S K Fagervold

A S Rodrigues

C Rohee

R Roé

M Bourrain

D Stien

Philippe Lebaron
email: lebaron@obs-banyuls.fr

Occurrence and environmental distribution of 5 UV filters during the summer season in different water bodies

Organic UV filters are used worldwide in various personal care products as well as textiles, paints, plastic, food and adhesives. They are main ingredients in sunscreen lotions that are used heavily by beachgoers in the summer season.

There is thus an increasing concern regarding the fate of organic UV filters in the environment and their impact on living organisms. Many of the UV filters in use are hydrophobic and are expected to accumulate in the sediment phase in aquatic systems, but this has yet to be validated in situ. We targeted the UV filters benzophenone 3 (BP3), butyl Methoxydibenzoylmethane (BMDBM), diethylhexyl butamido triazone (DBT), bis-ethylhexyloxyphenol methoxyphenyl triazine (BEMT) and methylene bis-benzotriazolyl tetramethylbutylphenol (MBBT) in a freshwater lake and in a coastal bay in order to understand their distribution during summer 2016. Further, we examined their environmental partitioning by collecting samples from the surface water, the sediment phase and water surface microlayer (SML). We show for the first time the presence of DBT, BEMT and MBBT in environmental matrices (water, SML, and sediment). Notably, these UV filters were detected at low amounts in surface waters with maximum concentrations of 9.9 ng/L for DBT, 18.4 ng/L for BEMT and below detection limits for MBBT, and somewhat higher concentrations in the SML, with maximum concentrations of 43.3 ng/L for DBT, 5625.4 ng/L for BEMT and 45.6 ng/L for MBBT. These filters were detected at even greater concentrations in the sediments, with maximum concentrations of 652.6 ng/g for DBT, 115.0 ng/g for BEMT and 75.2 ng/g for MBBT (dry weight sediment). We also performed controlled laboratory experiments to determine their partitioning behavior and we verified the actual solubility of many of the filters. This will help in determining the environmental fate and finally lead to a better risk assessment of these compounds. Together, these results corroborate the hypothesis that hydrophobic UV filters accumulate in the sediment phase and highlight the importance of discerning whether these UV filters impact the benthic community and their potential for bioaccumulation.

Introduction

Over the past fifteen years, public interest in sunscreens has increased because of their potential ecological risk in aquatic environments. Sunscreens use organic UV filters as ingredients in their formulations to protect human skin from sunburns, premature skin aging and skin cancer. Organic UV filters include a heterogeneous group of molecules characterized by conjugated aromatic groups that can absorb UV-A (320-400 nm) and UV-B (280-320 nm) radiation. These UV filters are also added to textiles, plastics, paints, food and adhesives to protect polymers and pigments against photo degradation and to prevent discoloring. In sunscreen lotions, formulations generally combine several filters and ingredients to cover a broad UV spectrum and to improve skin application and water resistance [START_REF] Osterwalder | Global state of sunscreens[END_REF]. More than 50 different UV filters are on the market [START_REF] Shaath | Ultraviolet filters[END_REF], and their usage varies depending on the regulations of the region.

UV filters enter the aquatic environment directly from recreational activities and indirectly from wastewater treatment plants (WWTPs), see [START_REF] Ramos | A review of organic UV-filters in wastewater treatment plants[END_REF] and references therein, or leaching from solid waste disposal sites on land. UV filters enter the sewage systems when people shower following bathing or from industrial discharge. Once in the environment, partitioning between water and sediment occurs depending on the lipophilicity of the compound, which is estimated by the octanol-water partition coefficient (log kow) value. Those compounds with low aqueous solubility tend to accumulate in sediments and sludge [START_REF] Ramos | Advances in analytical methods and occurrence of organic UV-filters in the environment -A review[END_REF]. Environmental concentrations of different UV filters are found in numerous reports, with recent reviews summarizing their concentrations in different matrices [START_REF] Montes-Grajales | Occurrence of personal care products as emerging chemicals of concern in water resources: A review[END_REF]) including wastewater treatment plants [START_REF] Ramos | A review of organic UV-filters in wastewater treatment plants[END_REF], sediments [START_REF] Tsui | Occurrence, distribution and ecological risk assessment of multiple classes of UV filters in marine sediments in Hong Kong and Japan[END_REF] and water bodies [START_REF] Ramos | Advances in analytical methods and occurrence of organic UV-filters in the environment -A review[END_REF][START_REF] Sánchez-Quiles | Are sunscreens a new environmental risk associated with coastal tourism?[END_REF].

The possibility that these compounds have negative effects on non-target organisms is one factor that raises concern about the release of UV filters into the environment. This is especially true in areas, like the Mediterranean, that are highly impacted by tourism [START_REF] Tovar-Sanches | Massive costal tourism influx the Mediterranean Sea: The environmental risk of sunscreens[END_REF]. Further, because UV filters have been detected in various biological materials, such as fish [START_REF] Balmer | Occurrence of Some Organic UV Filters in Wastewater, in Surface Waters, and in Fish from Swiss Lakes[END_REF][START_REF] Fent | Widespread occurrence of estrogenic UVfilters in aquatic ecosystems in Switzerland[END_REF][START_REF] Tsai | Optimization of matrix solid-phase dispersion for the rapid determination of salicylate and benzophenonetype UV absorbing substances in marketed fish[END_REF][START_REF] Langford | Environmental occurrence and risk of organic UV filters and stabilizers in multiple matrices in Norway[END_REF]Gago-Ferrero et al. 2015;[START_REF] Emnet | Personal care products and steroid hormones in the Antarctic coastal environment associated with two Antarctic research stations, McMurdo Station and Scott Base[END_REF][START_REF] Sang | Environmental occurrence and ecological risk assessment of organic UV filters in marine organisms from Hong Kong coastal waters[END_REF], dolphins [START_REF] Gago-Ferrero | First Determination of UV Filters in Marine Mammals. Octocrylene Levels in Franciscana Dolphins[END_REF][START_REF] Alonso | Toxic heritage: Maternal transfer of pyrethroid insecticides and sunscreen agents in dolphins from Brazil[END_REF] and bird eggs [START_REF] Molins-Delgado | A Potential New Threat to Wild Life: Presence of UV Filters in Bird Eggs from a Preserved Area[END_REF], biomagnification is a real concern [START_REF] Fent | Widespread occurrence of estrogenic UVfilters in aquatic ecosystems in Switzerland[END_REF][START_REF] Sang | Environmental occurrence and ecological risk assessment of organic UV filters in marine organisms from Hong Kong coastal waters[END_REF][START_REF] Molins-Delgado | A Potential New Threat to Wild Life: Presence of UV Filters in Bird Eggs from a Preserved Area[END_REF]. Many UV filters are thought to impact non-target organisms via endocrine disruption [START_REF] Díaz-Cruz | Chemical analysis and ecotoxicological effects of organic UV-absorbing compounds in aquatic ecosystems. Applying combinations of chemical analysis and biological effects to environmental and food samples -II[END_REF][START_REF] Sánchez-Quiles | Are sunscreens a new environmental risk associated with coastal tourism?[END_REF]. Recently, benzophenone 3 (BP3) was shown to have toxic effects on many living organisms [START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF][START_REF] Wnuk | Apoptosis Induced by the UV Filter Benzophenone-3 in Mouse Neuronal Cells Is Mediated via Attenuation of Erα/Pparγ and Stimulation of Erβ/Gpr30 Signaling[END_REF]). As a consequence, this compound was banned in the state of Hawaii, USA ("Office of the governor -news release -Governor David Ige signs bill making Hawaii first in the world to ban certain sunscreens" 2018).

Diethylhexyl butamido triazone (DBT), methylene bis-benzotriazolyl tetramethylbutylphenol (MBBT) and bis-ethylhexyloxyphenol methoxyphenyl triazine (BEMT) have very high predicted log Kow values (Table 1) and have not been reported in any environmental matrix to date. BP3 and butyl Methoxydibenzoylmethane (BMDBM), have lower log Kow values (Table 1) and are technically easier to detect: butyl methoxydibenzoylmethane (BMDBM), has been detected in various compartments, including seawater and freshwater [START_REF] Bratkovics | Determination of seven commonly used organic UV filters in fresh and saline waters by liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Giokas | Determination of UV-filter residues in bathing waters by liquid chromatography UV-diode array and gas chromatography-mass spectrometry after micelle mediated extraction-solvent back extraction[END_REF][START_REF] Sánchez Rodríguez | Occurrence of eight UV filters in beaches of Gran Canaria (Canary Islands). An approach to environmental risk assessment[END_REF][START_REF] Poiger | Occurrence of UV filter compounds from sunscreens in surface waters: regional mass balance in two Swiss lakes[END_REF]) and in WWTP effluents and influents [START_REF] Rodil | Non-porous membrane-assisted liquid-liquid extraction of UV filter compounds from water samples[END_REF][START_REF] Tsui | Seasonal occurrence, removal efficiencies and preliminary risk assessment of multiple classes of organic UV filters in wastewater treatment plants[END_REF]) and BP3 has been found in many environmental matrices (ex. (Montes-Grajales et al. 2017;[START_REF] Ramos | A review of organic UV-filters in wastewater treatment plants[END_REF]). However, for a proper assessment of the environmental fate of these filters, we also need to determine the solubility and the partitioning behavior of these compounds experimentally.

In the current study, we determined the concentration of the widely studied UV filter BP3 and compared the concentration of BP3 with that of less investigated but relatively broadly utilized compounds. We investigated a coastal marine site, the Bay of Banyuls-sur-mer, and a freshwater lake, Villeneuve de la Raho Lake.

Both sites experience a high number of sunbathers during the summer months (June to August). In addition to determining the dynamics of the filters during the summer months, we determined in which compartment these UV filters are found. For example, the surface microlayer (SML) has different chemical and biological characteristics than those of the underlying water [START_REF] Obernosterer | Enhanced heterotrophic activity in the surface microlayer of the Mediterranean Sea[END_REF]) and this air-sea boundary, in which hydrophobic compounds may accumulate and living organisms may be present [START_REF] Rahlff | Blue pigmentation of neustonic copepods benefits exploitation of a prey-rich niche at the air-sea boundary[END_REF], can be important in the aquatic environment. Further, given the fairly high log Kow of many UV filters; the expectation is that these compounds will eventually concentrate into the solid/sediment compartment. However, we do not know how long this partitioning will take and whether UV filters will accumulate in sediments over time. Therefore we also set out to resolve several factors important to better understand the fate of these compounds in aquatic environments.

Materials and methods

Chemicals and solvents

The molecular structures and physicochemical properties of the studied compounds are shown in Table 1. Analytical standards of MBBT, (97 %), BEMT (98 %), DBT (98 %), BMDBM (99 %) and BP3 (98%) were purchased from Sigma-Aldrich (Lyon, France). Analytical or LC-MS grade methanol was purchased from Biosolve (Biosolve Chimie France, Dieuze). Analytical-grade dichloromethane, 1-methyl-2-pyrrolidinone (NMP) and formic acid (98 %) were obtained from Sigma-Aldrich. Pure water was obtained from Elga Purelab Flex System (Veolia LabWater STI, Antony, France). In order to avoid contamination, glassware was cleaned with dichloromethane and dried at 450 °C for 2 hours.

Standard solutions of individual UV filters were prepared in NMP at a concentration of 1 mg/mL. These solutions were kept at -20°C and protected from light. A standard mixture solution was prepared by diluting each individual standard solution with NMP/water (8:2, volume/volume (v/v)) in order to get a concentration range from 1024 to 4 ng/mL. A calibration curve was built both with LC-MS/MS and LC/DAD using these solutions. These standard solutions were also used for spiking seawater and sediments for recovery studies.

Sampling sites

All samples were collected from April to October 2016 from two different sites: the main beach in Banyuls-sur-mer (Latitude 42°28'57.5" N, Longitude 3°07'55.4" E), and a nearby artificial lake in Villeneuve-de-la-Raho (Latitude 42°37'46.3" N, Longitude 2°54'18.0" E). Both these sites see highly increased tourism pressure during the summer months. Lake Villeneuve measures more than 200 ha, with 16 ha dedicated to touristic activities including a bathing zone and a 800 m-wide beach. Banyuls-sur-Mer is a small tourist town in the south of France, where the main beach measures 350 meters and is partially enclosed and protected from heavy ocean currents. We sampled the surface water, the water surface microlayer (SML) and the sediment in Villeneuve Lake and Banyuls Bay during the summer season from April to October. We sampled at 5 time points from Villeneuve Lake and at 9 from Banyuls Bay (Table S1). The first time point was before the bathing season for both sites.

Sampling procedures

All samples were collected using gloves and in order to minimize contamination, the people involved in sampling did not use any UV protection cream. The sampling was always performed in the afternoon where wind conditions were favourable. Water samples were collected in the middle of the bathing zones using an inflatable boat. For Banyuls Bay this was approximately 50 meters from the beach and for lake Villeneuve the sampling was preformed approximately 10 m from the beach. Samples from the water column (approximately 50 cm below the surface) were collected using a bucket and poured into glass bottles and closed with PTFE caps. The bucket and glass bottles were rinsed several times before final sampling and 3 x 1L were collected at each time point. The SML was collected with a glass plate system [START_REF] Harvey | A simple microlayer method for small samples[END_REF] resulting in 3 x 0.5 L SML samples per time point. The superficial layer (top 5 cm) of sediments from the same zone was collected in glass jars. This was done manually in Lake Villeneuve because the water depth was less than 1 meter, and by snorkeling/freediving in Banyuls bay, where the sediment depths were around 7-8 meters

Water extraction and LC-MS/MS analysis

Collected water samples were filtered through 47 mm glass microfiber filters with a pore size of 1.6 μm (GF/A Whatman, GE Healthcare, Little Chalfont, UK), using a glass filtration system. All samples were acidified with formic acid (0.1% v/v) and extracted using an automated liquid handler GX-271 ASPEC® (Gilson International, Villiers-le-Bel, France) using Hypersep TM phenyl SPE cartridges 1 g/6 mL (Thermo Fisher Scientific, Illkirch, France). All the eluents were acidified with formic acid (0.1% v/v) to pH 3. Before loading, the cartridges were conditioned with 10 mL of methanol followed by 10 mL of water. Next, an entire 1 L sample was percolated through the cartridge with a constant flow rate (8 mL/min). The cartridge was dried under nitrogen for 10 min (AirLiquide, Toulouse, France). Then, the analytes were extracted with 10 mL of methanol followed by 10 mL of methanol/dichloromethane (1:1, v/v). The extracts were evaporated to dryness under vacuum in an HT-4X system (Genevac, Biopharma Technologies France, Lyon, France) and dissolved in 500 µL of NMP/water (8:2, v/v) before analysis. LC-MS/MS is more sensitive than LC-DAD (lower limits of detection (LODs)) for the detection of these UV filters. Water samples were analyzed with LC-MS/MS because UV filter concentration was lower in these samples, while sediment samples were analyzed with LC-DAD. Here, chemical analyses were conducted with a Q Exactive Focus Orbitrap System coupled to an Ultimate 3000 TM UHPLC system (Thermo Fisher Scientific). Analyses of water samples and standards (5 µL injected) were performed in electrospray positive ionization mode in the 53.4-800 m/z range in profile mode. The parameters were as follow: spray voltage: 3 kV; sheath flow rate: 75; aux gas pressure: 20; capillary temperature: 350°C; heater temperature: 430°C. The analysis was conducted in FullMS data dependent MS2 mode (confirming ions). Resolution was set to 70,000 in Full MS mode, and the AGC (automatic gain control) target was set to 1x10 6 . In MS2, resolution was 35,000, AGC target was set to 1x5e 4 , isolation window was 3 m/z, and normalized collision energy was 35 eV, with an inclusion table. The UHPLC column was a Kinetex Biphenyl 2.6 µm, 150 x 4.6 mm column (Phenomenex).

The column temperature was set to 50°C, and the flow rate was 1 mL min -1 . The solvent system was a mixture of water (A) with increasing proportions of methanol (B), with both solvents modified with 0.1% formic acid. The gradient was as follows: 20% B 4 min before injection, then from 1 to 7 min, a gradient increase of B up to 100% (curve 2), followed by 100% B for 8 min. The flow was injected into the mass spectrometer starting from 6,5 min after injection. All data were acquired and processed using Trace Finder 3.1 software Trace Finder TM 3.1 software (Thermo Fisher Scientific). Obtained retention times (RT) were the following: 6,68 min for BP-3; 7,84 min for BM-DBM; 9,26 min for DBT; 13,68 min for BEMT; and 12,79 min for MBBT.

Sediment extraction and LC-DAD analysis

Collected sediments were frozen at -20°C and then freeze-dried for 24 h. The freeze-dried samples (5 g) were passed through a 1 mm pore sieve and stored at -20°C until extraction. Freeze-dried sieved sediment (1 g) were extracted twice with 5 mL of a mixture of methanol/dichloromethane (1:1, v/v), with 0.1% formic acid and a 10 min sonication. All the supernatants were combined and evaporated under vacuum in an HT-4X system (Genevac, Biopharma Technologies) and dissolved in 1 mL of NMP/water (8:2, v/v) before analysis. Samples (30 µL injected) were analyzed with an Ultimate 3000 TM HPLC system, equipped with a DAD detector (Thermo Fisher Scientific) and a Phenomenex Kinetex Biphenyl 2.6 µm, 150 x 4.6 mm column. The oven temperature was set to 50°C, and the flow rate was 1 mL min -1 . The solvent system was a mixture of water (A) with increasing proportions of methanol (B), with both solvents modified with 0.1% formic acid. The gradient was as follows: 10% B 4 min before injection, then from 1 to 15 min, a gradient increase of B up to 100% (curve 2), followed by 100% B for 10 min. The data acquisition software was Chromeleon™ 7.2 (Thermo Fisher Scientific). Standard solutions were injected to generate ten-point calibration curves for the quantification of target compounds.

Peak areas were within the linear range of the curve. The obtained retention times (RT) were the following: 9,4 min for BP-3; 11,76 min for BMDBM; 14,9 min for DBT; 20,6 min for BEMT; and 19,5 min for MBBT.

Sediment samples were also analyzed for organic carbon and granulometry.

Dried sediments were decarbonized with repeated additions of H3PO4 (1M) and HCl (2M) until no effervescence occurred. Decarbonated sediment were dried again at 40 °C at least 24 hours. POC content were then measured using an Elementar Variomax elemental analyzer. Particle size analyses (range: 0.1-3500 µm) was performed using a Malvern3000 laser diffraction particle size analyzer coupled with Hydro EV unit (Wet dispersion).

Method performance

Water and sediment extraction recovery was determined using spiked samples (100 ng/L for water samples or 100 ng/g dry sediment). Net recovery was calculated by subtracting the analyte concentration observed in unspiked samples from the concentrations in the corresponding spiked samples. The LODs were determined based on the concentrations in a signal-to-noise ratio of 3 and ranged from 1.3 to 5.2 ng/L. The LODs in the sediment samples ranged from 4.6 to 8.6 ng/g. The concentrations were corrected for the contributions from the blanks. Recovery rate and LODs of each compound are shown in Table 2. The values we report in the paper are not corrected for recovery rates.

Sediment/water distribution coefficient experiment

Dry sediment (3 g) was placed into 250 mL Erlenmeyer flasks. A mix of the different UV filters dissolved in acetone was added to the sediment and left overnight in a chemical hood to evaporate the acetone. The final amounts (total) added were 100 ng, 1 µg, and 10 µg of each of the UV filters tested, each in triplicate. MilliQ water, 100 mL, was then added, and the flasks were incubated in the dark for 24 h at 25°C with constant shaking. The liquid and solid phase were then separated by filtration and extracted, and the UV filters quantified as described above.

Results and discussion

Detection of hydrophobic UV filters in different environmental matrices and experimental solubility measurements.

The UV filters we tested had varying chemical characteristics, with predicted log Kow values from 3.79 to 14.06 (Table 1 and [START_REF] Ramos | A review of organic UV-filters in wastewater treatment plants[END_REF])) and many of them were very hydrophobic. Therefore, a method that effectively includes the wide range of chemicals targeted in this study had to be developed for the extraction and concentration from the different environmental matrices tested.

For testing the extraction efficiency from water samples, we used spiked seawater and tap water and tested different extraction protocols using different solvents (data not shown). The optimum extraction protocol determined for both the water and sediment samples was a compromise among the different filters.

The recovery of BP3, DBT, BEMT and MBBT filters from tap water, seawater and sediment samples ranged from 47 to 80%, but was only 10% for BMDBM extracted from tap water and seawater samples and 2% from sediment (Table 2). Therefore, we must consider these extraction efficiency values when interpreting our data, particularly for BMDBM.

For many filters, the only available data regarding solubility are predicted values.

To investigate the water solubility of the five UV filters in greater detail, a private and independent analytical laboratory (Intertek France) measured the water solubility by conducting a water saturation study in pure water and seawater.

Solubilities were low ranging from < 4 x 10 4 to 1.3. x 10 8 ng/L. The least water soluble UV filter is MBBT (2500 fold less than BP3), followed by BEMT, DBT and BMDBM (approximatively 500-, 300-and 10-fold less soluble than BP3 respectively; Table 1). Indeed, values for MBBT were not obtained because the saturated water concentration was below the defined limit of detection (LOD) for this study.

This study shows, for the first time, experimental values for the water solubility of BEMT, DBT and MBBT (Table 1). The experimental and predicted values are comparable for BP3 and BMDBM, but for BEMT and DBT, experimental values are much higher. This difference demonstrates the difficulty in predicting the fate of these extremely hydrophobic UV filters in the environment. The hypothesis that such compounds should not be detected in the water column [START_REF] Ramos | Advances in analytical methods and occurrence of organic UV-filters in the environment -A review[END_REF] was not supported by the experimental values. Although the solubility of the UV filters was very low in filtered seawater, one can expect that a portion of these compounds might also absorb onto planktonic microorganisms and suspended matter. Indeed, we filtered the water before we passed the water through SPE columns, so we did not measure any UV filter absorbed to particles bigger than 1.6 μm. Therefore, our values represent dissolved UV filter and filters absorbed to very small particles. For example, polycyclic aromatic hydrocarbons (PAHs) have been detected in water (water accommodated fraction), although they are also virtually insoluble in water [START_REF] Forth | Characterization of dissolved and particulate phases of water accommodated fractions used to conduct aquatic toxicity testing in support of the Deepwater Horizon natural resource damage assessment[END_REF]). In the water column, PAHs are predominantly associated with the solid-phase particulate matter that eventually settles in marine sediments [START_REF] Hassan | Baseline concentrations and distributions of Polycyclic Aromatic Hydrocarbons in surface sediments from the Qatar marine environment[END_REF][START_REF] Wang | Distribution and Partitioning of Polycyclic Aromatic Hydrocarbons (PAHs) in Different Size Fractions in Sediments from Boston Harbor, United States[END_REF]. This is most likely also the case for many of the hydrophobic UV filters.

Temporal dynamics of UV filter concentrations in Lake Villeneuve and Banyuls Bay

Surface water

In many samples from surface waters, we could not detect any of the UV filters in quantities above LOD. For clarification, the graphs in Figure 1 depict the sampling points where all three replicates from one timepoint contained the specific UV filter above the LOD, while figure 2 depict maximum values for any one sample (not triplicates). Generally, we could not detect any of the filters in surface waters in the early summer season. The highest concentrations in the lake were found for BMDBM and BEMT in July and August with average concentrations of 14.5 and 7.2 ng/L, respectively, whereas MBBT and DBT concentrations were below LOD. In Banyuls Bay, only BEMT was found at peak concentrations of approximately 12 ng/L, (Figure 1), DBT could be detected above the LOD in some samples with a maximum value of 9.88 ng/L (Figure 2) and we could only determine a concentration above LOD for BP in one sample, namely August 8 th with 1.52 ng/L. However, BMDBM, and MBBT were not detected.

Concerning the surface water, many studies detail different concentrations of UV filters, but of the five UV filters in this study, most data are available for the two least hydrophobic compounds, BP3 and to a small extent, BMDBM (see review article (Sánchez-Quiles and Tovar-Sánchez 2015)). The highest concentration of BP3 in the Villeneuve Lake surface water was found in September and was close to 3 ng/L (Figure 1a and Figure 2a). BP3 has previously been detected in lakes with maximum values of 125 ng/L [START_REF] Poiger | Occurrence of UV filter compounds from sunscreens in surface waters: regional mass balance in two Swiss lakes[END_REF]), 35 ng/L [START_REF] Balmer | Occurrence of Some Organic UV Filters in Wastewater, in Surface Waters, and in Fish from Swiss Lakes[END_REF]) and 40 ng/L [START_REF] Rodil | Non-porous membrane-assisted liquid-liquid extraction of UV filter compounds from water samples[END_REF]); thus, the maximum values we report here are not exceedingly high. BP3 concentrations in Banyuls Bay, which did not surpass LOD (Figure 1D and 2A), were also low compared with concentrations found in previous studies. For example, concentrations of BP3 in (filtered) seawater samples from recreational areas were as high as 241.9 ng/L in Mallorca [START_REF] Tovar-Sanchez | Sunscreen Products as Emerging Pollutants to Coastal Waters[END_REF]) and an extreme value of 1,390,000 ng/L has been reported in the US Virgin Islands [START_REF] Downs | Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands[END_REF]. These varying values can be partly explained because BP3 is more commonly used in the US due to the limited number of filters that are allowed, and the use of BP3 is perhaps more limited in Europe due to regulations (European Union 2009).

The peak value of BMDBM in the Villeneuve Lake surface water was approximately 14.5 ng/L, whereas BMDBM was not detected in Banyuls Bay.

Regarding BMDBM concentrations in lakes, there are only a few studies that can be used for comparison, and they report varying concentrations, from below detection level [START_REF] Poiger | Occurrence of UV filter compounds from sunscreens in surface waters: regional mass balance in two Swiss lakes[END_REF]) to a very high concentration of over 2000 ng/L [START_REF] Rodil | Non-porous membrane-assisted liquid-liquid extraction of UV filter compounds from water samples[END_REF]. For the coastal marine environment, BMDBM was detected in relatively high concentrations in beaches in Gran Canaria, with a median value of 59.4 ng/L [START_REF] Sánchez Rodríguez | Occurrence of eight UV filters in beaches of Gran Canaria (Canary Islands). An approach to environmental risk assessment[END_REF], and the maximum concentration of 321 ng/L was found in coastal water samples from South Carolina [START_REF] Bratkovics | Determination of seven commonly used organic UV filters in fresh and saline waters by liquid chromatography-tandem mass spectrometry[END_REF].

Notably, we found BEMT at significant concentrations in the lake and Banyuls Bay surface waters, with maximum concentrations of 13.8 and 18 ng/L, respectively, see figure 2a. Further, DBT was detected at the height of summer, albeit at concentrations very close to the calculated LOD. To our knowledge, this is the first report of detection of this UV filter in the water phase in the environment. This finding is notable considering the extremely low predicted and experimentally determined water solubility values of this compound. Indeed, this compound should be practically insoluble in the water phase (Table 1). However, we propose that, similar to PAHs, hydrophobic UV filters are in the water-accommodated fraction presumably associated with suspended particles.

SML

The concentrations of some of the UV filters in the SML in Villeneuve Lake showed a clear trend during the summer months, with a peak in July-August (Figure 1B). Furthermore, the SML contained higher concentrations of the UV filters than the surface water (note the difference in scale between Figure 1 A and B and Figure 2 A andB). The peak average concentrations of BEMT and BMDBM were 4281 and 531 ng/L, respectively. However, the variability was very high at these sampling points. Additionally, July and August samplings in the lake were both performed when many swimmers were present, and we observed an "oily layer" on the water during these peak sampling times. We know of no other reports on the SML in lakes.

The SML in Banyuls Bay contained consistently over 10 ng/L of BEMT, with a peak average concentration on June 28 at 101.2 ng/L (Figure 1e). The concentrations of the other UV filters were below detection level for most of the samples. The concentrations of BP3 remained below LOD, which is very low compared with previous values in Mallorca which exceeded 100 ng/L in many cases [START_REF] Tovar-Sanchez | Sunscreen Products as Emerging Pollutants to Coastal Waters[END_REF]. The variations reported between sites and over the season can be explained by variations in the environmental conditions and because the SML can only be established when the wind speed is below 5 m/s [START_REF] Romano | Photographic records of sea-surface microlayers as a survey of pollution daily rhythm in coastal waters[END_REF].

Sediment

The dynamics of UV filters in the sediment were very different from those we observed for the surface water and SML. Notably, we detected several of the UV filters in significant amounts in April, before the bathing season. In particular, in Lake Villeneuve BEMT, MBBT and DBT were found at average concentrations of 48.2, 47.8 and 22 ng/g (dw) sediment, respectively. Further, DBT and BEMT concentrations in Lake Villeneuve sediments increased during the summer season, and the highest detected average values were 628.8 and 87 ng/g (dw), respectively, at the end of September. MBBT, BP-3 and DBM concentrations were more or less stable and slightly decreased at the last sample point in October (Figure 1c).

The results regarding the concentrations of UV filters in the Banyuls Bay sediment were unexpected because the concentrations of many of the UV filters were actually higher in the beginning of May than those at the last time points (Figure 1f). This was the case for all UV filters except BEMT. BP3 was the only UV filter to follow a dynamic during the summer season with a maximum average peak of 49.4 ng/L in July, and this value is relatively high compared with that of other studies. Baron et al. (2013) found low concentrations of BP3 (max 2.96 ng/g) in estuary samples, and [START_REF] Tsui | Occurrence, distribution and ecological risk assessment of multiple classes of UV filters in marine sediments in Hong Kong and Japan[END_REF] found a maximum concentration of 39.8 ng/g. Indeed, the low concentrations of BP3 found in sediments from highly industrialized areas [START_REF] Peng | Persistence, temporal and spatial profiles of ultraviolet absorbents and phenolic personal care products in riverine and estuarine sediment of the Pearl River catchment, China[END_REF] suggest BP3 partitions less into the sediment phase or is degraded. One reason for these differences could be that the source of UV filters to sediments in Banyuls Bay is direct, through the swimmers at the beach, whereas the marine sediments from China described by [START_REF] Tsui | Occurrence, distribution and ecological risk assessment of multiple classes of UV filters in marine sediments in Hong Kong and Japan[END_REF] are affected by industrial and municipal discharge sources. BMDBM is also reported previously in marine sediments at concentrations up to 64.4 ng/g [START_REF] Tsui | Occurrence, distribution and ecological risk assessment of multiple classes of UV filters in marine sediments in Hong Kong and Japan[END_REF]), but we were only able to detect BMDBM above our LOD in two samples from lake sediment, presumably due to the low recovery rate of this molecule in the extraction process. It is also worth noting that the sediments from both sites contained a relatively low percentage of organic carbon (Table S2). The median organic carbon percentage of Banyuls Bay sediments was 0.032 % and 0.011 % for sediments from Lake Villeneuve, and samples from both these contained more than 95% sand (particles > 63 µm).

There are only a few studies that have examined the concentrations of UV filters in the sediments of lakes. One comparative study found both BP3 and BMDBM in several German lakes also at several time points during the year (Kaiser et al. 2012). These authors detected BP3 at a maximum concentration of 3.6 ng/g and BMDBM at 62.2 ng/g, which are within the same order of magnitude of concentrations we found in Villeneuve Lake for BP3. However, they observed more of a seasonal dynamic with a sharp increase in the peak summer season, whereas our results showed a more steady concentration of these compounds in the summer season (Figure 1c). This same study Kaiser et al. (2012) found octocrylene (OC) concentrations up to 652 ng/g in some lake sediments, and we found maximum detectable concentrations for DBT, BEMT and MBBT (629,88 and 57 ng/g, respectively). Furthermore, we found a higher concentration of the UV filters in the sediment in the lake than that in the bay sediment (Figure 1c), except for BP3.

Environmental distribution of UV filters

Previous studies assume that hydrophobic UV filters will partition into the sediment phase. Nevertheless, environmental concentrations of MBBT, BEMT and DBT in the different compartments (sediment, water phase and SML) have not been reported. To further explore the distribution of these compounds in the environment, we defined "environmental distribution coefficients (env Kd)" for sediment/water and SML/water. For this calculation, we used the median value over the entire season for each UV filter from both the lake and Banyuls Bay (Table 3).

We also experimentally determined the distribution sediment/water ratio by performing controlled laboratory experiments for 24 h. The distribution ratio between water and solid phase is determined by: Kd = Cs/Cw, where Cs is the concentration (ng/kg) in the solid phase and Cw is the concentration in the water phase (ng/L). Thus, we could easily and directly compare the Kd among the different UV filters. Furthermore, the sorption isotherms were determined after fitting the data to the Freundlich model in which plotting the log Cs to log Cw should give a linear response, which was the case in our study (Figure S2). The Freundlich model equation is Cs = Kf x Cw n , where Kf characterizes the absorption capacity of the solid and n measures the intensity of the absorption (or the nonlinearity). Indeed, we observed some nonlinearity of the sorption kinetics of the UV filters; the lowest concentration (100 ng added or 1 µg/L) gave a higher Kd than that of 10 and 100 µg/L (Table 3). Thus, the UV filters adsorbed to the sediment in a higher proportion at low concentrations.

Clearly, the experimental Kd values varied greatly among the different UV filters (Table 3); thus, although many of the UV filters partitioned into the sediment phase, the filters partitioned at different ratios or alternatively at different rates.

BEMT strongly distributed into the sediment phase, with the highest Kd value of over 3000, although MBBT and DBT also produced high values, whereas BP3, with the experimental Kd of approximately 10, was expected to distribute into the sediment at a comparatively low rate. We determined a Kd for BMDBM of 777, which is substantially different from a previous report [START_REF] Li | Sorption and degradation of selected organic UV filters (BM-DBM, 4-MBC, and OD-PABA) in laboratory water-sediment systems[END_REF] with values from approximately 11 to 45.8 depending on the sediment. These differences could also be due to differences in experimental design; for example, Li and colleagues used only an 8-hour contact time and did not actually measure the amount sorbed to the sediment [START_REF] Li | Sorption and degradation of selected organic UV filters (BM-DBM, 4-MBC, and OD-PABA) in laboratory water-sediment systems[END_REF].

We could not determine the "environmental Kd (env Kd)" values for many of the filters because they were not present above the methodological LOD in the surface water. For the UV filters that we could determine the sediment/water "env Kd", we see that the "env Kd" values were in the same order of magnitude as the experimental ones. Further, from the SML Kd, we can see that many of the filters are present at higher concentrations in the SML compared to the surface water.

Once the UV filters are adsorbed to the sediments, and if no degradation or dilution processes occur, the UV filters can potentially accumulate over time.

Data regarding the degradation of UV filters in sediments are scarce. [START_REF] Volpe | Biodegradation of UV-filters in marine sediments[END_REF] found that EH-DPAB degraded after approximately 60 days in aerobic microcosms with marine sediments but found even slower degradation of 4-MBC, with a half-life of over a year. Compared to [START_REF] Volpe | Biodegradation of UV-filters in marine sediments[END_REF], [START_REF] Liu | Degradation of Six Selected Ultraviolet Filters in Aquifer Materials Under Various Redox Conditions[END_REF] found shorter half-lives of 4-MBC in their experiments, but in a different environment, namely aquifer sediments. Of the UV filters we examined, BP3 degrades in aquifer sediments [START_REF] Liu | Degradation of Six Selected Ultraviolet Filters in Aquifer Materials Under Various Redox Conditions[END_REF] within 5-6 days. Li and colleagues followed the potential degradation of BMDBM in sediment microcosms, but whether BMDBM was degraded or adsorbed to the sediment to a greater extent with time was unclear [START_REF] Li | Sorption and degradation of selected organic UV filters (BM-DBM, 4-MBC, and OD-PABA) in laboratory water-sediment systems[END_REF].

We would expect that in an open environment, hydrodynamic processes would result in a dilution of the UV filters. Thus, an accumulation from year to year or even throughout the season would not be likely in Banyuls Bay. However, we observed a considerably higher concentration of DBT and MBBT in the April samples (beginning of the season) than that at the end of the season in Banyuls Bay. This means that, surprisingly, UV filters may accumulate from the previous season. This accumulation most likely occurred because tides in the Mediterranean are not significant and because the bay is relatively closed. In future research, the concentrations of UV filters over several years in both Banyuls Bay and the lake should be monitored to determine whether UV filters accumulate in the sediments with time.

Conclusions

This study showed for the first time the presence of DBT, BEMT and MBBT in environmental matrixes (water, SML and sediment), which is interesting because these compounds, with high predicted log Kow values, are presumed insoluble in water. Indeed, substantial concentrations of these compounds were measured during peak tourist time in the surface water and SML in Villeneuve Lake.

However, we hypothesize that lipophilic UV filters are adsorbed to suspended particulate matter and might not be fully solubilized in the water phase. We show, both by sampling efforts in the environment and laboratory experiments, that a large proportion of the hydrophobic UV filters predictably partitioned into the sediment phase. Further, although some of these compounds, such as BP3, degrade relatively quickly in sediment systems, many of the hydrophobic UV filters are most likely not easily biodegraded once sequestered in the sediment phase, although this has yet to be confirmed. Thus, future ecotoxicological evaluation of lipophilic UV filters should be performed on benthic marine species as it has already been done in freshwater systems (Kaiser et al. 2012).

Overall, our work improves comprehension of the partitioning of three highly water insoluble UV filters (BEMT, MBBT and DBT). The fact that BP3 and other UV filters are found at various trophic levels in the literature suggests that some of these compounds can biomagnify. Further tests are required for a better understanding of their bioaccumulation and the potential toxicological risks to marine organisms, starting with the zoobenthos. Importantly, we must investigate whether the uptake of very lipophilic UV filters by benthic organisms (bioaccumulation) occurs for a full understanding of the potential risk of these compounds to ecosystems.

  (n=6), b= Kd determined from median concentrations in sediments and median concentrations in the water column with a minimum of 3 samples over LOD, c= Kd determined from median concentrations in the SML and median concentrations in the water column with a minimum of 3 samples over LOD, N.D.=not detected

Fig. 1

 1 Fig. 1 Dynamics of selected UV filters in Villeneuve Lake and Banyuls Bay surface

Fig. 2

 2 Fig. 2 Box and whiskers plot of all values in the water phase (a), SML phase (b),

Table 1 :

 1 UV filters information

				Molecular
	Abbr.	Chemical names	CAS	weight
				(g/

mol) Log KOW Molecular structure Water solubility (ng/L)

  Predicted by EPI Suite TM[START_REF] Ramos | A review of organic UV-filters in wastewater treatment plants[END_REF] b Values obtained by Intertek France c The water solubility was less than the quantification limit of the method used by Intertek

						Experimental b	Predicted (tap
						tap water	sea water	water) a
	BP3	Benzophenone 3	131-57-7	228.2	3.79	1.3E+08	8.7E+07	6.9E+07
	BMD BM	Butyl methoxydibenzoylmethane	70356-09-1	310.4	4.51	1.9E+07	4.7E+07	1.5E+06
	DBT	Diethylhexyl butamido triazone	154702-15-5	765.9	14.06	2E+05	< 2E+05 c	1.3E-05
	BEMT	bis-ethylhexyloxyphenol methoxyphenyl triazine	187393-00-6	627.8	9.29	3E+05	< 6E+04 c	1.5E-10
		methylene bis-
	MBBT	benzotriazolyl tetramethyl	103597-45-1	658.8	12.5	< 4E+04 c	< 1E+05 c	4.5E-04
		butylphenol

a

Table 2 .

 2 Recoveries and method limits of detection of analysis of BP3, BMDBM, DBT, BEMT and MBBT in seawater and sediment samples.

		Water samples	Sediment samples
	Compound Recovery	LOD	Recovery	LOD
		(%)	(ng/L)	(%)	(ng/g)
	BP3	67	1.3	47	4.6
	BMDBM	10	2.3	2	6.8
	DBT	71	2.3	75	8.6
	BEMT	80	2.3	60	7.5
	MBBT	47	5.2	80	3.9

Table 3 .

 3 Distribution coefficients (L/kg) of UV filters determined experimentally and calculated from environmental samples Kd determined experimentally from an average of triplicate tubes and 2 different concentrations

			Exp. a		Lake		Banyuls bay
		Kd	Kf	R 2	Kd sed b	Kd SML c	Kd sed b	Kd SML c
	BMDBM	777±150	463	0.9777	N.D.	9.14	N.D.	N.D.
	BP3	10±0.8	19.6	0.985	3765	1.23	N.D.	N.D.
	MBBT	2212±453	1492	0.9921	N.D.	N.D.	N.D.	N.D.
	BEMT	3506±641	3129	0.9837	9939	6.81	3756	2.76
	DBT	2829±467	1848	0.9870	N.D.	N.D.	7678	1.26
	a=

Funding

This work was financially supported by the Pierre Fabre Dermo-Cosmetic Laboratories in France.

Conflict of interest statement

We need to disclose that this work has been financially supported by the Pierre Fabre Laboratories, but this does not influence the results. The company was not involved in the work plan and they wanted to have independent data on the occurrence and concentration of some filters that have never been quantified in the natural environment.

Figure Captions


Simone Pinto Carneiro

Laurence Moine

Barbara Tessier

Valérie Nicolas

Orlando David

Henrique Dos Santos

Elias Fattal
email: elias.fattal@u-psud.fr.

Simone Pinto Carneiro

Pyrazinoic acid-Poly(malic acid) biodegradable nanoconjugate for efficient intracellular delivery

Keywords: nanoconjugate, pyrazinoic acid, poly(malic acid), tuberculosis

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

Introduction

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, a pathogen that resides partially within alveolar macrophages [START_REF] Russell | Tuberculosis: What We Don't Know Can, and Does, Hurt Us[END_REF]. TB is considered worldwide as one of the leading causes of death produced by a single infectious agent. It is regarded by many countries as public health and social concerns [START_REF]Global tuberculosis report[END_REF]. The World Health Organization (WHO) recommends a treatment guideline that comprises the association of four anti-tuberculosis drugs within a minimum six-month period [START_REF]Global tuberculosis report[END_REF]. However, daily high doses of each drug required for an effective patient response are associated with the occurrence of systemic and severe adverse effects, mostly hepatotoxicity, affecting consequently patient compliance [START_REF] Du Toit | Tuberculosis chemotherapy: current drug delivery approaches[END_REF], [START_REF] Kaur | Nanostructured drug delivery for better management of tuberculosis[END_REF]. Failed therapy contributes to the emergence of bacteria multidrug-resistant TB that additionally demands the introduction of second-line anti-tuberculosis drugs into the therapeutic regimen, elongating the period of treatment and culminating in side-effects much more toxic [START_REF] Maartens | Tuberculosis[END_REF].

Pyrazinamide (PZA) is one of the four first-line anti-tuberculosis drugs used in the standard protocol of TB treatment. Since PZA was introduced, TB therapy was shortened from 12 to 6 months, due to its specific ability to act against non-growing tubercle bacilli resident inside the acidic pH of phagosome that cannot be killed by other anti-tuberculosis drugs [START_REF] Zhang | The curious characteristics of pyrazinamide: a review[END_REF], [START_REF] Mitchison | The near future: Improving the activity of rifamycins and pyrazinamide[END_REF].

When combined with rifampicin or isoniazid, PZA exhibits unique sterilizing and synergistic effects making this drug essential for a successful treatment. The PZA mechanism of action is still unknown, however, Zhang and co-workers [START_REF] Zhang | The curious characteristics of pyrazinamide: a review[END_REF]. Produced the hypothesis that PZA is a prodrug that enters the bacilli, and is converted into its active moiety, the pyrazinoic acid (PA), through the action of the mycobacterial enzyme pyrazinamidase. Although PA is known as the active PZA metabolite, the use of this drug alone is not recommended, because of its poor lipophilicity and high-ionized state at physiological pH that limit its diffusion and passage through the bacilli cell wall, leading to the drug low bioavailability [START_REF] Cynamon | Pyrazinoic Acid Esters with Broad Spectrum in Vitro Antimycobacterial Activity[END_REF], [START_REF] Fernandes | Synthesis and evaluation of a pyrazinoic acid prodrug in Mycobacterium tuberculosis[END_REF]. Recently, the synthesis of alternative pyrazinoic acid prodrugs which do not require bioactivation by an enzyme arose as a smart strategy for overcoming the above limitations. Cynamon et al. [START_REF] Cynamon | Antimycobacterial activity of a series of pyrazinoic acid esters[END_REF] have proposed novel PA esters with better anti-mycobacterial activity than PZA itself; the same encouraging result with the additional finding of an improved drug stability in serum was demonstrated by Simões et al [START_REF] Simões | Lipophilic pyrazinoic acid amide and ester prodrugs: Stability, activation and activity against M. tuberculosis[END_REF] after evaluating conjugates of PA esters and amide derivatives, reinforcing the potential of these ester products to be more effective than PZA against mycobacteria.

Direct administration of anti-tuberculosis drugs to the lungs offers major advantages over traditional oral therapy [START_REF] Courrier | Pulmonary drug delivery systems: recent developments and prospects[END_REF]- [START_REF] Pham | Pulmonary drug delivery systems for tuberculosis treatment[END_REF]. First, the pulmonary route allows delivering drugs directly on TB target site -the alveolar macrophages. Then, local delivery can achieve a high drug concentration at the site of infection with the intention of improving therapeutic effect and reducing systemic exposure. Sung et al. [START_REF] Sung | Formulation and Pharmacokinetics of Self-Assembled Rifampicin Nanoparticle Systems for Pulmonary Delivery[END_REF] corroborated this hypothesis by developing a dry powder composed of rifampicin porous-nanoparticle aggregate particle (PNAP) and performing a pharmacokinetic study in vivo. Formulations administered by intratracheal insufflation were effective in maintaining rifampicin amounts in the lungs over a longer period of 8 hours, whereas after oral or intravenous delivery, no more rifampicin was detected. Interestingly, the same pulmonary route was tested for the administration of an aerosol without nanoparticle formulation and rifampicin was not detected in the lungs after 8 hours, emphasizing the advantageous strategy of delivering nanoparticles via the pulmonary route.

Numerous studies on the design of nanoparticle-based anti-TB drugs over the past decade report their potential to improve drug bioavailability and reduce drug dosing frequency [START_REF] Pham | Pulmonary drug delivery systems for tuberculosis treatment[END_REF],

[16]- [START_REF] Pandey | Nanoparticle encapsulated antitubercular drugs as a potential oral drug delivery system against murine tuberculosis[END_REF]. However, despite promising results, physical loading of anti-TB drugs in nanoparticles was characterized by a poor loading efficiency and a burst release. As an alternative strategy, in this work, drug-polymer conjugation has been considered to improve the absorption and bioavailability of anti-tuberculosis-type medications. Compared to conventional nanoparticles, nanoconjugates reveal attractive properties such as high-loading capacity and sustained release without drug leakage among others [START_REF] Abioye | Polymer-Drug Nanoconjugate -An Innovative Nanomedicine: Challenges and Recent Advancements in Rational Formulation Design for Effective Delivery of Poorly Soluble Drugs[END_REF], [START_REF] Seidi | Designing Smart Polymer Conjugates for Controlled Release of Payloads[END_REF]. After administration, these nanoconjugates can be easily degraded through an enzymatic or hydrolytic mechanism, releasing the active molecule directly on the target site.

In the field of drug delivery systems, several (bio)degradable polymers have been studied to formulate polymer-drug conjugates. Among them, poly(malic acid) (PMA), an aliphatic polyester, has been proven to be a promising candidate [START_REF] Loyer | Natural and synthetic poly(malic acid)-based derivates: a family of versatile biopolymers for the design of drug nanocarriers[END_REF]. In addition to these excellent degradation and biocompatibility properties, presence of carboxyl group on each repeating unit allows for chemical modification with a large spectrum of therapeutic molecules. It has been extensively investigated as a water-soluble, biodegradable, and biocompatible polymer, with remarkable advantages in clinical use [START_REF] Ljubimova | Poly(malic acid) nanoconjugates containing various antibodies and oligonucleotides for multitargeting drug delivery[END_REF].

The nanoconjugate, we designed, is based on poly(malic acid), a water-soluble polyester. PA was chemically conjugated to the polyester backbone via a hydrolyzable carboxylic acid ester linker. To improve the stability and afford self-assembly of the conjugate system, a hydrophobic spacer was introduced between the polymer and the drug. After synthesis, the self-assembly behavior was studied and characterized and in vitro release was investigated.

Cytotoxicity and nanoconjugate cell-uptake were evaluated on macrophages, the cell target.

Materials and Methods

For synthesis experiments, all solvents were of analytical or HPLC grade. L-malic acid, N,Ndicyclohexylcarbodiimide (DCC) and 4-dimethyl aminopyridine (DMAP) were purchased from Sigma-Aldrich (France). Pyrazinoic acid (PA) and 1,8-octanediol were provided by TCI (Europe, N.V.). Rhodamin B-PLGA was supplied from PolySciTech (USA). All reagents were used as received. For cell culture experiments, Dulbecco's Modified Eagle Medium (DMEM), 3-[4,5-dimethylthiazol-2-yl]-3,5 diphenyl tetrazolium bromide (MTT), penicillin, streptomycin, and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (France). Fetal bovine serum (FBS) was obtained from Thermo Fischer Scientific (USA).

Experiments

Synthesis of PMA

PMA was obtained by polycondensation of L-malic acid [START_REF] Su | Functionalization of biodegradable hyperbranched poly(α,β-malic acid) as a nanocarrier platform for anticancer drug delivery[END_REF]. 5 g of L-malic acid (37 mmol) was added into a schlenk flask and heated at 110 ºC during 72h under 0.7-1 mmHg. The viscous product was dissolved in tetrahydrofuran (THF) and purified twice by precipitation into a diethyl ether:petroleum ether (60:40). The precipitated polymer was dissolved in purified water and freeze-dried for 24h (Alpha 2-4 LD Plus, Christ). L-malic conversion = 76% ( 1 H NMR).

Synthesis of PA conjugates

PA conjugate was obtained by esterification reactions, performed in two steps.

Step 1: Synthesis of intermediate PA:Oct product. 2 g of PA (16.1 mmol) and 2.3 g of 1,8octanediol (16.1 mmol) were dissolved in 80 ml of anhydrous THF. In another round bottom flask, 2.5 g of DCC (12.1 mmol) and 0.2 g of DMAP (1.6 mmol) were dissolved in 40 ml of anhydrous THF. After complete dissolution, DCC:DMAP solution was added dropwise into the PA:1,8-octanediol solution and the reaction went over overnight at room temperature. The reaction mixture was filtered and the solvent was evaporated. The crude product was purified by column chromatography, using ethyl acetate as eluent. Yield = 50%.

Step 2: Synthesis of PA conjugate. In a round bottom flask, 614 mg of PMA and 466 mg of PA:Oct (molar ratio: n MA unit = 2.9 n PA:Oct ) were added and dissolved in 20 ml of anhydrous THF. In another flask, 572 mg of DCC (2.78 mmol) and 64.5 mg of DMAP (0.53 mmol) were dissolved in 20 ml of anhydrous THF. Both solutions were maintained under argon flow and magnetic stirring. After complete dissolution, the DCC:DMAP solution was added dropwise to the PMA-OctPA solution, at 0 °C for 30 min. The reaction was carried overnight at room temperature, then filtered and the product was condensed by solvent evaporation. The crude product was dissolved in chloroform and purified twice by precipitation into a diethyl ether:petroleum ether solution (60:40). PMA:OctPA was dried under vacuum and recovered as a slightly brown powder.

PMA and conjugate characterization

Preparation of PA-nanoconjugate

The labeled NC-PA was obtained by the nanoprecipitation method, which is based on nanoparticle formation through rapid diffusion of the solvent into the aqueous phase [START_REF] Fessi | Nanocapsule formation by interfacial polymer deposition following solvent displacement[END_REF].

Briefly, 5 mg of PMA:OctPA was dissolved in 0.5 ml of THF. This organic phase was placed into a syringe-pump apparatus and then slowly added to an aqueous phase, composed of 5 ml of purified water, at a constant rate of 7 ml/min and under moderate magnetic stirring. The solvent was evaporated, yielding nanoparticles at 1.0 mg/ml. In addition, fluorescent nanoparticles labeled with Rhodamin B-PLGA were also prepared following the same procedure and replacing the total amount of conjugate to a mixture of 5% of Rhodamin B-PLGA and 95% (w/w) of PMA:OctPA, labeled NC-PAFLU.

Nanoconjugate characterization

Particle size and zeta potential analysis

NC-PA was characterized by means of particle size and polydispersity index (PDI) through dynamic light scattering technique and zeta potential by electrophoretic mobility. For size distribution measurements, each sample was diluted in purified water (1:10 ratio), while for surface charge measurements, the same dilution ratio was performed in a 1 mM NaCl solution. Analyses were carried out in triplicate on three different batches at 20 ºC, using a Zetasizer Nano Zs (Malvern Instruments, UK).

Transmission electron microscopy (TEM)

Morphology of NC-PA was analyzed by TEM images. 5 µl of nanoconjugate was deposited onto copper grids covered with a formvar film (400 mesh) for 2 min. Then, NC-PA were covered with 5 µl of a 0.5% (w/v) uranyl acetate solution, for 30 s. Colorant excess was removed with a filter paper and samples were observed in a JEOL JEM-1400 microscope operating at 80 kV with a filament current of about 55 µA, a post-column high-resolution (11 megapixels) and a high-speed camera (SC1000 Orius; Gatan). Data were acquired from Digital Micrograph (Gatan).

Stability tests

NC-PA was stored at refrigerated (4 ºC) and physiological (37 ± 2ºC) conditions for 60 days. Periodically, a sample was collected and evaluated as particle size and PDI. Analyses were done in triplicate on three different batches at 20 ºC.

In vitro drug release

2 ml of NC-PA at 1.0 mg/ml were separately dispersed into 8 ml of PBS (pH 7.4).

Then, aliquots of 0.5 ml were stored into vials, incubated at 37 ºC under shaking and protected from light. During a period of 5 days, at each 12 h, a sample from each nanoconjugate was collected and frozen. At the end of the experience, samples were unfrozen and the amounts of PA released at each time point were quantified by an HPLC method described below. Free PA solutions were also prepared in the same conditions and analyzed using the same procedure to follow the potential degradation of the drug.

HPLC system

HPLC was employed to quantify PA amounts. The system was composed of a Waters 1525 Binary HPLC Pump, an injector Waters 2707 Autosampler and an UV-visible Waters 2998 Photodiode Array Detector. Data acquisition was done using the Empower software. PA was assessed following the protocol described by Simões et al [START_REF] Simões | Lipophilic pyrazinoic acid amide and ester prodrugs: Stability, activation and activity against M. tuberculosis[END_REF]. An RP18 Hydro (250 mm x 4.6 mm x 5.0 µm, Phenomenex) column was used at a temperature of 35 ºC. The eluent was composed of a mixture of acetonitrile and 25 mM phosphate buffer (pH 2.0 adjusted with phosphoric acid) in a proportion of 5:95% (v/v). The isocratic flow rate was 1.0 ml/min and UV detection was set at λ = 267 nm. PA retention time was 5.47 min. Values reported on each time point analysis were plotted on a PA calibration curve (5 -200 µg/ml) and quantified following the equation y = 65936x -92640 (R² = 0.9999).

Cell culture assays

Cell culture

Murine alveolar macrophages -RAW 264.7 cells (ATCC ® TIB-71™) were cultured in DMEM supplemented with 10% FBS, 50 U/ml penicillin and 50 U/ml streptomycin. Cells were incubated at 37 ºC in a 5% CO 2 humidified atmosphere and passaged twice a week at a 1:10 ratio.

Cytotoxicity

Cytotoxicity of nanoconjugate was evaluated using the MTT test, as previously described by Mosmann [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays[END_REF]. Cells were seeded at a density of 4000 cells per well in 96-well plates and pre-incubated for 24 h. Culture medium was discarded and cells were treated with a different range of nanoconjugate concentration diluted in fresh medium. PA and pure PA were incubated at 1, 5, 10, 25, 50 and 100 µg/ml with cell culture. Untreated cells were used as control. Plates were incubated for 48 h and then 20 µl of a 5 mg/ml MTT solution was added in each well for more 2 h to enable crystals formation. Culture medium was removed and 200 µl of DMSO was added to dissolve formazan crystals. Plates were shaken for 10 minutes at room temperature (20 ± 2 ºC) and the absorbance was measured at 540 nm in a microplate reader. Cell viability, expressed as a percentage, was calculated as the absorbance ratio of treated cells to untreated cells (control). The IC 50 , expressed as the concentration required by each treatment to induce cell death of 50%, was calculated using a sigmoidal regression and GraphPad Prism 6 software (USA).

Nanoconjugate cellular uptake

The kinetics of nanoconjugate internalization by RAW 264.7 macrophages was investigated by confocal microscopy, following an adapted protocol described by Grabowski et al [START_REF] Grabowski | Surface coating mediates the toxicity of polymeric nanoparticles towards human-like macrophages[END_REF]. Briefly, cells were seeded at a density of 80000 cells per well in 6-well plates, containing a glass coverslip in each well. 24h after incubation at 37 ºC in a 5% CO 2 humidified atmosphere, non-adherent cells were washed off and all other cells were treated with fluorescent-labeled PA nanoconjugate (NC-PAFLU) at 10 µg/ml diluted in a fresh culture medium. Untreated macrophages were used as control. Cells re-incubated at the same conditions and, at pre-determined intervals (0.5, 1.5, 3 and 6h), they were assessed with an LSM 510 (Zeiss-Meta) confocal microscope equipped with Argon (488 nm, 300 mW) and Helium-Neon (543 nm, 5 mW) lasers, and a plan-apochromat 63x objective. Data were acquired from LSM 510 software, version 3.2. In addition, for each pre-determined time evaluated on a microscope, an average of 20 cells was selected and fluorescence was quantified inside them, using ImageJ software.

Statistical analysis

Statistical analysis data are presented as mean values with standard deviation (SD).

Statistical analysis was performed with the Prism Graph-Pad 7.0 software and the significance was calculated using a one-way ANOVA method, in which p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.005, *** p < 0.0005, **** p < 0.0001).

Results and Discussion

Conjugate synthesis and characterization

In this study, it was important to make a judicious choice of the degradable polymer backbone to allow the self-assembly and loading capacity of nanoconjugates. Poly(malic acid) (PMA) has been extensively investigated as a water-soluble, biodegradable, and biocompatible polymer, with remarkable advantages in clinical use [START_REF] Ljubimova | Poly(malic acid) nanoconjugates containing various antibodies and oligonucleotides for multitargeting drug delivery[END_REF]. PMA is a non-toxic polymer since it degrades into malic acid after administration, which is an intermediate product of carbohydrates metabolism and therefore, non-toxic to cells [START_REF] He | Synthesis and degradation of biodegradable photo-cross-linked poly(α,β-malic acid)-based hydrogel[END_REF]. In addition, PMA contains multiple carboxylic functions that can be used to introduce molecules of interest via ester hydrolyzable linkages affording high payload nanoconjugates.

The copolymer of malic acid carrying pyrazinoic acid was synthesized via a simple coupling of the drug on the polymer backbone through an ester cleavable bond, enabling degradation of PMA-conjugate into biocompatible metabolites.

PMA was first prepared by direct polycondensation of L-malic acid which was carried out under reduced pressure without the use of any catalyst [START_REF] He | Synthesis and degradation of biodegradable photo-cross-linked poly(α,β-malic acid)-based hydrogel[END_REF]. After purification, a high yield was achieved, up to 80%, demonstrating that conditions set for this reaction were suitable to obtain large batches. Formation of PMA was confirmed by 1 H NMR analysis through the presence of CH 2 at 2.9 ppm and CH at 5.4 ppm [START_REF] Ouchi | Synthesis of poly(αmalic acid) and its hydrolysis behavior in vitro[END_REF], [START_REF] Kajiyama | Improved synthesis with high yield and increased molecular weight of poly(α,β-malic acid) by direct polycondensation[END_REF]. The molecular weight of the polymer was in the range of 2,000 -4,000 g/mol.

Then, PA conjugate was synthesized following a two steps process. Prior to PA conjugation onto PMA, a hydrophobic spacer was coupled to the drug through its carboxylic function. The use of pyrazinoic acid ester instead of PA provides two additional ester groups that can be further hydrolyzed to release the drug from the conjugate. Moreover, in a recent study pyrazinoic acid ester analogs have shown a higher efficacy than pyrazinamide [START_REF] Simões | Lipophilic pyrazinoic acid amide and ester prodrugs: Stability, activation and activity against M. tuberculosis[END_REF]. 1,8-Octanediol was coupled to PA through a classical esterification reaction as shown in Fig. 1.

Formation of n-octyl pyrazinoic acid (PA:Oct) was confirmed by mass spectrometry through m/z fragment at 252.2 and 1 H NMR by the appearance of the signal at 4.4 ppm attributed to COO-CH 2 -protons (Fig. S1, supporting information).

Nanoconjugate formation

Nanoconjugates were prepared by a simple process of nanoprecipitation taking advantage of the amphiphilic character of PA conjugate which easily self-assembled into nanoparticles in aqueous solution. This method is very accurate to produce monodisperse and stable nanoparticles, without the addition of surfactant and without the use of toxic solvents [START_REF] Martínez Rivas | Nanoprecipitation process: From encapsulation to drug delivery[END_REF]. DLS analyses showed uniform distributions of size with an average diameter around 145nm and a low polydispersity index of 0.13. Mean zeta potential evidenced a negatively surface charge of -58mV, consistent with the presence of non-conjugated carboxylic residues on the final product which considerably influences the surface charge of the resulting nanoconjugate.

To visualize their morphology and size, nanoconjugates were imaged by TEM with negative staining. As shown in Figure 2, a rather homogenous particle size population was observed with an average size between 150 and 200nm in diameter, which was consistent with DLS data. Moreover, TEM results also evidenced the spherical shape of nanoconjugates and the absence of particle aggregation even without the use of stabilizers. Stability of the nanoconjugates was evaluated at low (4°C) and physiological conditions (37°C) over a period of 60 days. Interestingly, NC-PA exhibited excellent colloidal stability independently of the storage temperature (Fig. 3). No aggregation was observed and the distribution remained narrow over the whole period.

The loading rate of pyrazinoic acid, is similar to the feed composition determined for PMA:OctPA conjugate since the nanoconjugates are completely formed from the esterified poly(malic acid), no additive has been introduced during the nano-precipitation step.

Therefore, it corresponds to approximately 16 wt% of PA in the final NC-PA. This loading is much higher than the one obtained with a simple physical encapsulation of pyrazinamide, the esterified drug, in PLGA nanoparticles (around 4wt%) [START_REF] Pham | Pyrazinamide-loaded poly(lactide-co-glycolide) nanoparticles: Optimization by experimental design[END_REF].

Release study

Drug release study of the nanoconjugate NC-PA was conducted at 37°C and physiological pH in PBS over a period of 10 days. Free PA was also assessed as control, no drug degradation was observed in this elapsed time. As shown in Fig. 4, a gradual and complete drug release was achieved in almost 6 days from the nanoconjugate. The initial release of 10% at 15 min can be attributed to free PA adsorbed on nanoconjugate surface and not attached to the polymer. As expected with this kind of drug delivery system, a sustained release of pyrazinoic acid with no burst release was observed through the hydrolysis of the ester linker between PA and PMA polymer. These drug-controlled release results are desirable in overall TB treatment [START_REF] Costa | The formulation of nanomedicines for treating tuberculosis[END_REF], once a unique dose is able to maintain therapeutic concentration over a long period and consequently, minimize the drug toxic effects during TB therapy, which may help in increasing patient compliance.

T im e ( h )

In vitro internalization and cytotoxicity studies

Cytotoxicity of PA nanoconjugates was monitored by MTT assay. NC-PA formulation was incubated with macrophages cells in different ranges of increasing concentrations for 48h. Results (Fig. 5) expressed by means of percentage of cell viability showed that nanoconjugates did not exhibit a toxic potential for the majority of concentrations tested (cell viability over 70%). Toxicity observed must come exclusively from drugs inherent in the constitution of nanoconjugates since previous studies carried out by our team have shown that PMA has no toxicity to these cells even at concentrations much higher than those used in this work (unpublished work, see Fig. S3, supporting information). Both free drug and PA nanoconjugates did not present a toxic potential up to 25µg/ml. Above this concentration, cell viability decreased achieving 52 and 40% for free PA and NC-PA, respectively, at 100µg/ml. The higher cell viability observed for free PA could be explained by drug physicochemical properties. As PA is weakly hydrophobic and highly ionized at physiological pH [START_REF] Fernandes | Synthesis and evaluation of a pyrazinoic acid prodrug in Mycobacterium tuberculosis[END_REF], it is less likely that this molecule will cross the cell membrane, causing a lower cell death than PA nanoconjugates which are internalized by macrophages as showed below. Similar behavior was described by Heifets and co-workers [START_REF] Heifets | Does pyrazinoic acid as an active moiety of pyrazinamide have specific activity against Mycobacterium tuberculosis?[END_REF], who reported unexpected higher minimum inhibitory concentration results for PA than for pyrazinamide against susceptible M. tuberculosis strain. The authors associated this finding to inefficient solubilization of PA stock solution into water, which leads to the formation of insoluble precipitates after dilution in the culture medium. Similarly to this study, the toxic effect of free PA used in this work may be underestimated. The main triggering factor for tuberculosis management is the administration of antituberculosis drugs in therapeutic quantities at the target sites i.e. alveolar macrophages. These cells are an interesting target in TB treatment, once after inhalation of M. tuberculosis, bacteria infect alveolar macrophages and inhibit the phagosome-lysosome fusion, deactivating the defensive mechanisms of these cells and using them as a reservoir to replicate and survive for a long period [START_REF] Russell | Tuberculosis: What We Don't Know Can, and Does, Hurt Us[END_REF]. Therefore, in order to propose an efficient nanocarrier to fight against TB, it is relevant to assure the capacity of macrophages to uptake nanoparticles. For this reason, PA nanoconjugates developed in this work were fluorescently labeled (NC-PAFLU)

and incubated with macrophages at 10 µg/ml (subtoxic concentration) from 0.5 to 6 hours, at 37ºC. Internalization of NC-PAFLU by cells was followed by confocal microscopy and results are reported on Fig. 6. According to the observations, red fluorescence inside cells demonstrated that nanoconjugates were effectively taken up by macrophages over time.

Besides, these results pointed out that nanoconjugates were located within the cytoplasm rather than merely attached on the cell membrane. This ability to spread within cell content expressed by NC-PAFLU is particularly interesting in TB therapeutic since nanoconjugates can easily face mycobacteria and release drugs directly on the disease target site. Finally, NC-PA and PA toxicity were quantitatively expressed by means of IC 50 . In order to induce 50% cell death, doses of 85.42 and 127.4 µg/ml are required for NC-PA and PA respectively. According to these results, NC-PA can be considered as a safe nanoconjugate since its IC 50 is far higher than the concentration necessary for an efficient cell uptake (10 µg/ml). This finding corroborates the main goal of this work in proposing safe and efficient nanoconjugates able to effectively be taken up by macrophages and improve TB treatment.

h 1.5 h 3 h 6 h Control

Conclusions

In this work, we demonstrated that PA biodegradable nanoconjugates were successfully synthesized as a novel and smart strategy for lung administration. Lateral carboxylic groups of PMA were directly conjugated with PA by a simple esterification reaction, ensuring conjugates to self-assemble into nanometric, negatively surface charged and spherical nanoconjugates. NC-PA evidenced a sustained drug release, suggesting the ability of this nanocarrier to maintain drug concentration over time and consequently, to reduce doses and frequency of administration. Allying these findings with the relevant cell culture results, which evidenced that nanoconjugates were effectively taken up by cells for 6 hours and did not present a cytotoxic profile for a wide range of concentrations, we concluded to the discovery of an innovative material with a promising potential in reducing side effects and improving TB therapy. These preliminary results encourage to move with these nanoconjugates to the perspective of entrapping them into trojan microparticles to enable pulmonary administration and delivery on alveolar macrophages.

1 H

 1 NMR spectroscopy was performed in 5 mm diameter tubes in CDCl 3 on a Bruker Avance-300 (300 MHz) spectrometer. Intermediate product from PA conjugate synthesis (PA:Oct) was characterized by electrospray ionization mass spectrometry (LTQ-Velos Pro Thermofisher Scientific). The molar mass and dispersity of polymers were measured by size-exclusion chromatography (SEC). Measurements were performed at 30°C with two columns from Malvern Panalytical (Viscotek LT4000L Mixed, Low 300 x 8 mm), a triple detection system (Viscotek 270 Dual Detector and Waters 2414 Refractive Index Detector) coupled with a Waters 515 HPLC pump and Waters 717plus Autosampler. THF was used as the eluent with a flow rate of 1 mL/min.

Fig. 1 :

 1 Fig. 1: Synthesis route for pyrazinoic acid ester conjugate (PMA:OctPA).

Fig. 2 :

 2 Fig. 2: TEM images with negative staining of NC-PA (scale bar = 200 nm).

Fig. 3 :

 3 Fig. 3: Stability tests performed for NC-PA stored at 4 and 37 ºC for 60 days, represented in terms of (A) particle size, and (B) PDI. Data represent the mean ± standard deviation (n = 3).

Fig. 4 :

 4 Fig. 4: In vitro drug release of NC-PA in PBS buffer at 37 ºC. Data represent the mean ± standard deviation (n = 3).

Fig. 5 :

 5 Fig. 5: Percentage of cell viability over ranges of nanoconjugates concentrations incubated with RAW 264.7 macrophages for 48h. Results express the cytotoxic profiles of PA and NC-PA. Data represent the mean ± standard deviation (n = 3).

Fig. 6 :Fig. 7 :

 67 Fig. 6: Uptake kinetics of NC-PAFLU incubated with RAW 264.7 macrophages cell line at 10 µg/ml for different time intervals. (A) Control (cells without treatment), (B) 0.5 h, (C) 1.5 h, (D) 3 h, (E) 6 h. Mean fluorescence inside cells was quantified to investigate the kinetics of the nanoconjugate. Data in Fig. 7 express a general profile for NC-PAFLU with increasing fluorescence over time to a maximum value after 3h of incubation and then no significant change was observed at 6h. At the optimum time point, the internalization of nanoconjugates by macrophages was maximal.

Acknowledgements

Authors would like to thank Stéphanie Denis (Institut Galien Paris-Sud) for her help with cell culture and Valérie Nicolas from MIPSIT for helping with confocal microscopy. The present work has benefited from the core facilities of Imagerie-Gif, (http://www.i2bc.paris-saclay.fr), member of IBiSA (http://www.ibisa.net), supported by "France-BioImaging" (ANR-10-INBS-04-01), and the Labex "Saclay Plant Science" (ANR-11-IDEX-0003-02).

Acknowledgements

The authors thank the financial support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes, Brazil) and Institut Galien Paris-Sud. Thanks to Anne Beilvert and Emeline Servais for their chemical help with the synthesis procedure, Audrey Solgadi for mass spectrometry analysis, BioCis platform for 1 H NMR analysis. The present work has benefited from the core facilities of Imagerie-Gif, (http://www.i2bc.paris-saclay.fr),

member of IBiSA (http://www.ibisa.net), supported by "France-BioImaging" (ANR-10-INBS-04-01), and the Labex "Saclay Plant Science" (ANR-11-IDEX-0003-02) with the precious help of C. Boulogne and C. Gillet.


Yifei Ma
email: mayifei718@163.com

Xuetao Li
email: lixuetao1979@163.com

Wenjie Zhang
email: wenjiezhang509@hotmail.com

Xixiang Ying
email: yingxixiang@163.com

Didier Stien
email: didier.stien@cnrs.fr

Prof Ying Xixiang

Prof. Didier Stien

A trace alkaloid, oleraisoindole A from Portulaca oleracea L. and its anticholinesterase effect

Keywords: Portulaca oleracea L., alkaloids, anticholinesterase effect

A new trace alkaloid possessing the lignan structure, named oleraisoindole A, was obtained from the extract of the Portulaca oleracea L.. The structure of oleraisoindole A was elucidated by 1D and 2D NMR and high resolution electrospray ionization time-of-flight mass spectroscopic methods. The compound presented an anticholinesterase effect with the IC50 value of 60.4 μM.

Introduction

Portulaca oleracea L., a member of the Portulacaceae family has been used as a vegetable, spice and medicine since the times of the ancient Egyptian and was popular in England during the Middle Ages 33 [START_REF] Okafor | A review on Portulaca oleracea (Purslane) plant -Its nature and biomedical benefits[END_REF]. In China, P.

oleracea is a traditional Chinese medicine well-known for its pharmacological function of removing heat, counteracting toxicity, cooling blood, hemostasia and antidysentery (The Pharmacopoeia Commission of PRC, 2015) owing to its many bioactive compounds, including alkaloids [START_REF] Li | Two new similar alkaloids from Portulaca oleracea L[END_REF][START_REF] Xu | A novel alkaloid from Portulaca oleracea L[END_REF][START_REF] Zhao | A new lactam alkaloid from Portulaca oleracea L. and its cytotoxity[END_REF][START_REF] Xiu | A new alkaloid from Portulaca oleracea L. and its antiacetylcholinesterase activity[END_REF], flavonoids (Yang et al. 2018;Yang et al. 2018), coumarins [START_REF] Awad | Lipid content and antimicrobial activity of phenolic constituents of cultivated Portulaca oleracea L[END_REF], lignans [START_REF] Ma | Four lignans from Portulaca oleracea L. and its antioxidant activities[END_REF], phenolic acids [START_REF] Erkan | Antioxidant activity and phenolic compounds of fractions from Portulaca oleracea L[END_REF], terpenoids [START_REF] Elkhayat | Portulene, a new diterpene from Portulaca oleracea L[END_REF]. As alkaloids are the main components of the plant and presents many bioactivities, such as antiinflammatory [START_REF] Meng | The anti-inflammation and pharmacokinetics of a novel alkaloid from Portulaca oleracea L[END_REF][START_REF] Jiang | An isoindole alkaloid from Portulaca oleracea L[END_REF], antimalarial [START_REF] Frederich | Potential antimalarial activity of indole alkaloids[END_REF]) and antioxidant activities [START_REF] Racková | Antiradical and antioxidant activities of alkaloids isolated from Mahonia aquifolium. Structural aspects[END_REF]), etc.. Therefore, the aim of the study is to isolate alkaloids, a trace new alkaloid, named oleraisoindole A (Figure 1) was finally obtained from the extract of P. oleracea. In addition, its anticholinesterase effect was studied as Alzheimer's diseasehas (AD) related to acetylcholinesterase (AChE) [START_REF] Howes | Plants used in chinese and indian traditional medicine for improvement of memory and cognitive function[END_REF].

Results and discussion

Oleraisoindole A (1 mg, purity of > 99% with UHPLC) was obtained as a pale yellow powder, which turned orange and cyan after spraying Dragendorff reagent and ferric chloride on a TLC plate. UV-Vis (MeOH) λ max : 284 nm. IR (KBr) ν max : 3425, 1756, 1706, 1600, 1515, 1400, 1265, 1212 cm -1 . Its molecular formula was identified as S1, in supplementary material). A 3-spin system was found, which linked to carbon C-4 at δ137.0 and defined a 1,3,4-trisubstituted phenyl moiety based on 1 H-1 H coupling pattern, COSY and HMBC experiment. There is also a typical of para-disubstituted phenyl ring, in which C-2'' was linked to C-1''' as the HMBC spectrum shows the correlation H-2'''/C-2''. The 1 H-1 H COSY and DEPT spectrum shows the correlation position 1"(δ H 3.66, δ C 39.0)/2"(δ H 2.74, δ C 32.9). The highfield chemical shift of position 2'' was obviously different with that of the oleraisoindole (Table S2 and Figure S1 in supplementary material) previously isolated in our lab [START_REF] Jiang | An isoindole alkaloid from Portulaca oleracea L[END_REF]) meaning that CH 2 in oleraisoindole A only connected to position 1'' that was typical of CH 2 connected to N, and N linked to C-1 and C-3. In HMBC, H-1'' correlated to carbonyl groups C-1 and C-3 at δ166.9 and 167.4, respectively. In 1 H-NMR, only three singlet CH signals and nine quaternary carbons were remained and attached to the established peripheral groups. Protons H-8 and H-9 were not coupled to each other. Nevertheless, they correlated in ROE indicating that they were close to each other but not attached to a single phenyl ring. Altogether, these indications led to position two periprotons on a naphthalene moiety. Based on the strong ROE correlation between methyl group and H-8, the methoxy substituent at the C-7 position was determined. H-9 correlated to C-4a and H-8 which based on HMBC and ROE, respectively. This further confirms the relative positions of H-8 and H-9. H-9 also correlated to C-1 and C-3a. It was therefore to link C-1 to C-9a, and the C-3 linked C-3a is reasonable based on chemical shift of the quaternary carbon C-3a (δ121.6). Since the singlet proton H-5 at δ7.12 correlated to both C-6 and C-7, it should to be placed in paraposition with respect to H-8. H-5 also correlated to C-4 and C-8a from their weak correlation, and the ROE correlation between H-5 and H-6' confirmed the position of the tetrasubstituted phenyl ring. The last carbon C-9a remained was δ124.9 compared with that of oleraisoindole (Table S2). As a result, the compound was identified as the 6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-2-(4hydroxyphenethyl)-7-methoxy-1H-b enzo[f]isoindole-1,3(2H)-dione and was given the trivial name oleraisoindole A (Experimental section, in supplementary material).

Among these natural products, alkaloids are considered to be the most promising candidates for use in the treatment of AD, such as physostigmine (eserine) have been long recognized as acetyl-and butyrylcholinesterase (BChE) inhibitors, due to their complex nitrogen-containing structures [START_REF] Pereira | Pharmacological effects of catharanthus roseus root alkaloids in acetylcholinesterase inhibition and cholinergic neurotransmission[END_REF]. The inhibition of AChE, the key enzyme in the breakdown of acetylcholine, is currently the main pharmacological strategy available for AD [START_REF] Konrath | Alkaloids as a source of potential anticholinesterase inhibitors for the treatment of alzheimer's disease[END_REF]. Consequently, the anticholinesterase effect of oleraisoindole A was determined using a microplate assay with the sample of concentrations from 2.5 to 40 μM, and inhibited 50% of AChE activity (IC50) were presented in Table S3. Figure S14 showed that the anticholinesterase effect of oleraisoindole A was dose-dependent. Anticholinesterase effect was determined according to the modified Ellman method (Ellman et al. 1961).

The sample solutions in methanol was diluted into five different concentrations and the AChE solution in buffer of PBS (pH 8.0, containing 0.1 mol/L Na 2 HPO 4 and NaH 2 PO 4 ) were mixed in wells a 96-well microplate. Then incubated for 10 minutes at 37°C and added 5,5-dithiobis-2-nitrobenzoic acid (DTNB) to the buffer. Finally, acetylthiocholine iodide (ATCI) was added and the absorbance was monitored spectrophotometrically at 405 nm. The same concentration of eserine was used as a positive control. Instead of samples, methanol was included in the blank control.

Each sample was measured in parallel five times and the percentage of inhibition of AChE was calculated using the following formula (Šinko et al. 2007):

Anticholinesterase effect (%) = [(Ablank -Asample)/Ablank] × 100% where, Asample is the absorbance of the test compound, and Ablank is the absorbance of the blank control. The anticholinesterase effect was evaluated by the value of IC50, and values are expressed as the means ± SD for n = 5.

Conclusion

A trace (1 mg) new alkaloid, oleraisoindole A possessing the lignan structure was isolated from the extract of P. oleracea of 250 kg, and its structure was complicated that only can be determined by 600 MHz NMR with cryogenic probe. In addition, oleraisoindole A presented anticholinesterase effect with an IC 50 value of 60.4 μM.

Figure 1 .

 1 Figure 1. Structure of Oleraisoindole A.

C

  28 H 23 NO 7 with 18 degrees of unsaturation based on a molecular ion peak at m/z 484.1404 [M+H] + in the HR-ESI-TOF-MS (calcd. for C 28 H 23 NO 7 , 484.1401) and the NMR data (Table

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant 81573546).

Supplementary material

Supporting information can be found in the online version of this article.

Disclosure statement

The authors have no conflicts of interest to disclose.