RESEARCH Open Access Antioxidant Polyphenol-rich Extracts .

Marimoutou et al. Journal of Inflammation (2015) 12:10Page 3 of 15polyphenols depended on their chemical nature, dose andtime of exposure [22,23]. There is more and more evidence that the consumption of medicinal plants couldcontribute to increase the daily intake of polyphenols [18].Three medicinal plants from the Indian Ocean area aretraditionally used in order to reduce the incidence ofobesity and diabetes, namely Antirhea borbonica J.F.Gmelin (Rubiaceae), Doratoxylon apetalum (Poir.)Radlk. (Sapindaceae) and Gouania mauritiana Lam.subsp. Mauritiana (Rhamnaceae). Even if antioxidantand anti-inflammatory properties have been attributedto some medicinal plants from the same species orgenus [24-26], there is still a lack of data regardingtheir effect on adipose cell biology.Our objective was to evaluate for the first time the antioxidant and anti-inflammatory properties of polyphenolrich extracts from A. borbonica, D. apetalum and G.mauritiana medicinal plants on preadipocytes exposed toH2O2, TNFα or LPS. We determined their effects on cellviability, the production of ROS, IL-6 and MCP-1 proinflammatory markers, as well as on the expression ofgenes coding for SOD and catalase antioxidant enzymes,and for NF-κB transcription factor.MethodsDetermination of antioxidant polyphenol content inmedicinal plant extractsPlants were selected according to their endemic and indigenous status at Réunion Island based in the IndianOcean area. All plants tested are commonly used intraditional medicine, although there is a lack of published data concerning their biological effects. Table 1lists the botanical terms, the voucher number and theparts used. Plant materials were collected during August2009 and March 2010. They were harvested from various locations in Réunion Island. Botanists of the University of Réunion Island confirmed the identity of all plantmaterials. After airflow drying (45 C), plant organs werereduced to powder.Table 1 Global description of the medicinal plants testedBotanical nameGouania mauritianaaFamilyVoucher numberParts usedRhamnaceaeRUN-081ELeaf, stemRubiaceaeRUN-052 FLeaf, stemSapindaceaeRUN-055ELeafLam. Subsp. mauritianaAntirhea borbonicabJ.F. GmelinDoratoxylon apetalum c(Poir.) RadlkCommon names:a: Liane Montbrun.b: Bois d’Osto.c: Bois de gaulette.Quantification and identification of polyphenols inmedicinal plant extractsPolyphenol-rich extracts from medicinal plants were obtained after dissolving each plant powder (2 g) in 20 mLof an aqueous acetonic solution (70%, v/v). After incubation at 4 C for 90 min, the mixture was centrifuged at3500 rpm at 4 C for 20 min and polyphenol-rich supernatants were collected and stored at 80 C until analysis.To determine polyphenol contents in plant extracts,Folin-Ciocalteu test was used [27]. Briefly, 25 μL plantextract, 125 μL Folin-Ciocalteu’s phenol reagent (SigmaAldrich, Germany) and 100 μL sodium carbonate wereadded in a 96-well microplate and incubated at 54 Cfor 5 min and then at 4 C for 5 min. The absorbancewas measured at 765 nm (FLUOstar Optima, BmgLabtech, Germany). A calibration curve was preparedusing a standard solution of gallic acid (Sigma-Aldrich,Germany). Total phenol content was expressed as ggallic acid equivalent (GAE) / 100 g plant powder. Totalflavonoids were also measured using a colorimetric assayadapted from Zhishen et al. [28]. For this experiment,100 μL of each polyphenol-rich plant extract wereplaced in a 96-well microplate with 6 μL of 5% aqueousNaNO2. After 5 min, 6 μL of 10% aqueous AlCl3 wereadded and the mixture was vortexed. Then, after 1 min,40 μL of 1 M NaOH were added. The absorbance wasmeasured at 510 nm (FLUOstar Optima, Bmg Labtech,Germany). A calibration curve was prepared using astandard solution of catechin (Sigma-Aldrich, Germany).Total flavonoid contents were expressed as g catechinequivalent (CE) / 100 g plant powder. The identificationof polyphenols was achieved by an UPLC-ESI-MSanalysis (Agilent Technologies, USA) according to themethod previously described by Mossalayi et al. withslight modifications [29].Free radical-scavenging activity of polyphenol-rich extractsfrom medicinal plantsOxygen radical absorbance capacity (ORAC) assayORAC is based on the decrease of fluorescein fluorescence in presence of the chemical oxidant 2,2′-azobis[2-methyl-propionamidin] dihydrochloride (AAPH). Theassay was done as described by Huang et al. with somemodifications [30]. Briefly, 25 μL sample were placed ina 96-well black microplate, and 150 μL of 8 10 5 mMfluorescein (Sigma-Aldrich, Germany) were added.After 15 min at 37 C, 25 μL of 153 mM AAPH (SigmaAldrich, Germany) were added to each well. Then, thefluorescence was measured for 1 h 40 min at a wavelength of excitation and emission of 485 nm and530 nm, respectively (Infinite 200, Tecan, USA). Theresults were based on the area under the curve of fluorescence decay over time and compared with a Trolox(Sigma-Aldrich, Germany) calibration curve. Free

Marimoutou et al. Journal of Inflammation (2015) 12:10Page 4 of 15radical-scavenging activities of polyphenol-rich plantextracts were expressed as mM Trolox equivalent.Finally, preadipocytes were counted using Trypan Bluesolution (Sigma-Aldrich, Germany).2,2-Diphenyl-1-picrylhydrazyl (DPPH) assayLactate dehydrogenase (LDH) release assayThe scavenging activity on DPPH radical was measuredaccording to the method described by Yang et al.with slight modifications [31]. Briefly, 0.25 mM DPPH(Sigma-Aldrich, Germany) diluted in methanol was incubated with each polyphenol-rich plant extract or 100 μMantioxidant standard (vitamin C, the flavonoids catechinor quercetin, and the phenolic acids gallic, caffeic, ferulicor chlorogenic acids, Sigma-Aldrich, Germany). After25 min at 25 C, the optical density (OD) was read at517 nm (FLUOstar Optima, Bmg Labtech, Germany).The percentage of free radical-quenching activity ofDPPH was determined according to the followingformula: Antioxidant capacity ð%Þ ¼ ðOD control – OD sampleÞ OD control 100Evaluation of the effect of polyphenol-rich plant extractson preadipocyte viabilityMouse preadipocytes obtained from ATCC CL-173 werecultivated in Dulbecco’s Modified Eagle’s Medium containing 25 mM glucose, 10% heat-inactivated fetal bovineserum, L-glutamin (5 mM), streptomycin (2 μg/mL) andpenicillin (50 μU/mL). The cell culture condition was ina humidified 5% CO2 incubator at 37 C.MTT assayMitochondrial metabolic activity of cells was determinedby 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenenyl tetrazolium bromide (MTT) reduction assay. 3T3-L1 cells wereseeded overnight in 96-well plates at a density of 6 103cells per well. The day after, the medium was removedand cells were treated with each polyphenol-rich plantextract (0–200 μM GAE) for 24, 48, and 72 h. Five hoursbefore the end of the incubation, 20 μL MTT dye(5 mg/mL, Sigma-Aldrich, Germany) were added intoeach well to allow the formation of the dark blue formazan crystals generated by living cells. Then, the mediumwas removed and 100 μL of DMSO were added to dissolve the crystals. Absorbance was read at 595 nm(FLUOstar Optima, Bmg Labtech, Germany).Trypan blue exclusion method3T3-L1 cells were seeded overnight in 12-well plates at adensity of 50 103 cells per well. The day after, themedium was removed and cells were treated with eachpolyphenol-rich plant extract (25 μM GAE) for 24 and48 h. Then, cells were detached with trypsin, collected inthe medium and centrifuged (2000 rpm, 24 C, 4 min).LDH activity was measured using a commercial cytotoxicity assay kit (Sigma-Aldrich, Germany). For this test,3T3-L1 cells were treated with each polyphenol-richplant extract (25 μM GAE) for 24 and 48 h. Accordingto the manufacturer’s instructions, 50 μL of cell culturemedium were added to 100 μL of LDH reagent in 96well plates, then the mixture was incubated at roomtemperature during 30 min. The reaction was stoppedby adding 15 μL of 1 M HCl. The absorbance was readat 490 nm (FLUOstar Optima, Bmg Labtech, Germany).Evaluation of the effect of polyphenol-rich plant extractson ROS production from preadipocytesThe level of intracellular ROS was assessed by measuring the oxidation of DCFH-DA, according to the methodpreviously published [22,23]. DCFH-DA diffuses throughthe cell membrane and is deacetylated by cellular esterases to the non-fluorescent DCFH. Intracellular ROS areable to oxidize DCFH to the fluorescent 2,7-dichlorofluorescein (DCF), whose intensity of fluorescence is directly proportional to the levels of intracellular ROS.Briefly, cells were cultured in 96-well black microplates(6 103 cells/well) for 24 h. Then, the medium was removed and replaced by PBS containing 10 μM ofDCFH-DA (Sigma-Aldrich, Germany) and cells werekept in a humidified atmosphere (5% CO2, 37 C) for45 min. Next, cells were exposed to each polyphenolrich plant extract (25 μM GAE) or caffeic acid standardas a positive control (25 μM), or treated with H2O2(200 μM, Sigma-Aldrich, Germany), TNFα (5 ng/mL,eBioscience, UK) or LPS from E. coli K-235 (1 μg/mL,Sigma-Aldrich, Germany) in the presence or not of eachplant extract (25 μM GAE) or caffeic acid standard(25 μM). After 1 h, fluorescence was measured at an excitation wavelength of 492 nm and an emission wavelength of 520 nm (FLUOstar Optima, Bmg Labtech,Germany).Evaluation of the effect of polyphenol-rich plant extractson the production of pro-inflammatory cytokines frompreadipocytesCells were pre-incubated overnight in 24-well plates at adensity of 37 103 cells/well. The day after, they weretreated with H2O2 (200 μM), TNFα (5 ng/mL) or LPS(1 μg/mL) in the presence or not of each polyphenolrich plant extract (25 μM GAE). After 24 h, cell culturemedia were collected and stored at 20 C until analysis.Levels of the pro-inflammatory markers IL-6, TNFα andMCP-1 were determined by using specific ELISA kits

Marimoutou et al. Journal of Inflammation (2015) 12:10(eBioscience, UK) and normalized according to total cellular protein amounts determined by Bradford assay [32].Page 5 of 158G. mauritianaA. borbonicaD. apetalum6Evaluation of the effect of polyphenol-rich plant extractson the expression of SOD, catalase and NF-κB genes frompreadipocytesCells were pre-incubated overnight in 6-well plates at adensity of 150 103 cells/well. The day after, they weretreated with H2O2 (200 μM), TNFα (5 ng/mL) or LPS(1 μg/mL) in the presence or not of each polyphenol-richplant extract (25 μM GAE). After 24 h, total RNA was isolated with TRIzol (Invitrogen, France). An amount of2 μg of total RNA were reverse-transcribed (RT) usingRandom hexamer primers (Eurogentec, Belgium) withSuperscript II (Invitrogen, France). As previously described by Awada et al. [33], the quantitative polymerasechain reaction (QPCR) was conducted using SYBR green master Mix (Eurogentec, Belgium). Primer and probesequences are listed on Table 2. Results were analyzedusing 7500 system SDS software (Applied Biosystems,France) and the relative expression of SOD, catalaseand p50 NF-κB genes was normalized against the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene.Statistical analysisData were expressed as means SEM from three independent experiments (three different passages), themselvesbased on triplicates. Statistical analysis was performed byusing one-way ANOVA followed by the Tukey’s multiplecomparisons test. Significant differences were consideredfor p value 0.05 (PRISM software, USA).ResultsPolyphenol content and antioxidant activities ofmedicinal plant extractsTotal polyphenol content from medicinal plant acetonicextracts was evaluated by using Folin-Ciocalteu assay.As shown on Figure 1, D. apetalum extract exhibited thehighest polyphenol content (7.0% GAE, w/w) followedby A. borbonica (3.8% GAE, w/w) and G. mauritiana extracts (1.0% GAE, w/w). As flavonoids are the mostabundant polyphenols in the human diet and may account for about two thirds of the total intake [18,34],total flavonoid content from plant extracts was determined. Data reported on Figure 1 demonstrated the420Total polyphenols(GAE)Total flavon

No effective pharmacological treatments against obesity-associated oxidative stress and inflammation have been found yet. Thus, the identification of natural compounds able to increase the antioxidant and anti-inflammatory capacity of the body is of high interest. Plant polyphenols constitute