted at either 0% US (only maceration) or 100% US. The experimental results are then compared with the theoretical values obtained from the developed models, as shown in Table 4. For the extraction yield, the first test was performed at 0% US under the predicted optimal conditions (time: 47.49 min; temperature: 47.84°C; volume to mass ratio: 40 mL/g), giving a maximum predicted extraction yield of 81.7%. The average extraction yield of three repeated experiments at 0% US was 81.7% ± 0.1%. The second test was performed at 100% US, which

D C B A
allowed to obtain a maximum predicted extraction yield of 82.7% under the optimal conditions (time: 30.03 min; temperature: 39.62°C; volume to mass ratio: 30.02 mL/g). The average extraction yield of three repeated experiments at 100% US was 82.5% ± 0.1%.

In the case of the polysaccharides yield, at 0% US the maximum predicted polysaccharides yield was 74.8% under the optimum conditions (time: 42.12 min; temperature: 47.1°C; volume to mass ratio: 37.2 mL/g). The average yield of three repeated experiments was 74.7 ± 0.1%. At 100% US, the maximum predicted polysaccharides yield was 75.8% under the optimum conditions (time: 32.41 min; temperature: 41.2°C; volume to mass ratio: 30.8 ml/g). The average polysaccharides yield of three repeated experiments was 75.7 % ± 0.1%.

The model was then used to achieve the yield maximum under the mildest conditions. A theoretical extraction yield of 83.0% was predicted with the following conditions: 23.7 min, 39°C, 22.6 mL/g and 69.5 % US. The experiences gave an average yield of 83.0 ± 0.1%, which is nearly the same as the predicted value. This technique leads to significant decrease in time by 6.3 min, temperature by 0.7°C and volume to mass ratio by 7.4 mL/g, while a small increase in extraction yield by 0.4% is obtained as compared to 100% US extraction (Table 4). Similarly, an optimal polysaccharide yield of 76.3% is predicted under the following conditions: 23.1 min, 35°C, 21.3 mL/g and 65.3% US. Extraction performed three times under these conditions gave a polysaccharide yield of 76.1% ± 0.1%. Comparison between the combined system and extraction with 100% US indicates that the former allows a small increase in yield by 0.4%, while decreasing significantly the time, temperature, and volume to mass ratio by 9.3 min, 6.2°C, and 9.5 mL/g, respectively (Table 4).

It is also of interest to compare the purity of the extract which is defined as the ratio of the polysaccharides yield to the total extraction yield. Under the optimum conditions of maceration, 100% US, and combined system, the purity of polysaccharides was 91.4 ± 0.1 %, 91.8 ± 0.2 %, and 96.6% ± 0.1%, respectively. Thus, the combination of maceration and US allows improving the purity of the extract. The extract with optimal purity of polysaccharides, namely OP%US, was obtained under the following conditions: time 37.1 min, temperature 44.2°C, volume to mass ratio 33.8 mL/g, and US% 51.7%. OP%US was used for further analyses.

Structural characterization

FT-IR analysis

The FT-IR spectrum of OP%US is presented in Fig. S2 (Supporting information). The large band at 3405 cm -1 is assigned to the stretching vibration of O-H groups, and the band at 2941 and 2896 cm -1 were assigned to the stretching of -CH3 and -CH2 groups respectively. The band in the range of 1420 cm -1 belongs to the deformation vibration of C-H bond. The monosaccharide of OP%US has pyranose rings as evidenced by two strong absorption bands of C-O-C asymmetric stretching at 1132 cm -1 and 1027 cm -1 . The band at 931.2 cm -1 s attributed to the symmetric stretching of furan rings. And the band detected at 599 cm -1 is assigned to the presence of the skeletal -CH2 of pyranose rings. An α-configuration in the polysaccharide is confirmed by the presence of characteristic absorptions at 818 cm -1 .Therefore, OP%US was an α-configuration polysaccharide and consisted of pyranoside and funanside rings [START_REF] Chen | Chemical characterization and immunostimulatory effects of a polysaccharide from Polygoni Multiflori Radix Praeparata in cyclophosphamide-induced anemic mice[END_REF][START_REF] Wang | Structure and antioxidant activity of polysaccharide POJ-U1a extracted by ultrasound from Ophiopogon japonicus[END_REF].

Size-exclusion chromatography analysis

From the results obtained in the RSM experiments, it appears that chain cleavage of polysaccharides occurred to some extent during extraction. GPC analysis was then performed for the corners of the design expert model in order to figure out the effect of extraction parameters on the molar masses of the extracts, as summarized in Table 5. The effect of extraction time is evidenced from the comparison between runs 17 and 18 since different extraction times were used while keeping the three other parameters constant. Similarly, the effect of extraction temperature and US % is evidenced from the comparison between runs 19 and 20, and runs 23 and 24, respectively.

In runs 17 and 18, the average molar mass (Mw) decreases from 5190 to 2940 Da when the extraction time is raised from 10 to 60 min at fixed temperature (45°C), volume to mass ratio (30 mL/g), and US% (50%). In the meantime, the dispersity (Ð = Mw/Mn) slightly increases from 1.41 to 1.53. The negative effect of ultrasonic time on the molar mass of schizophyllan has been also reported by Zhong et al. [START_REF] Zhong | Molecular weight degradation and rheological properties of schizophyllan under ultrasonic treatment[END_REF].

In runs 19 and 20, with the extraction temperature increasing from 25 to 65°C, the Mw of the extract slightly decreases from 3180 to 2830 Da, whereas the dispersity remains almost unchanged.

Finally, in runs 23 and 24, with increase of US treatment from 0% to 100%, the Mw slightly decreases from 3350 to 3030 Da, and the dispersity remains almost unchanged. These results

suggest that long extraction time, high extraction temperature, and ultrasound treatment provoke chain cleavage and molar mass decrease of the extract, which could affect the extract yield as shown in the RSM experiments.

NMR Spectroscopy

Various NMR analyses, including 1 H , 13 C, HSQC, HMBC, and COSY NMR were performed to determine the composition of the extract. Fig. 3B presents the 1 H NMR spectrum of the extract. Small signals are detected around 2.4, 2.5 and 2.7 ppm, which could be assigned to the presence of amino acids, carboxylic acids, alcohols or phenols, in agreement with literature [START_REF] Ailiesei | NMR detected metabolites in complex natural fluids. Quinic acid in apple juice[END_REF]. The signals at 3.8-3.7, 4.27, 4.13, 3.89, and 3.86-3.68 ppm belong to the protons of fructose ring from H1 to H6, whereas the signals at 3.50, 3.59, and 3.98 ppm are attributed to the protons H4, H2 and H5 of glucose, respectively. The signal at 5.45 ppm indicates the presence of a proton on α of anomeric carbon. This proton belongs to glucose named as residue A as fructo-ketose has no anomeric proton, which is in agreement with FTIR results [START_REF] Liu | )]3-(2 ↔ 1)-α-d-Glcp (nystose) and [β-d-Fruf-(2 → 1)]4-(2 ↔ 1-α-d-Glcp (1,1,1-kestopentaose) from two-dimensional NMR spectral measurements[END_REF]. The chemical shifts for H1, H3, H4 of residues B, C, D, and E of fructose presented in Table 6 were determined from 1 H, HSQC, 1 H-1 H COSY spectra, and the assignment of H5 and H6 realized from the 1 H-1 H COSY and HMBC spectra.

The HQSC spectrum fig. 3C [START_REF] Zhang | Antioxidant and Immunomodulatory Activities and Structural Characterization of Polysaccharides Isolated from Lobelia chinensis Lour[END_REF].

The sequence among residues and the linkage sites were achieved using the HMBC spectrum which provides signals correlations between protons and carbons (Fig. 3D). The various signals Mo (VI) is able to combine with proteins at the metal-binding site and to cause DNA and protein damage [START_REF] Pratt | Transformation of Glucocorticoid and Progesterone Receptors to the DNA-Binding State[END_REF], based on the reduction of Mo (VI) to Mo (V) by the formation phosphate/Mo (V) complex at acidic pH. Fig. 4B presents absorbance changes of OP%US as a function of concentration compared to BHA and -tocopherol, two commonly used food additives. High absorbance indicates high antioxidant activity. When the concentration increases from 0.025 to 5 mg/mL, the absorption increases from 0.14 to 2.04, from 0.02 to 0.9 and from 0.01 to 1.4

for -tocopherol, BHA and OP%US respectively. It is noticed that the absorption of OP%US becomes higher than that of BHA beyond 4 mg/mL. These results suggest that OP%US presents a remarkable TCA capacity.

Metal-chelating power

The chelating agents are reported as secondary antioxidants because they can reduce the redox potential, thereby stabilizing the oxidized form of the metal ion [START_REF] Pavithra | Evaluation of free radical scavenging activity of various extracts of leaves from Kedrostis foetidissima ( Jacq .)[END_REF]. The ferrous chelating capacity was evaluated from absorption measurement at 562 nm. Fig. 4C presents the chelating capacity changes of OP%US as a function of concentration, using EDTA as a reference. The chelating activity of OP%US gradually increases with increasing concentration, reaching a maximum of 79.7 % at 10 mg/mL. The IC50 of OP%US is 3.7 mg/mL. In contrast, the chelating activity of EDTA rapidly increases with concentration to reach 100% at 0.4 mg/mL. Data show that the maximum activity is 36% and 10% for highly or less sulfated polysaccharides at 2 mg/mL, respectively [START_REF] Qi | Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa ( Chlorophyta ) in vitro[END_REF]. Comparison with literature data indicates that OP%US exhibits an acceptable Fe 2+ chelating activity.

β-carotene bleaching test

Linoleic acid generates peroxide radicals that will oxidize highly unsaturated β-carotene, known as provitamin A which turns from red color to transparency. Antioxidant compounds are able to neutralize free radicals, and thus preventing the oxidation and bleaching of β-carotene [START_REF] Unten | Antidiscoloring Activity of Green Tea Polyphenols on -Carotene[END_REF].

This test is of major importance for human safety as it allows to determine the ability of samples to neutralize lipophilic free radicals that can easily enter human cells causing serious damage to DNA [START_REF] Amandine | Plantes médicinales et antioxydants[END_REF]. Two other samples, namely OP M (only maceration) and OP 100%US (only ultrasound), were prepared under the same conditions as OP%US but with or without ultrasound. polysaccharides from Russula senecis Elicits at 0.5mg/ml, and an IC50 value of 0.49 mg/mL [START_REF] Khatua | Water Soluble Antioxidative Crude Polysaccharide From Russula senecis Elicits TLR Modulated NF-κ B Signaling Pathway and Pro-inflammatory Response in Murine Macrophages[END_REF].

In contrast, a value of 13.0 % and 19.3% was obtained by Rukiye et al. for polysaccharides from tragacanth gum at 20 mg/mL and from locust bean gum at 10 mg/mL, respectively [START_REF] Boran | Investigation of Hyaluronidase, Collagenase and Elastase Inhibitory Potentials and Comparative Evaluation of the Antimicrobial, Antioxidant and Homeostatic Activities of Two Natural Polysaccharides[END_REF]. And Li et al. reported an IC50 of 0.14 mg/mL for polysaccharides from Lycium barbarum fruits [START_REF] Li | POLYMER Evaluation of antioxidant activity of the polysaccharides extracted from Lycium barbarum fruits in vitro[END_REF].

Interestingly, in comparison with the literature, the extract obtained in the present work displays a more efficient antioxidant activity against lipophilic radicals compared to hydrophilic radicals, suggesting that it could have an anti-cancer activity [START_REF] Amandine | Plantes médicinales et antioxydants[END_REF].

Conclusions

Optimization an efficient process to extract polysaccharides from Ornithogalum billardieri with remarkable anti-oxidant properties. Additionally, the combined system enhances the anti-lipophilic radical activity of the extract. These natural compounds could be promising as alternative to synthetic antioxidants like BHA for applications in agro-food and pharmaceutical industries.

  ANOVA tests are performed to evidence the significance of the studied parameters on the responses. The insignificant terms are removed in order to refine the developed models. As shown in Fig. 1, the experimental data are scattered around the theoretical line (Predicted vs Actual values), which indicates that the results obtained from the model well agree with the experimental data.

Fig. 1

 1 Fig. 1 Diagnostic plot (predicted vs actual values) for the adequacy of proposed model for extraction yield (A) and polysaccharides yield (B). The simplified equations for the extraction yield Y1 and polysaccharides yield Y2 are presented in terms of coded factor as follows:

  Fig. 2B presents the effect of temperature and US % while keeping the time fixed at 22.5 min and volume to mass ratio at 20 mL/g. At low temperature (35°C), the polysaccharides yield increases from 70.1 to 75.3% with US% increasing from 25 to 75. It appears that there is little destructive effect of US under mild extraction conditions.

Fig. 2

 2 Fig. 2 3D response surface plots showing the interactive effects of (A) time and volume to mass ratio at (-1, -1), (B)

Chen

  et al. reported an optimized polysaccharide yield of 36.8 ± 1.8% for extraction from Ornithogalum Caudatum Ait under the following conditions: frequency 40 kHz, temperature 60 °C, extraction time 60 min, ultrasound power 500 W, solvent to raw material 30 mL/g, 3 times extraction [26]. The yields obtained in the present study (frequency 35 kHz, ultrasound power 120 W) are significantly higher, in agreement with successful optimization of ultrasound assisted extraction. The combined extraction process allowed to reduce the sonication time, and thus to prevent excessive polysaccharide degradation.

The 13 C

 13 NMR in Fig. 3A presents signals in the region of 59.0-61.48, 73.3-75.15, 57.3-77.77, 79.05-82.5, and 103.0-104.2 related to the C1, C6, C4, C3, C5, and C2 of fructose rings respectively. Additionally, four anomeric C2 carbon signals are detected (103.11, 103.76, 103.90, and 103.95 ppm), which indicates the presence of four fructose residues, i.e. β-d-Fruf-(2→, →1,6)-β-d-Fruf-(2→ , →1)β-d-Fruf-(2→, and→ 2)β-d-Fruf-(6→respectively respectively designated as residues B, C, D, and E.

Fig. 3 1

 3 Fig. 3 1 H (A), 13 C (B), HSQC (C), and HMBC(D) NMR spectra of OP%US, sample obtained with optimal purity of polysaccharides.

Fig. 4D presents

  Fig.4Dpresents the β-carotene bleaching data of the three samples as a function of concentration in comparison with BHA as reference. The results show that the antioxidant activity of OP M, OP%US and OP 100%US are enhanced with increasing concentration from 0.05 to 5 mg/mL. A maximum capacity of 62.5% ,98.1%, and 82.8% are obtained at 5 mg/mL for OP M, OP%US and OP 100%US, respectively. BHA exhibits slightly higher activity to βcarotene bleaching than OP%US in the 0.05 to 0.5 mg/mL concentration range. No significant difference is noticed between OP%US and BHA in the range from 1.0 to 5.0 mg/mL. The IC50 of OP M, OP%US and OP 100%US are 0.82 ± 0.05, 0.131 ± 0.03 and 0.29 ± 0.03 mg/mL, respectively. Chen et al.,Guo et al. and Shang et al. reported that ultrasound treatment enhances the antioxidant activity [18][35]]. Tang et al. observed that the antioxidant activity of polysaccharide from Cyclocarya paliurus slightly increases with US treatment from 55.5% and

  of polysaccharides extraction was carried out for the first time via combination of two extraction methods, i.e. maceration and ultrasound treatment by using the Surface Response Methodology. Four influencing parameters were considered, namely total extraction time, extraction temperature, ratio of water volume to raw material mass, and time percentage of US treatment in the extraction process. The interaction between parameters and their effects on the extraction yield and polysaccharides yield were investigated. The models to predict the optimal conditions have been validated by additional experiments. The combination of both extraction methods improves the extraction and polysaccharides yields. The combined system allows to significantly reduce the time of extraction, the temperature and volume to mass ratio, and to improve the purity which is of major importance for potential applications. Finally, the optimum extraction process consists of a maximum duration of 20 min maceration and 20 min US treatment at relatively low temperature (ca. 44°C) using only water as solvent. The extract is identified as fructo-polysaccharides the RMN analysis indicate that the OP%US possessed a backbone of (2→6)-linked β-d-fructofuranosyl (Fruf), with (2→1)-linked β -d-Fruf branched chains, and terminated with glucose and fructose residues. It presents good antioxidant activities as evidenced by ABTS radical scavenging activity, reducing activity of molybdate, metal-chelating power, and β-carotene bleaching tests. The RSM allowed designing

Table 1

 1 Independent variables and their levels used in the response surface design

	Coded value	α = -2	-1	0	+1	α = +2
		Real Value
	Time (min)	10	22.5	35	47.5	60
	Temperature (°C)	25	35	45	55	65
	Volume to mass ratio (mL/g)	10	20	30	40	50
	% US (%)	0	25	50	75	100

Table 2

 2 the Design Expert software (Version 11) used in this study. 275The extraction and polysaccharides yields obtained in the 29 experiments vary from 76.2 to 276 86.1% and from 70.1 to 82.9%, respectively. Such relatively high values in narrow ranges could 277 be explained by the fact that large amounts of polysaccharides can be easily extracted from the 278 RSM centrale composite design and results for extraction yield (Y1) and polysaccharides yield (Y2).

	279	superficial layer of the particles. However, harsher extraction conditions are required to extract
	280	species located well inside the particles.
	281
	282
		RUN	X1	X2	X3	X4	Y1	Y2
		1	22.5 35 20	25 76.2 70.1
		2	47.5 35 20	25 77.2 71.0
		3	22.5 55 20	25 76.5 71.6
		4	47.5 55 20	25 76.9 72.2
		5	22.5 35 40	25 78.7 72.2
		6	47.5 35 40	25 83.5 77.4
		7	22.5 55 40	25 79.4 72.8
		8	47.5 55 40	25 83.0 77.3
		9	22.5 35 20	75 81.9 75.2
		10	47.5 35 20	75 78.1 72.3
		11	22.5 55 20	75 80.0 73.4
		12	47.5 55 20	75 76.6 71.0
		13	22.5 35 40	75 82.7 75.0
		14	47.5 35 40	75 83.3 76.5
		15	22.5 55 40	75 78.8 72.8
		16	47.5 55 40	75 80.7 74.3
		17	10	45 30	50 76.7 71.1
		18	60	45 30	50 80.1 72.9
		19	35	25 30	50 78.7 74.3
		20	35	65 30	50 80.2 73.1
		21	35	45 10	50 76.7 70.1
		22	35	45 50	50 80.8 77.3
		23	35	45 30	0	78.5 72.5
		24	35	45 30 100 82.0 76.1
		25	35	45 30	50 85.3 82.6
		26	35	45 30	50 86.1 82.9
		27	35	45 30	50 85.8 81.7
		28	35	45 30	50 84.6 82.9
		29	35	45 30	50 85.2 82.0
	283
	284	In order to highlight the significance of models, statistical indicators such Analysis of Variance
	285	(ANOVA) and correlation coefficients (R 2 , adjusted R 2 and predicted R 2 ) are determined, as

The fitting of response functions Y1 (extraction yield) and Y2 (polysaccharides yield) with the generated by

Table 3

 3 Analysis of variance (ANOVA) for Response Surface Quadratic Model of extraction and polysaccharides yields

				Extraction yield			Polysaccharides yield
	Source	Sum of Squares	df	Mean Square	F Value	p-value	Sum of Squares	df	Mean Square	F Value	p-value
	Model	263.55 12	21.96	25.13	< 0.0001	448.3	12	37.36	121.3	< 0.0001
	Residual	13.99	16	0.87	-	-	4.93	16	0.31	-	-
	Lack of Fit	12.65	12	1.05	3.15	0.1388	3.72	12	0.31	1.02	0.5444
	Pure Error	1.34	4	0.33	-	-	1.21	4	0.3	-	-
	C.V.%	1.16					0.74
	3.1.3 Regression equations and significant terms analysis

Table 4

 4 Comparison of parameters, predicted and experimental values for optimization of extraction yield (Y1) and

	polysaccharides yield (Y2) by mixed system, maceration and US treatment
	X1	X2	X3	X4	Predicted	Experimental
	(min)	(°C)	(mL/g)	(%)	(%)	(%)
				Y1
	38.5	43.5	35.3	52.8	85.9	85.7 ± 0.2%
	47.5	47.8	40	0	81.7	81.7 ± 0.1%
	30.0	39.6	30.0	100	82.6	82.5 ± 0.1%.
	23.7	38.9	22.6	69.5	83.0	83.0 ± 0.1%.
				Y2
	37.1	44.2	33.8	51.7	82.7	82.8 ±0.1%
	42.1	47.1	37.1	0	74.8	74.7 ± 0.1%
	32.4	41.2	30.8	100	75.8	75.7 ± 0.1%
	23.1	35	21.3	65.3	76.3	76.1 ± 0.1%

Table 5

 5 Molar mass (Mw) and dispersity (Ð) of the extract obtained in selected runs

	Run Time (min)	Temperature (°C)	% US (%)	Ratio (mL/g)	Mw (Da)	Ð
	17	10	45	50	30	5 190	1.41
	18	60	45	50	30	2 940	1.53
	19	35	25	50	30	3 180	1.49
	20	35	65	50	30	2 830	1.52
	23	35	45	0	30	3 350	1.50
	24	35	45	100	30	3 030	1.52

  of the extract presents typical signals at 81.08/3.9 and 80.28/3.98 ppm assigned to the C5 of →2) β-d-Fru-(1→ and →2) β-d-Fru-(6→, respectively [24]. The signals of H1, H3, H4, H5 and H6 of fructose are related to the carbon signals at 60.46, 76.62, 74.46, 81.08, and 63.37 ppm, respectively. Meanwhile, the signals at 3.51, 3.59, 3.79, 3.98, 3.9-3.7, and 5.45 ppm are linked to C4 69.32, C2 71.18, C3 72.52, C5 71.67, C6 61.9, and C1 of anomeric carbon 92.15 ppm for glucopyranose ring, respectively

Table 6 1

 6 H and13 C NMR chemical shifts of OP%US Fig.4Apresents the ABTS radical scavenging activity changes of OP%US as a function of concentration, in comparison with ascorbic acid as a reference. The antioxidant activity of OP%US increases with concentration to reach a maximum of 96.4% at 10 mg. The half maximal inhibitory concentration (IC50) is 1.28 ± 0.13 mg/mL. In the case of ascorbic acid, a very sharp increase of the antioxidant activity is observed with concentration to reach 100% at 0.1 mg/mL.

		C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
	-d-Glcp-(1 →residue A	92.15 / 5.45 71.18/3.59	72.52/3.79	69.32/3.51	71.67/3.98	61.9/3.74
	β-d-Fruf-(2 →residue B	60.55/3.78	103. 11/-	76.57/4.28	74.42/ a 4.12 81.28/3.92	62.65/3.8
	→1,6) β-d-Fruf-(2 →residue C	57.25/3.71	103.76/-	76.98/4.28 a 74.34/4.14	80.31/3.88	62.19/3.73
	→1) β-d-Fruf-(2 →residue D	60.46/3.91	103.93/-	76.62/4.27	74.46/4.15 a 81.08/ 3.9	63.37/3.74
	→2) β-d-Fruf-(6 →residue E	60.04/3.8	103.95/-	76.85/4.16 a 74.77/4.01	80.28/ 3.98	62.31/3.89
	a Unresolved from other signals.
	3.3 Antioxidant activity
	3.3.1 ABTS radical scavenging activity
	Luo et al. reported a maximum scavenging activity of 28.0% and 82.6% for different fractions
	of polysaccharides extracted from Dendrobium nobile Lindl against ABTS .+ at 2 mg/mL [27]. Hu
	et al. studied the ABTS .+ scavenging activity of different fractions of polysaccharides extracted
	from Flammulina velutipes. The maximum activity was 36.7%, 42.4%, and 61.8% at a
	concentration of 3 mg/mL, with an IC50 value of 2.8 mg/mL [28]. Comparison with literature
	data suggests that OP%US has a good scavenging activity against ABTS .+ .
	3.3.2 Total antioxidant capacity (TCA)

  Nobre et al. investigated the chelating activity of various polysaccharides. The authors obtained a maximum chelating activity of 69.9, 57.8, 46.1, and 43.3% for polysaccharides extract from C. Prolifera, C. Sertularioides, D. Cervicornis, and D. Mertensis at 2 mg/ml, respectively [31]. Qi et al. determined the chelating activity of sulfated polysaccharides extracted from Ulva pertusa.

Acknowledgements M. K. Medlej benefits of Baalbeck's municipality fellowship. The work was financially supported by Platform for Research and Analysis in Environmental Sciences (Lebanon) and by Institut Européen des Membranes (France).

Conflicts of interest

There are no conflicts of interest to declare.


MD, PhD Daniele De Luca
email: dm.deluca@icloud.com

Shivani Shankar-Aguilera

Chiara Autilio

Roberto Raschetti

Luca Vedovelli

Catherine Fitting

Christine Payré

Louise Jeammet

Jesus Perez-Gil

Paola E Cogo

Virgilio P Carnielli

Gérard Lambeau

Lhousseine Touqui

Surfactant-secreted phospholipase A2 interplay and respiratory outcome in preterm neonates

Keywords: 14.3 Neonatal Lung Disease & BPD phospholipase A2, surfactant, RDS, prematurity, lung injury, newborn infant

Secreted phospholipase A2 hydrolyses surfactant phospholipids and is crucial for the inflammatory cascade; preterm neonates are treated with exogenous surfactant but the interaction between surfactant and phospholipase is unknown. We hypothesize that this interplay is complex and the enzyme plays a relevant role in neonates needing surfactant replacement. We aimed to: 1) identify phospholipases A2 isoforms expressed in preterm lung; 2) study the enzyme role on surfactant re-treatment and function and the effect of exogenous surfactant on the enzyme system;

3) verify if phospholipase A2 is linked to respiratory outcomes. In bronchoalveolar lavages of preterm neonates, we measured enzyme activity (alone or with inhibitors), enzyme subtypes, surfactant protein-A and inflammatory mediators. Surfactant function and phospholipid profile were also tested. Urea ratio was used to obtain epithelial lining fluid concentrations. Follow-up data were prospectively collected. Subtype-IIA is the main phospholipase isoform in preterm lung, although subtype-IB may be significantly expressed. Neonates needing surfactant retreatment have higher enzyme activity (p=0.021) and inflammatory mediators (p always0.001), and lower amounts of phospholipids (p always<0.05). Enzyme activity was inversely correlated to surfactant adsorption (=-0.6; p=0.008; adjusted p=0.009), total phospholipids (=-0.475; p=0.05) and phosphatidylcholine (=-0.622; p=0.017). Exogenous surfactant significantly reduced global phospholipase activity (p<0.001), subtype -IIA (p=0.005), and increased dioleoylphosphatidylglycerol (p<0.001) and surfactant adsorption (p<0.001). Enzyme activity correlated with duration of ventilation (=0.679, p=0.005; adjusted p=0.04), respiratory morbidity score at 12 months postnatal age ( -b =0.349, p=0.037; adjusted p=0.043), but was not associated with mortality, bronchopulmonary dysplasia or other long-term respiratory outcomes.

INTRODUCTION

Surfactant is a cornerstone of neonatal critical care and has significantly improved clinical outcomes of preterm neonates with respiratory distress syndrome (RDS), that is, primary surfactant deficiency related to prematurity, which is treated with one or, less often, multiple surfactant doses [START_REF] Academy | Surfactant Replacement Therapy for Preterm and Term Neonates With Respiratory Distress[END_REF]. Surfactant is a complex mixture of phospholipids (90%) and proteins (10%) and its adsorption at the air/liquid interface is essential to create a surfactant layer able to lower alveolar surface tension. Despite surfactant replacement, preterm neonates may still experience negative long-term outcomes requiring a significant burden of care [START_REF] Academy | Surfactant Replacement Therapy for Preterm and Term Neonates With Respiratory Distress[END_REF].

Secreted phospholipases A2 (sPLA2; phosphatide 2-acylhydrolase, EC3.1.1.4) are the main enzymes which hydrolyze surfactant phospholipids and participate in the first step of the arachidonic acid inflammatory cascade. Different sPLA2 subtypes exist with various substrate specificity. Increased levels of sPLA2 are associated with the occurrence of acute respiratory distress syndrome (ARDS) [START_REF] Touqui | A role for phospholipase A2 in ARDS pathogenesis[END_REF] and sPLA2 correlates with lung function and clinical outcomes, both in adults and children affected by ARDS [START_REF] De Luca | Clinical and biological role of secretory phospholipase A2 in acute respiratory distress syndrome infants[END_REF][START_REF] Nakos | Phospholipases A2 and platelet-activating-factor acetylhydrolase in patients with acute respiratory distress syndrome[END_REF]. ARDS is characterized by a complex qualitative and quantitative injury of endogenous surfactant, but the exogenous surfactant may also be damaged if it is administered in an environment with increased levels of sPLA2 and other inflammatory agents [START_REF] Murakami | A new era of secreted phospholipase A2[END_REF][START_REF] Touqui | A role for phospholipase A2 in ARDS pathogenesis[END_REF].

Despite exogenous surfactant being routinely used in preterm infants, there are no data about its interactions with sPLA2 in humans. We only know that sPLA2 is expressed irrespective of gestational age and that its activity correlates with ventilatory requirements and lung compliance [START_REF] De Luca | Secretory phospholipase A2 and neonatal respiratory distress: pilot study on broncho-alveolar lavage[END_REF][START_REF] Schrama | Pulmonary Secretory Phospholipase A2 in Infants with Respiratory Distress Syndrome Stimulates in vitro Neutrophil Migration[END_REF]. Preterm neonates may sometimes need repeated surfactant administrations. Therefore, they represent the ideal setting to study the sPLA2-surfactant interactions and their possible consequences. We hypothesize that sPLA2 may play a relevant role in neonates needing surfactant replacement. Our aims were: 1) to identify sPLA2 subtypes expressed in preterm neonates with RDS; 2) to study the role of sPLA2 on surfactant retreatment and function, and, conversely, the effect of exogenous surfactant on sPLA2 and its inhibitors; [START_REF] Berger | Dioleylphosphatidylglycerol inhibits the expression of type II phospholipase A2 in macrophages[END_REF] to investigate the relationship between sPLA2 and respiratory outcomes. Some partial results of this study have been previously presented as abstracts at the 58 th annual congress of the European Society for Pediatric Research (Venice, Italy -Oct 30, Nov 3 2017).

MATERIALS AND METHODS

Design and hypothesis

This was a prospective, translational, pragmatic, international, long-term, cohort study, which is part of a larger project investigating the role of sPLA2 in various pediatric respiratory disorders [START_REF] Sspp | Secretory phospholipase A2 pathway in various types of lung injury in neonates and infants: a multicentre translational study[END_REF]. STrengthening the Reporting of OBservational studies in Epidemiology guidelines were followed [START_REF] Vandenbroucke | Strengthening the Reporting of Observational Studies in Epidemiology (STROBE): explanation and elaboration[END_REF]. The study has been conducted in four laboratories collaborating with one tertiary academic neonatal intensive care unit (NICU). We hypothesize that: 1) preterm lungs may express at least the four sPLA2 isotypes previously found in adult lungs [START_REF] Lambeau | Biochemistry and Physiology of Mammalian Secreted Phospholipases A2[END_REF]; 2) sPLA2 activity affects the need for surfactant and its function and that surfactant replacement influences sPLA2 activity; 3) sPLA2 may influence respiratory outcomes. The study protocol was approved by the local institutional review board (n.13/046) and informed consent was obtained from parents upon NICU admission.

Patients

Eligible babies were inborn preterm neonates with gestational age 32 weeks needing intubation and surfactant replacement for RDS [START_REF] Sspp | Secretory phospholipase A2 pathway in various types of lung injury in neonates and infants: a multicentre translational study[END_REF]. Exclusion criteria were: 1) lung or airway malformations; 2) any need for surgery; 3) chromosomal abnormalities, genetic syndromes or complex congenital malformations; 4) septic shock, as defined earlier (18); 4) other previously defined neonatal lung disorders: a] meconium or blood aspiration syndrome [START_REF] De Luca | Clinical and biological role of secretory phospholipase A2 in acute respiratory distress syndrome infants[END_REF][START_REF] De Luca | Secretory phospholipase A2 pathway during pediatric acute respiratory distress syndrome: A preliminary study[END_REF]; b] pulmonary hemorrhage or maternal blood aspiration [START_REF] Raimondi | Point-of-care lung ultrasound in neonatology: classification into descriptive and functional applications[END_REF]; c] congenital pneumonia [START_REF] Dell'orto | Cell Count Analysis from Nonbronchoscopic Bronchoalveolar Lavage in Preterm Infants[END_REF]; d] any type of neonatal ARDS [START_REF] De Luca | The Montreux definition of neonatal ARDS: biological and clinical background behind the description of a new entity[END_REF]. Finally, babies enrolled but having bloody bronchoalveolar lavage (BAL) samples were excluded from the study.

Neonates received full prenatal care, including antenatal steroids, delayed cord clamping and MgSO 4 , when appropriate, following current guidelines [START_REF] Sweet | European Consensus Guidelines on the Management of Respiratory Distress Syndrome -2016 Update[END_REF][START_REF]Committee Opinion No 652: Magnesium Sulfate Use in Obstetrics[END_REF][START_REF]Committee Opinion No. 684: Delayed Umbilical Cord Clamping After Birth[END_REF]. Respiratory care was provided, as described earlier [START_REF] Dell'orto | Cell Count Analysis from Nonbronchoscopic Bronchoalveolar Lavage in Preterm Infants[END_REF]. In short, neonates are started on variable-flow, nasal mask-delivered continuous positive airway pressure (CPAP; 6 cmH 2 O) in the delivery room and continued during the transfer to the NICU, which is adjacent to the delivery suite. Surfactant (200 mg/kg poractant-, CUROSURF ® , Chiesi Farmaceutici, Parma, Italy) was administered in the NICU, when CPAP failed, based on inspired oxygen fraction (FiO 2 ) thresholds derived from the European guidelines [START_REF] Sweet | European Consensus Guidelines on the Management of Respiratory Distress Syndrome -2016 Update[END_REF] and using intubation-surfactant-extubation technique. However, neonates were kept ventilated if they had hemodynamic troubles needing inotropes or severe anemia (hemoglobin 8g/L) or if oxygenation was not rapidly improved by surfactant administration (4). Anyhow, invasive ventilation was shortened as much as possible and noninvasive ventilatory techniques were extensively applied following a strict protocol [START_REF] Dell'orto | Cell Count Analysis from Nonbronchoscopic Bronchoalveolar Lavage in Preterm Infants[END_REF]. A second 100 mg/kg poractant- dose was administered, if the FiO 2 was higher than the threshold FiO 2 , at least 10h after the first administration. Retreatment was not performed earlier, as the median half-life of dipalmitoylphosphatidylcholine (DPPC) is 10h in preterm neonates requiring multiple surfactant doses [START_REF] Cogo | Pharmacokinetics and clinical predictors of surfactant redosing in respiratory distress syndrome[END_REF].

Sampling

Non-bronchoscopic BAL was performed following European Respiratory Society advices [START_REF] Blic | Bronchoalveolar lavage in children. ERS Task Force on bronchoalveolar lavage in children[END_REF] and using a well standardized technique [START_REF] De Luca | Secretory phospholipase A2 and neonatal respiratory distress: pilot study on broncho-alveolar lavage[END_REF]. In short, 1 mL/kg 0.9%NaCl (37°C) was injected through a Y-piece connected to the endotracheal tube to keep the patients ventilated during the lavage. Sedation was provided as described earlier [START_REF] Dell'orto | Cell Count Analysis from Nonbronchoscopic Bronchoalveolar Lavage in Preterm Infants[END_REF]. BAL was performed before the first surfactant dose and repeated after at least 10h from the administration, only if, according to clinical conditions, the neonate still needed invasive ventilation at that time. If this was the case, BAL was performed as soon as the neonate needed airway suction for secretions removal. BAL samples were immediately divided: 0.2 mL were sent for standard microbiological cultures, as per our policy of microbiological surveillance. The remaining volume was immediately centrifuged (800g;10'). Supernatant was divided in several tubes and frozen (-80°C). Tubes were sent in dry ice to the different laboratories; because of their small volume, each sample was not used for each assay. All measurements were performed in duplicate and laboratory investigators were blinded to patients' clinical data. Due to the small volume of BAL samples, it was not possible to perform all assays and experiments with each sample. However, we used a fair mix of patients' samples for each assay, paying attention to be representative of the whole population, according to its basic characteristics.

sPLA2 activity

Total sPLA2 activity was measured as previously described [START_REF] Pernet | Staphylococcus aureus Adenosine Inhibits sPLA2-IIA-Mediated Host Killing in the Airways[END_REF]. Briefly, 30 L of BAL samples were incubated with substrate ( 3 H-OA-labeled Escherichia coli membrane phospholipids) at C in 150 L of assay buffer (0.1 M Tris [pH 8], 1 mM CaCl 2 , 0.2% sodium azide). After incubation, the released 3 H-OA-labelled free fatty acids were counted and the sPLA2 activity was expressed as % of radioactivity. This assay has an average sensitivity of 0.1 pmol (i.e.: can detect the activity of as low as 1 pg of enzyme) [START_REF] Pernet | Staphylococcus aureus Adenosine Inhibits sPLA2-IIA-Mediated Host Killing in the Airways[END_REF].

Differential sPLA2 isoforms assays

sPLA2 activity was also measured as above, but in the presence of various sPLA2 inhibitors as described earlier [START_REF] Hernandez-Anzaldo | Identification of a Novel Heart-Liver Axis: Matrix Metalloproteinase-2 Negatively Regulates Cardiac Secreted Phospholipase A 2 to Modulate Lipid Metabolism and Inflammation in the Liver[END_REF]. Characteristics of the inhibitors are described in the supplementary data (Figshare -10.6084/m9.figshare.12148686). Control recombinant human sPLA2-IB, -IIA, -V and -X were prepared [START_REF] Singer | Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2[END_REF] and time-resolved fluoroimmunoassays for these subtypes were performed [START_REF] Nevalainen | Time-resolved fluoroimmunoassays of the complete set of secreted phospholipases A2 in human serum[END_REF]. Moreover, concentrations of these subtypes were measured using specific ELISA (Biomatik, Cambridge, Canada; Cayman Chemical, Ann Arbor, USA; Cusabio Technology, Houston, USA; Cloud Clone Corp., Katy, USA, respectively) and normalized for total proteins.

Inflammatory mediators and other assays

Multiple inflammatory mediators were measured using customized Luminex assay (Thermofisher scientific, Waltham, MA, USA) [START_REF] Moncunill | Performance of Multiplex Commercial Kits to Quantify Cytokine and Chemokine Responses in Culture Supernatants from Plasmodium falciparum Stimulations[END_REF]. Surfactant protein A (SP-A) was measured with ELISA (BioVendor, Brno, Czech Republic). Total proteins and urea in BAL were measured using Lowry's and colorimetric methods, respectively [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF][START_REF] Moncunill | Performance of Multiplex Commercial Kits to Quantify Cytokine and Chemokine Responses in Culture Supernatants from Plasmodium falciparum Stimulations[END_REF]. Epithelial lining fluid concentrations were obtained by correcting for serum-to-BAL urea ratio, using urea measured on the first day of life with routine blood tests. Measurements were expressed as epithelial lining fluid concentrations, unless otherwise indicated. sPLA2 was also normalized for SP-A, as it is a main natural inhibitor [START_REF] Touqui | A role for phospholipase A2 in ARDS pathogenesis[END_REF].

Phospholipid profile

Total phosphatidylcholine was assayed with an enzymatic method [START_REF] Nanjee | Enzymatic fluorometric procedure for phospholipid quantification with an automated microtiter plate fluorometer[END_REF]. A BAL volume containing 50 g of phosphatidylcholine was dried to analyze the phospholipid profile with LC/MS, as described earlier [START_REF] Remesal | Effect of prenatal steroidal inhibition of sPLA2 in a rat model of preterm lung[END_REF] and data were normalized to specific standards per each analyzed lipid (purity=±2%; error<5ppm; Avanti Polar Lipids; Merck KGaA, Darmstadt, Germany). Lyso-phosphatidylcholine and lyso-phosphatidylethanolamine have been also

normalized for phosphatidylcholine and phosphatidylethanolamine amount. Limit of detection for the various phospholipids are reported in the supplementary methods.

Surfactant adsorption test

Samples were diluted to a phosphatidylcholine concentration of 0.043 mg/mL (corresponding to a total of 3 g of surfactant in a volume of 70 L), using 5 mM Tris buffer (pH 7.4), containing 150 mM NaCl. Surfactant adsorption was then measured as previously described and reported as relative fluorescence unit (RFU) [START_REF] Autilio | A Noninvasive Surfactant Adsorption Test Predicting the Need for Surfactant Therapy in Preterm Infants Treated with Continuous Positive Airway Pressure[END_REF].

Clinical follow up

Clinical data were recorded in real time during the NICU stay. Broncho-pulmonary dysplasia (BPD) was defined according to common definition [START_REF] Jobe | Bronchopulmonary Dysplasia[END_REF]. According to our routine clinical protocol, patients were followed with regular visits for 24 months and follow-up data were collected on a dedicated database. The clinician taking care of the follow-up was blinded to laboratory data. Data collected as long-term endpoints were: respiratory infections, use of respiratory drugs, need for re-hospitalization for respiratory disorders and home oxygen therapy.

Moreover, respiratory morbidity during the first year of life was evaluated using a respiratory morbidity score, which is known to correlate with late respiratory outcomes [START_REF] Parad | Prediction of Respiratory Outcome in Extremely Low Gestational Age Infants[END_REF]. In detail, we scored 0 if there was no cough, wheezing or need of respiratory drugs; Relationship between surfactant adsorption and sPLA2 activity has been investigated using multiple curve estimation procedure to find the best-fitting model; R 2 values were considered to assess the model goodness-of-fit and the model with the highest R 2 was chosen [START_REF] Cameron | An R-squared measure of goodness of fit for some common nonlinear regression models[END_REF].

Correlations between sPLA2, surfactant adsorption and clinical outcomes were adjusted for gestational age or birth weight using partial correlation after Log-normalization ( adj r) or using multinomial regression with backward method, as appropriate. SPSS 15.0 (SPSS, Chicago, IL, USA) was used and p<0.05 was considered to be significant. Dataset used are available to readers on reasonable request.

RESULTS

Eighty-three preterm neonates were eligible; BAL procedure was uneventful and lasted 4

(1.5)'. Five patients had bloody BAL samples and were excluded. Mean retrieved volume was 1.5 (0.1) mL. Basic data of neonates finally studied are shown in Tab.1.

sPLA2 subtypes identification

The concentration of sPLA2-IIA was the highest amongst enzyme subtypes, followed by that of sPLA2-IB while other enzyme subtypes were much less present (Fig. 1A). Time-resolved fluoroimmunoassays overall detected sPLA2-IIA and -IB in a similar amount of samples, that is, in 25 (69.4%) and 24 (66.6%) BALs, respectively; sPLA2-V was never detected and sPLA2-X was detected in 2 (5.6%) BAL samples (n=36) (data not shown). Fig. 1B depicts results of enzymatic activity assays in the absence and presence of various sPLA2 inhibitors for four illustrative patients with different subtype expression profiles: sPLA2-IIA appears to be the predominant subtype in some cases (patients n.1 and 2), sPLA2-IB also contributes to the enzyme activity (patient n.3), but other phospholipases may also be present (patient n.4).

Consistently, when incubating samples with increasing varespladib concentrations, the enzymatic activity was almost completely or only partially blocked (residual activity 10% for patients n.1-2 and 45% for patient n. was the more efficacious inhibitor (overall p<0.0001; Fig. 2).

Role of sPLA2 on surfactant retreatment and function

Neonates needing surfactant retreatment showed higher sPLA2 total activity and higher expression of sPLA2-IIA (Tab.2) in their epithelial lining fluid before receiving any surfactant treatment. Consistently, these neonates also had more lung tissue inflammation, as they showed higher inflammatory mediators (Tab.2). There were significant positive correlations between sPLA2-IIA and TNF (=0.505; p=0.027; n=19) and between sPLA2-IIA and GM-CSF (=0.416; p=0.006; n=19).

Neonates needing multiple surfactant administrations had significantly lower total phospholipids and various phospholipid species in their epithelial lining fluid (prior to any surfactant treatment) (Fig. 3). Lyso-phospholipids were only present in small amounts: lysophosphatidylcholine levels were similar in neonates receiving one or two surfactant doses (one dose: 0. There was a significant inverse correlation between sPLA2 activity and the adsorption of total surfactant available in the first day of life, that is, endogenous and exogenous pool, in babies undergoing BAL before and after surfactant replacement (= -0.6; p=0.008; n=18; Fig. 3).

This correlation remains significant upon adjustment for gestational age ( adj r= -0.61; p=0.009).

There were also significant correlations between sPLA2 activity and total phospholipids (= -0.475; p=0.05; n=17) or phosphatidylcholine (= -0.622; p=0.017; n=29) and between sPLA2-IIA and lysophosphatidylethanolamine (= 0.686; p=0.002; n=17), all measured prior to any surfactant treatment.

Effect of exogenous surfactant on sPLA2 and its natural inhibitors

In neonates undergoing BAL before and after surfactant replacement (Fig. 4), poractant- significantly reduced sPLA2 activity and sPLA2-IIA expression and significantly increased dioleoylphosphatidylglycerol (DOPG). Surfactant administration did not significantly change SP-A levels (p=0.247). sPLA2 total activity significantly correlated with the concentration of sPLA2-IIA both before (=0.89; p<0.0001; n=34) and after surfactant replacement (=0.826; p<0.0001; n=40).

Relationship between sPLA2 and respiratory outcomes

The ratio between the sPLA2 activity measured before and after surfactant replacement was considered to better describe the enzymatic activity present during the first day of life.

Enzyme activity correlated with the duration of invasive ventilation (=0.679; p=0.005; n=15) and this remained significant upon adjustment for gestational age ( adj r=0.46; p=0.04; n=15). As 12 babies died and 18 were lost to follow up (for family relocations), 48 patients were finally followed-up after NICU discharge. sPLA2 activity was not significantly different between patients followed (1.

DISCUSSION

This study addressed three open key points in human surfactant biology. We found that:

1) sPLA2-IIA is the main subtype expressed in the preterm lung and its concentration is higher than other enzyme isoforms. sPLA2-IB or other phospholipases may also be significantly expressed and contribute to the enzyme activity, while sPLA2-V and -X are marginal. However, varespladib is able to inhibit most of the enzymatic activity. These data are interesting because they have been produced in a homogeneous population of surfactant-treated human neonates affected by RDS treated with a homogeneous ventilatory policy. This represents an ideal clinical model to study sPLA2-surfactant interactions and a pragmatic picture of common NICU patients. Moreover, these findings are consistent with the data produced so far, mainly in animal models [START_REF] Murakami | A new era of secreted phospholipase A2[END_REF][START_REF] Tingay | Aeration strategy at birth influences the physiological response to surfactant in preterm lambs[END_REF], and foster future steps in research and development.

The presence of sPLA2-IIA as the main alveolar subtype [START_REF] Murakami | A new era of secreted phospholipase A2[END_REF] and the expression of other subtypes in airway cells were already known [START_REF] Seeds | Cell-Specific Expression of Group X and Group V Secretory Phospholipases A 2 in Human Lung Airway Epithelial Cells[END_REF]. Our sampling technique may also recover some material from the airways (9) and this may explain the finding of small amounts of sPLA2-V and -X, which are unlikely to play a relevant role, given their marginal concentrations. It is difficult to clarify the relative importance of sPLA2-IB, although it seems to influence the enzyme inhibition profile, at least in some cases. However, sPLA2-IIA is the main pulmonary subtype even in expectoration specimens obtained without any deep sampling in cystic fibrosis patients [START_REF] Pernet | Pseudomonas aeruginosa eradicates Staphylococcus aureus by manipulating the host immunity[END_REF]. The presence of sPLA2-IB is consistent with data showing its production by activated alveolar macrophages in adults or children with ARDS [START_REF] De Luca | Clinical and biological role of secretory phospholipase A2 in acute respiratory distress syndrome infants[END_REF][START_REF] Kim | Bronchoalveolar lavage fluid phospholipase A2 activities are increased in human adult respiratory distress syndrome[END_REF] or when lung tissue inflammation is relevant [START_REF] Kitsiouli | Phospholipase A2 subclasses in acute respiratory distress syndrome[END_REF]. In fact, some of our patients may have been exposed to fetal inflammation since it triggers preterm labor [START_REF] Gomez-Lopez | Inflammasome activation during spontaneous preterm labor with intraamniotic infection or sterile intra-amniotic inflammation[END_REF]. Fetal inflammation, if significant, is known to impair response to surfactant replacement, negatively affecting respiratory outcomes (2) and this might be mediated by the increased presence of sPLA2.

There seems to be three different inhibition profiles, as ex vivo treatment with varespladib (the sPLA2 inhibitor with largest clinical experience ( 25)), inhibits sPLA2 activity in some cases, while inhibition is less evident in others for the presence of sPLA2-IB or other phospholipases. Nonetheless, the importance of sPLA2-IIA is also confirmed by the strong correlation between this isoform and the total enzymatic activity. As varespladib is particularly able to inhibit sPLA2-IIA, -V and -X in the nanomolar range ( 14), the different inhibition profile is likely to be due to the presence of sPLA2-IB or other phospholipases, which are less sensitive to varespladib. These data confirm that inhibiting sPLA2-IIA is a potential target to protect surfactant and reduce lung tissue inflammation [START_REF] Tingay | Aeration strategy at birth influences the physiological response to surfactant in preterm lambs[END_REF]. This is consistent with our previous experiments showing ex vivo potency of varespladib in mixed populations of neonates and infants, with various respiratory disorders and higher levels of lung inflammation [START_REF] Schrama | Pulmonary Secretory Phospholipase A2 in Infants with Respiratory Distress Syndrome Stimulates in vitro Neutrophil Migration[END_REF][START_REF] Seeds | Cell-Specific Expression of Group X and Group V Secretory Phospholipases A 2 in Human Lung Airway Epithelial Cells[END_REF], where varespladib was efficient at higher doses. This may be caused by the higher protein content related to the inflammation, since the inhibitor may bind to proteins preventing it to reach the sPLA2 catalytic site, or to the different assay conditions [START_REF] De Luca | Ex Vivo Effect of Varespladib on Secretory Phospholipase A2 Alveolar Activity in Infants with ARDS[END_REF].

The need for surfactant retreatment seems to be associated to sPLA2 and lung inflammation, as retreated neonates had significantly higher enzyme activity and sPLA2-IIA and cytokines concentrations. This is consistent with the dual role of sPLA2, which regulates the first step of the inflammatory cascade and hydrolyses surfactant phospholipids [START_REF] Lambeau | Biochemistry and Physiology of Mammalian Secreted Phospholipases A2[END_REF]. Moreover, correlations between sPLA2-IIA, TNF and GM-CSF confirm our previous findings, as TNF is the main inducer of sPLA2-IIA gene transcription in alveolar macrophages [START_REF] Singer | Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2[END_REF][START_REF] Sweet | European Consensus Guidelines on the Management of Respiratory Distress Syndrome -2016 Update[END_REF].

The link between sPLA2 and need for surfactant retreatment is confirmed by the differences in lipid profile between patients receiving one or two doses, and by the correlations between sPLA2 and various phospholipids. In fact, babies needing more surfactant have less phospholipids both in terms of total amount and various species; furthermore, the higher is the sPLA2, the lower are total phospholipids and phosphatidylcholine. Interestingly, the amount of sPLA2-IIA is also correlated to lysophosphatidylethanolamine. As phosphatidylethanolamine is a preferential substrate for sPLA2-IIA [START_REF] Hite | Lysophospholipid generation and phosphatidylglycerol depletion in phospholipase A(2)mediated surfactant dysfunction[END_REF], this correlation also confirms the main role played by this subtype in the endogenous surfactant hydrolysis. Furthermore, sPLA2 activity negatively influences the efficiency of surfactant in terms of air/liquid interface adsorption, since there is an inverse relationship between surfactant adsorption and sPLA2 activity. This can be explained since sPLA2 can modify the phospholipid profile, as shown by correlations between sPLA2 and phospholipids. Our data show this negative effect on the total surfactant available on the first day of life (endogenous plus exogenous pool), as measurements were done in babies undergoing BAL before and after surfactant replacement. Thus, the relationship between sPLA2 and surfactant adsorption is evident within a relatively narrow range of values, as surfactant adsorption is overall concentrated on intermediate values with only few negative ones. These points indicate a globally poor surfactant function and they actually represent the few sickest babies who do not have a good surfactant function even after surfactant replacement (in fact, four of them out of six needed surfactant retreatment). We unfortunately did not have a population large enough to reliably distinguish the effect of sPLA2 on endogenous and exogenous surfactant. Moreover, more datapoints would be needed to better detail the decay of surfactant function with increasing sPLA2 activity.

One dose of exogenous surfactant increased the air/liquid interface adsorption and reduces both sPLA2 activity and sPLA2-IIA expression. Surfactant also increased DOPG, which was almost undetectable in our patients before surfactant replacement. The relative absence of DOPG in preterm neonates with RDS seems logical, as DOPG is only a minor component of endogenous surfactant and these patients have primary surfactant deficiency. DOPG and SP-A are important, as they are natural modulators of sPLA2 activity and expression [START_REF] Touqui | A role for phospholipase A2 in ARDS pathogenesis[END_REF]. Poractant- contains small amounts of DOPG [START_REF] Pelizzi | Different electrospray tandem mass spectrometric approaches for rapid characterization of phospholipid classes of Curosurf®, a natural pulmonary surfactant: Different ES-MS/MS approaches for characterization of phospholipids[END_REF] but it is SP-A-free, thus its administration resulted in an increase of DOPG only.

sPLA2 activity in the first day of life correlates with short-term ventilatory needs and this is consistent with a previous pilot study in a mixed population of term and preterm neonates with various respiratory disorders [START_REF] De Luca | Secretory phospholipase A2 and neonatal respiratory distress: pilot study on broncho-alveolar lavage[END_REF]. Analogous correlations with similar short-term respiratory outcomes have been found in adults and children affected by ARDS [START_REF] Brix | Predictors for an unsuccessful INtubation-SURfactant-Extubation procedure: a cohort study[END_REF][START_REF] Cogo | Pharmacokinetics and clinical predictors of surfactant redosing in respiratory distress syndrome[END_REF]. We add here the correlation between the sPLA2 activity on the first day of life and long-term respiratory outcomes in a homogeneous population of preterm babies with RDS. In fact, neonates exposed to higher sPLA2 activity early in life have a worse respiratory score at 12 th month of age. This may be related to the need for a longer and/or more aggressive ventilation, increasing the risk of secondary lung injury; the confirmation of this hypothesis would require a dedicated and larger follow-up study. These results are however interesting since the respiratory morbidity score is known to be more predictive of later respiratory performance than the diagnosis of BPD [START_REF] Parad | Prediction of Respiratory Outcome in Extremely Low Gestational Age Infants[END_REF].

It is unlikely that our results would be due to the effect of gestational age. In fact: 1) in our population, gestational age does not significantly differ between babies needing one or multiple surfactant doses; 2) correlations between sPLA2 and surfactant efficiency or respiratory outcomes remain significant upon multivariate adjustments. Consistently, we already knew that the expression of sPLA2 is unrelated to gestational age and that correlations between sPLA2 and short term respiratory outcomes were unrelated to patients' age [START_REF] Brix | Predictors for an unsuccessful INtubation-SURfactant-Extubation procedure: a cohort study[END_REF][START_REF] Dargaville | Comparison of two methods of diagnostic lung lavage in ventilated infants with lung disease[END_REF].

We acknowledge some study limitations. Ours is a pragmatic study willing to integrate clinical and biological data obtained with several techniques. Due to the low samples volume, not all techniques were applied to every sample: this is a common issue of studies on newborn BAL specimens, but these studies still represent an useful and pragmatic tool to study neonatal lung biology. Animal studies may provide larger samples volume, but these specimens are coming from controlled experimental situations that are far from the clinical reality. Given the low cellularity of our samples, we could not study other phospholipases. Some data suggest that cytosolic phospholipase might be more involved in inflammation than sPLA2 [START_REF] Murakami | A new era of secreted phospholipase A2[END_REF], but the relative contribution of different phospholipases to the inflammation during neonatal respiratory failure should be further investigated. We performed non-bronchoscopic BAL and limitations of this technique are well-known (9): however, this technique is the only one applicable to preterm neonates and these samples are more suitable to describe the alveolar biology than tracheal aspirates [START_REF] Dargaville | Comparison of two methods of diagnostic lung lavage in ventilated infants with lung disease[END_REF]. Phospholipases are not the only inflammatory mediators able to injure surfactant:

our findings may also be partially due to other agents, however, sPLA2 has a well-recognized role in surfactant injury and our findings are consistent with the literature data [START_REF] Lambeau | Biochemistry and Physiology of Mammalian Secreted Phospholipases A2[END_REF]. Quadratic regression performed better than others to describe the relationship between sPLA2 activity and surfactant adsorption, however there is no pathobiological reason to prefer quadratic over linear regression. It is physiopathologically sound to think that, at low sPLA2 activity value, surfactant adsorption tends to plateau but more datapoints are needed to clarify this. We analyzed BALs taken after a relatively short ventilation but we cannot totally exclude that the ventilation itself might have also influenced surfactant function and local inflammation, as shown in animal models [START_REF] Tingay | Aeration strategy at birth influences the physiological response to surfactant in preterm lambs[END_REF]; however, our patients are all ventilated with a homogenous gentle ventilation policy [START_REF] Dell'orto | Cell Count Analysis from Nonbronchoscopic Bronchoalveolar Lavage in Preterm Infants[END_REF]. Our population is large enough to study relationship between the sPLA2 system, surfactant efficiency and various perinatal-neonatal variables. This could not have been presented in an unique paper: we preferred a pragmatic design, analyzing a homogeneous population of preterm babies including all cofactors commonly influencing the clinical picture (such as chorioamnionitis or antenatal steroids). Future works should also investigate if perinatal cofactors may trigger the production of sPLA2-IB, as this might identify subgroups of patients with a different inhibition profile and therapeutic possibilities. The study was subjected to a certain follow-up drop out and other variables (such as nutritional or environmental factors), may have influenced the clinical outcomes. This part of results should be considered preliminary for a larger cohort long-term study. Moreover, ours is the first study investigating the relationship between sPLA2 and long-term respiratory outcomes and results are consistent with those shown for short term outcomes [START_REF] De Luca | Secretory phospholipase A2 and neonatal respiratory distress: pilot study on broncho-alveolar lavage[END_REF][START_REF] De Luca | Clinical and biological role of secretory phospholipase A2 in acute respiratory distress syndrome infants[END_REF][START_REF] Nakos | Phospholipases A2 and platelet-activating-factor acetylhydrolase in patients with acute respiratory distress syndrome[END_REF].

In conclusion, sPLA2-IIA seems to be the main sPLA2 subtype in the lung of preterm neonates with RDS and linked to the need for surfactant retreatment. Surfactant administration partially inhibits sPLA2 activity. sPLA2 activity may negatively influences surfactant adsorption, short-and long-term respiratory outcomes. sPLA2 may be a potential target to protect surfactant and increase efficacy of surfactant replacement.

3 ;

 3 (Figshare -supplementary results-110.6084/m9.figshare.12148686). Pooling down the data of all samples (n=12) treated with different sPLA2 inhibitors and analyzed under the same experimental conditions, varespladib

2 -Figshare - 10 .

 210 Figshare -10.6084/m9.figshare.12148686).

2 )

 2 Preterm neonates needing repeated surfactant administrations have significantly higher sPLA2 activity, sPLA2-IIA expression and inflammation. Neonates needing re-treatment have a different phospholipid profile which correlates to sPLA2 activity, while the enzymatic activity negatively influences surfactant adsorption. The administration of one poractant- dose reduces sPLA2 activity and sPLA2-IIA expression through, at least partially, the increment of DOPG. 3) sPLA2 activity in the first day of life correlates with short-term ventilatory needs and with long-term respiratory morbidity score.

Figure 1 .Figure 2 .

 12 Identification of sPLA2 subtypes responsible for enzyme activity in BAL samples.Panel A shows the results of ELISA normalized for total proteins (n=36). Panel B shows inhibition of enzymatic activity for four illustrative patients versus human sPLA2-IIA by different sPLA2 inhibitors. Results in panel B are expressed as % of sPLA2 residual activity after the addition of various inhibitors as listed in the legend; recombinant human sPLA2-IIA is used as control and the first column represents the raw sPLA2 global activity without any inhibitor. Characteristics of each inhibitor are described in the online supplement. Experiments have been performed in BAL samples obtained prior to any surfactant administration. All measurements have been performed in duplicate. Abbreviations: anti-hGIB: rabbit polyclonal antibody anti-sPLA2-IB; anti-hGIIA: rabbit polyclonal antibody anti-sPLA2-IIA; anti-hGX: rabbit polyclonal antibody anti-sPLA2-X; BAL: broncho-alveolar lavage; EDTA: ethylenediaminetetraacetic acid; r-hu-sPLA2-IIA: recombinant human sPLA2-IIA; sPLA2: secretory phospholipase A2.Residual enzymatic activity (% of control withouth inhibitors) sPLA2 activity of BAL samples and inhibition with various sPLA2 inhibitors under the same experimental conditions. Results have been obtained in an unique assay session performed on BAL samples obtained prior to any surfactant administration (n=12). All measurements have been performed in duplicate. Results are expressed as specific radioactivity (dpm). Grey lines represent median and interquartile range. Control represents sPLA2 activity measured in BAL samples prior to the addition of any inhibitor. Characteristics of each inhibitor are described in the online supplement. Data were compared with Kruskal-Wallis and Mann-Whitney tests. sPLA2 activity obtained with varespladib addition is lower than that obtained with RO092906A (p=0.043), anti-hGIB (p<0.001) and anti-hGX antibodies (p<0.001) and is lower than control (p<0.001). Abbreviations: anti-hGIB: rabbit polyclonal antibody anti-sPLA2-IB; anti-hGIIA: rabbit polyclonal antibody anti-sPLA2-IIA; anti-hGX: rabbit polyclonal antibody anti-sPLA2-X; BAL: broncho-alveolar lavage; EDTA: ethylenediaminetetraacetic acid; sPLA2: secretory phospholipase A2.

A

  and B represent zwitterionic and anionic lipids, respectively; to increase visibility, the data clouds within the hatched red lines are depicted and zoomed in panel A1 and B1, respectively. Black and grey dots represent measurements in neonates receiving one (n=21) or two (n=12) surfactant doses, respectively. Horizontal thin lines represent median and interquartile range. Epithelial lining fluid concentrations have been obtained correcting for serum-to-BAL urea ratio. Data were compared with Mann-Whitney test and significant p-values are shown on top of the data. Abbreviations: BAL: broncho-alveolar lavage; DPPC: dipalmitoylphosphatidylcholine; LysoPC: lyso-phosphatidylcholine; Lyso-PE: lyso-phosphatidylethanolamine; PC: total phosphatidylcholine; PE: phosphatidylethanolamine; PG: phosphatidylglycerol; PL: total phospholipids.

Figure 4 . 29 0 5 .

 4295 Correlation between adsorption of surfactant available in the first day of life (endogenous plus exogenous pool) and epithelial lining fluid sPLA2 activity in neonates undergoing BAL before and after surfactant replacement (n=18). The adsorption of surfactant available on the first day of life is measured as surface accumulation of fluorescently labelled surfactant (read at 9 min as relative fluorescence unit) and reported as ratio of the values measured in BAL samples obtained after and before surfactant (200 mg/kg poractant-) replacement. sPLA2 activity is expressed (Log scale) as epithelial lining fluid corrected for serum-to-BAL urea ratio and normalized for surfactant protein-A content. All measurements have been performed in duplicate. The hatched line is the best-fitting data curve generated with quadratic regression (Y = -0.015 x X 2 -0.28 x X + 1.8). Correlation analysis was performed calculating Spearman coefficient. Abbreviations: BAL: broncho-alveolar lavage; RFU: relative fluorescent units; sPLA2: secretory phospholipase A2. Figure Effect of one poractant- administration on epithelial lining fluid sPLA2, its natural inhibitors and surfactant function. Measurements have been performed in BAL samples obtained before the first surfactant dose (PRE) and after at least 10h from the administration (POST). All measurements have been performed in duplicate. Panel A, B, C, D and E represent sPLA2 activity (n=55), sPLA2-IIA (n=55), SP-A (n=27), DOPG (n=36) and surfactant adsorption (n=34), respectively. Grey horizontal lines represent medians and interquartile range. Panel A, B, C and D show epithelial lining fluid concentrations obtained correcting for serum-to-BAL urea ratio; sPLA2 activity is also normalized for surfactant protein-A content. Adsorption of surfactant (Panel E) is measured as surface accumulation of fluorescently labeled surfactant (read at 90' as relative fluorescence unit). Data were compared with Mann-Whitney test and significant p-values are shown on top of the data. Abbreviations: BAL: broncho-alveolar lavage; DOPG: dioleoylphosphatidylglycerol; RFU: relative fluorescent units; SP-A: surfactant protein-A; sPLA2: secretory phospholipase A2.

Table 1 .

 1 Basic details of studied neonates. Data are expressed as mean (standard deviation), median [interquartile range] or number (%), as appropriate. Antenatal steroids were considered complete when two doses of 12 mg-betamethasone were given, 24h apart from each other and at least 24h before the delivery. The respiratory morbidity score describes the respiratory condition

	during the first year of life.(33) Apgar, CRIB-II and respiratory morbidity scores are
	dimensionless numbers. Abbreviations: BAL: broncho-alveolar lavage; BPD: broncho-
	pulmonary dysplasia; CRIB-II: Critical Risk Index for Babies-II.
	Neonates	78
	Gestational age (weeks)	28 (2.2)
	Birth weight (grams)	1168 (333)
	Complete antenatal steroids	49 (62.8%)
	Cesarean section	44 (56.4%)
	Male sex	44 (56.4%)
	5' Apgar score	8 [7-9]
	CRIB-II score	9.2 (3.9)
	Postnatal age at 1 st BAL (h)	4 [1-5]
	Postnatal age at 2 nd BAL, if any (h)	12 [10-14]
	Need for surfactant re-treatment	32 (41%)
	Duration of invasive ventilation (h)	96 [24-192]
	In-hospital mortality	12 (15.4%)
	BPD	31 (39.7%)
	Respiratory morbidity score	0 [0-1]

Table 2 . Epithelial lining fluid sPLA2 and inflammatory mediators in neonates receiving one (n=46) or two (n=32) poractant- doses.

 2 Measurements have been performed in BAL samples obtained prior to any surfactant administration. Surfactant treatments have been managed according to our protocol;(17) more details in the text. All measurements have been performed in duplicate. Data are reported as median [interquartile range] and compared with

	Mann-Whitney test. Epithelial lining fluid concentrations have been obtained correcting for
	serum-to-BAL urea ratio; sPLA2 total activity and sPLA2-IIA are also normalized for surfactant
	protein-A. Abbreviations: BAL: broncho-alveolar lavage; GM-CSF: granulocyte macrophage-
	colony stimulating factor; IL1-: interleukine-1; IL6: interleukine-6; sPLA2: secretory
	phospholipase A2; TNF: tumor necrosis factor-.
		Single dose (46)	Two doses (32)	p
	GM-CSF (pg/mL)	261 [70-367]	832 [352-1514]	<0.0001
	TNF (pg/mL)	128 [77-324]	5481 [152-20867]	0.005
	sPLA2 activity (nmol x min x mL/ng)	0.9 [0.3-2.5]	3.6 [0.8-13.3]	0.021
	sPLA2-IIA (ng/ng)	57.4 [13-198]	270 [34-2259]	0.039
	IL1- (ng/mL)	1.05 [0.4-2.2]	41.6 [15.2-211.4]	0.01
	IL6 (ng/mL)	18.2 [10.1-54.9]	101.5 [55.7-204.1]	0.012
	Total proteins (mg/mL)	17 [7.3-28.5]	23.3 [15.6-34]	0.

05 Figure 3. Epithelial lining fluid phospholipid profile of neonates receiving one or two poractant- doses.

  Measurements have been performed in BAL samples obtained prior to any surfactant administration. Surfactant treatments have been managed according to our protocol;[START_REF] Dell'orto | Cell Count Analysis from Nonbronchoscopic Bronchoalveolar Lavage in Preterm Infants[END_REF] more details in the text. All measurements have been performed in duplicate. Panel

ACKNOWLEDGEMENTS

Authors are grateful to all physicians and nurses who helped to collect broncho-alveolar lavage samples. The study has received funded (1/2017-2018) by ADRN (Association pour le Dévéloppement et la Recherche en Néonatologie) charity, a non-profit organization who had no role in design, preparation, review, approval of the manuscript or decision to submit it for publication. The corresponding author confirms that he had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Funding. ADRN (Association pour le Dévéloppement et la Recherche en Néonatologie) 1/2017-2018. A/Prof.De Luca has received research and educational grants from Chiesi Pharmaceuticals spa and ABBVIE inc. He served as consultant and lecturer for Airway Therapeutics, Chiesi Pharmaceuticals spa and ABBVIE inc. Finally, he has been member of advisory boards for Chiesi Pharmaceuticals spa and ABBVIE inc. Dr.Shankar-Aguilera received a travel grant from Chiesi Pharmaceuticals spa. A/Prof.Cogo and Proff.Perez-Gil, Carnielli and Touqui have received research grants and assistance and/or served as consultant for Chiesi Pharmaceuticals spa. Prof.Perez-Gil also received research grants and assistance from Airway Therapeutics.