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Paronychia argentea Lam. protects renal endothelial cells against oxidative injury
Journal of Ethnopharmacology 248 (2020) 112314
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Paronychia argentea Lam. protects renal endothelial cells against oxidative injury
T
L. Arkoub-Hamitouchea, V. González-del-Campob, M.E. López-Olivac, B. Fatihaa, O.M. Palominob,∗ a
Laboratoire de Biothechnologie Végétale et Ethnobotanique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, 06000, Bejaia, Algeria Department of Pharmacology, Pharmacognosy and Botany, Faculty of Pharmacy, University Complutense of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain c Departmental Section of Physiology, Faculty of Pharmacy, University Complutense of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain b
A R T I C LE I N FO
A B S T R A C T
Keywords: Renal endothelial cells Radical scavenging ability Polyphenols Quercetin derivatives HPLC
Ethnopharmacological relevance: Paronychia argentea Lam. (Arabic tea), a species spontaneously growing in the Mediterranean area, has been used in folk medicine for renal diseases. Aim of the study: To assess the antioxidant and protective potentials of different extracts from P. argentea in the renal endothelial NRK-52E cell line by several in vitro models, including a H2O2-induced oxidative stress model. Material and methods: Aerial parts of P. argentea were collected in Algeria and ethanolic, chloroform and aqueous-chloroform extracts were obtained from dried plant. The antioxidant capacity was first evaluated by the Oxygen Radical Absorbance Capacity (ORAC) and the free radical scavenging activity (DPPH) methods. Cellular viability was assessed by MTT method assay after 24 h pretreatment with each extract concentration in order to measure protection from H2O2 in NRK-52E cells. Furthermore, the intracellular ROS formation (DCFH-DA method), was determined. Results: P. argentea showed in vitro antioxidant activity as evidenced by the ORAC and DPPH assays. No cell toxicity was observed for concentrations ranging from 0.1 to 100 μg/mL of each extract. These extracts also exerted a protective effect on renal endothelial cells simultaneously treated with 1 mM H2O2. Chemical composition for the aqueous-chloroform extract was assessed by HPLC, as it showed the strongest antioxidant ability, revealing three quercetin derivatives as the main phenolic compounds. Conclusion: P. argentea is endorsed with antioxidant activity and protects renal endothelial cells against oxidative damage which indicate this plant constitutes a potential treatment for renal diseases.
1. Introduction Paronychia argentea Lam., a plant belonging to the Caryophyllaceae family, is widely distributed in the Mediterranean area and commonly known as Arabic tea. Extracts from its aerial parts have been frequently used in folk Algerian medicine as diuretic, to treat kidney stones or prostate discomfort, stomach ulcer, anorexia and as an antimicrobial agent (Abu-Irmaileh and Afifi, 2000; Al-Bakri and Afifi, 2007; Beloued, 1998; Bouanani et al., 2010; Ferreira et al., 2006). P. argentea has also been largely used in Portugal as a carminative remedy and in Israel, Jordan or Morocco to treat diabetes (Afifi and Al-Khalidi, 2005; Bellakhdar, 1997; Dafni et al., 1984; Ferreira et al., 2006; Hudaib et al.,
2008; Kim et al., 2004; Yaniv et al., 2002). Its reported hypoglycemic effect has been related to its content in glycosides and flavonoids aglycones which shown a marked inhibitory effect on alpha-amylase activity (Williamson et al., 1996). In vivo and in vitro antioxidant activities have also been reported for the aqueous and alcoholic extracts of P. argentea. They have shown potent inhibition of lipid peroxidation and a chemoprotective effect by quenching free radicals. This antioxidant efficiency is mostly attributed to their phenolic components (phenolic acids, tannins and flavonoids) which seem to be crucial considering the key role of free radicals in the origin and pathogenesis of degenerative diseases like arthritis, cancer or Alzheimer disease, among others (Sait et al., 2015; Yadav et al., 2011).
Abbreviations: DCF, Dichlorofluorescein; DCFH, 2′,7′-dichlorohydrofluorescein; DCFH-DA, 2ʹ, 7ʹ-dichlorohydrofluorescin diacetate; DMEM, Dulbecco's modified eagle's medium; DPPH, 2, 2-diphenyl-1-picrylhydrazyl; FBS, Fetal bovine serum; GAE, Gallic acid equivalents; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide; ORAC, Oxygen radical absorbance capacity; ROS, Reactive oxygen species ∗ Corresponding author. Pza Ramón y Cajal s/n, 28040, Madrid, Spain. E-mail addresses: [email protected], [email protected] (O.M. Palomino). https://doi.org/10.1016/j.jep.2019.112314 Received 24 June 2019; Received in revised form 14 October 2019; Accepted 15 October 2019 Available online 17 October 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
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evaporated under reduced pressure by means of rotary evaporation at 40 °C until dryness. The ethanolic extract was splitted by using a Chloroform:Water (3:1, v/v) mixture to obtain three different extracts: ethanolic, chloroform and aqueous-chloroform. Dried extracts were stored at 5 °C and protected from light until use.
The chemical composition of P. argentea aerial parts has been studied by Sait et al. (2015), who used HPLC coupled with diode array detection and electrospray ionization mass spectrometry to report the flavonoids content. Eleven compounds were identified; among them, isorhamnetin-3-O-dihexoside, quercetin-3-O-glucoside, quercetin-methyl-ether-O-hexoside, quercetin, jaceosidin and isorhamnetin, were described for the first time in this species. These flavonoids, along with some phenolic acids and tannins, are accountable of the observed antioxidant and chemoprotective effect (Cartea et al., 2010). One of the most frequent renal pathologies for which there is no fully satisfactory therapy yet is kidney stones. Cell models are currently utilized to investigate regular renal anatomy and physiology, as well as morphometric changes derived from age and the development of kidney diseases. Cell lines such as NRK-52E (rat), LLC-PK1 (pig), MDCK (dog), VERO (monkey), BHK (hamster) and OK (opossum) are frequently used for toxicological studies (Heussner and Dietrich, 2013). In particular, NRK cells from kidneys of Rattus norvegicus show epithelial morphology and their main function is storage and subsequent secretion of excretory materials. Biological effects of several bioactive compounds on renal system have been studied in NRK-52E cells (Heussner and Dietrich, 2013). Tandon et al. (2010) showed that an extract from Achyranthes aspera was able to prevent the oxalate-induced cell damage on NRK-52E cells, suggesting a chemoprotective effect on the renal epithelial cells. The effect of several herbs traditionally used for kidney and urinary system disorders has been tested on normal renal mammalian fibroblasts (NRK49F) and NRK-52E cells, where Dioscorea villosa showed renotoxicity, while Angelica sinensis, Centella asiatica, Glycyrrhiza glabra, Scutellaria lateriflora, and Olea europaea extracts exerted potent antifibrotic and antioxidant activity (Wojcikowski et al., 2009). Previous studies have shown the beneficial effects of P. argentea butanolic extract in the prevention of kidney stones growth in experimental calcium oxalate nephrolithiasis in Wistar rats (Bouanani et al., 2010). The results suggest that butanolic extract might prevent and probably remove the pre-exisiting kidney stones, although the precise mechanism of action remains to be described. When butanolic and aqueous extract effects were compared, the former showed a more pronounced anti-litiasic effect which could be related to the differences in their chemical composition, mainly flavonoids and saponins derivatives (Braca et al., 2008; Lakshminarasimhan et al., 2002). In this study, different extracts from aerial parts of spontaneous populations of P. argentea collected in Algeria were assayed for the in vitro antioxidant potential and biological properties in renal endothelial NRK-52E cell line.
2.3. Reagents Fluorescein (3′, 6′-dihydroxyspiro [isobenzofuran-1[3H], 9′ [9H]xanthen]-3-one), 2, 2′-azobis (2-amidinopropane) dihydrochloride (AAPH), 2, 2-Diphenyl-1-picrylhydrazyl (DPPH), Folin – Ciocalteu's phenol reagent, gentamicin, penicillin G and streptomycin were purchased from Sigma-Aldrich (Spain). Dulbecco's modified Eagle's medium (DMEM), RPMI1640 medium, foetal bovine serum (FBS), PBS were purchased from Gibco (Invitrogen, Paisley, UK). Dimethyl sulphoxide (DMSO), Hydrogen peroxide solution (30% w/w), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,7dichloro-dihydrofluorescein diacetate (DCFH-DA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Standards of Quercetin-3-O-(glucosyl) galactoside, Quercetin-3-Ogalactoside and Quercetin-methylether-O-hexoside of HPLC grade were purchased from Extrasynthese (Genay, France). 2.4. Polyphenol quantification Total polyphenol content was determined by the spectrophotometric method of Folin-Ciocalteu (Montreau, 1972) using gallic acid (GA) as standard with small modifications. A UVIKON 930 Kontron instruments spectrophotometer was used. Briefly, 0.5 mL solution of each sample (1 mg/mL concentration) was mixed with 0.5 mL FolinCiocalteu reagent; after 3 min, a saturated sodium carbonate reagent (75 g/L) was added to the solution. The mixture was then incubated for 1 h at room temperature protected from light, and the absorbance was measured at 760 nm. A standard curve was obtained for GA and results were expressed as mg of gallic acid equivalents (GAE)/mg dry weight (DW). 2.5. Antioxidant capacity and oxygen scavenging activity The antioxidant capacity of the three extracts of P. argentea (ethanol, chloroform and aqueous-chloroform extracts) was evaluated by the oxygen radical absorbance capacity (ORAC) method (Dávalos et al., 2004). Briefly, 20 μL of sample (at different concentrations), blank (phosphate saline buffer) and Trolox calibration solutions (12.5–200 μM) were placed on black 96-well untreated microplate and mixed with 200 μl of working fluorescein solution (10 nM, in 10 mM phosphate buffer, pH 7.4). Samples, blank and Trolox were tempered at 37 °C during 10min; then, 60 μl of peroxyl generator AAPH was added to initiate the oxidation reaction., causing a decrease in fluorescence (excitation wavelength 485 nm and emission wavelength 528 nm), which is measured every 60 s for 90 min at 37 °C in a multiwell plate reader (FLUOstar OPTIMA fluorimeter, BMG LABTECH). Results calculate the relationship of the areas under the curve between blank and samples and are expressed as micromoles of Trolox equivalents per gram. The free radicals scavenging ability of the samples was determined by the DPPH assay according to Sharma and Bhat (2009) in a multiwell plate reader (FLUOstar OPTIMA fluorimeter, BMG LABTECH). Briefly, a stock solution of DPPH of 2.3 mg/1 mL MeOH was kept at 5 °C until use; then increasing concentrations of each sample were added and absorbance was recorded at 517 nm. Assays were done in triplicate. The free radical-scavenging activity of each solution was then calculated as the percentage of inhibition and results are expressed as IC50 value, which is defined as the concentration of extract (lg/mL) required to scavenge 50% DPPH radicals, according the following expression:
2. Materials and methods 2.1. Plant material Aerial parts of P. argentea spontaneously growing in Chemini (SidiAich, Bejaia, Algeria; geographic coordinates 36° 36′ 30″ North, 4° 41′ 2″ East) were harvested in April 2015. The plant was identified by a botanist from the University of Abderramane Mira and a voucher specimen was kept for internal control. The plant name has been checked with http://www.theplantlist.org June 2019. Samples were dried at room temperature, protected from light, grind down and sieved to obtain a powder of 250 μm diameter. The powder was kept at room temperature protected from light and moisture until use. 2.2. Extraction process Extracts were obtained according to Chiang et al. (1994) with small modifications. Briefly, 1 g of the powder was macerated in 4 mL of ethanol for 24 h under shaking, at room temperature. The suspension was then poured and centrifuged at 3000g for 10min. Then, it was 2
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was determined. In order to evaluate the protective effect against an oxidative insult, cells were treated with different concentrations of P. argentea extract for 24 h. Cells were then washed and 1 mM H2O2 (diluted in DMEM) was added to all the cultures except for controls for 3 h. DCFH-DA was added and the generation of ROS was determined as previously described.
% Scavenging = [(AC-AS)/AC]*100 AC: Absorbance shown by control (Trolox) AS: Absorbance of the tested sample 2.6. HPLC analysis P. argentea samples (10 mg of dry extract in 1 ml DMSO and then filtered through a 0.45 μm cellulose acetate filter) were analyzed by HPLC according to previous studies with small modifications (Sait et al., 2015) within a Jasco modular 2065 system, equipped with a vacuum degasser, quaternary pump PU-2080, thermostated autosampler, column oven and UV/VIS detector (UV-2070 Plus). The analyses were carried out on a Mediterranean Sea column (250 nm × 4.6 nm, 5 μm) (Teknokroma, Spain) with a mobile phase consisting in A: water/formic acid (0.1%) and B: ACN. The separation was achieved by gradient elution as follows: 0–15 min from 10% to 20%B; 15–35 min from 20% to 30% B; 35–45min from 30% to 50% B, and kept for 5 min. Flow-rate was set at 1 mL/min. Sample injection volume was 10 μl and column temperature was 25 °C. Phenolic compounds were identified according to their retention time and their purity checked throughout their absorption spectra at 352 nm and 372 nm. Every sample was analyzed in triplicate.
2.11. Statistical analysis Each experiment was carried out in triplicate. Data are expressed as Mean ± SD values of at least three experiments. For multiple comparisons, one-way ANOVA was followed by a Bonferroni test when variances were homogeneous or by Tamhane test when variances were not homogeneous. The level of significance was p < 0.05. In order to study the correlationship between the total polyphenol content and the antioxidant activity assessed by ORAC and DPPH assays, Pearson's correlation coefficient (p < 0.05) was applied. The statistical analyses were conducted using the statistical package SPSS version 22.0 for Windows (SPSS, Chicago; IL, USA). 3. Results and discussion 3.1. Polyphenol content
2.7. Cell culture
The highest total polyphenol content was found in the aqueouschloroform extract (278.76 mg GAE/g), followed by the ethanol and chloroform extracts (120.35 mg GAE/g and 29.72 mg GAE/g, respectively (Fig. 1), these results being probably related to the differences in the solvent polarity. Moreover, total content is much higher than that reported for the methanolic extract of 15.5 mg GAE/g (Alali et al., 2007). These differences may be attributed to the harvesting season and related environmental conditions (Stypinski, 1997; Luczkiewicz et al., 2001; Vicas et al., 2008).
NRK-52E renal endothelial line was a kind gift from Dr Sonia Pascual and Dr Luis Goya, ICTAN, CSIC (Madrid, Spain). Cells were grown in a humidified incubator at 5% CO2 and 95% air at 37 °C in Dulbecco's Modified Eagle's medium (DMEM) pyruvate free, from Invitrogen (Madrid, Spain), supplemented with 10% foetal bovine serum (FBS) (Biowhittaker) and 50 mg/L of each one of the following antibiotics: gentamicin, penicillin and streptomycin. 2.8. Cell treatment
3.2. Antioxidant capacity and oxygen scavenging activity
For all the experiments, different concentrations of P. argentea extract were dissolved in DMEM and added to the 96 multi-well cell plates for 24 h. Every sample was analyzed in triplicate, with 3 different experiments.
Different methods can be used to evaluate the antioxidant capacity of phenolic compounds. In this work, total polyphenol content, antioxidant capacity (ORAC) and free radical scavenging activity (DPPH) assays were used to assess to the ethanol, chloroform and aqueouschloroform extracts from aerial parts of P. argentea. Trolox, as a water-soluble analogue of vitamin E, was selected as a positive control in all the assays performed in this study. Trolox has been shown to decrease ROS production, to prevent cytotoxicity in human cell lines and to rescue cells from apoptotic death (Kello et al., 2014; Schoeneberger et al., 2014). The antioxidant capacity in P.
2.9. MTT assay Cell viability (cell growth inhibition) was determined by MTT assay (Mosmann, 1983; Carmichael et al., 1987) with some modifications. Cells were incubated in 96-well plates, at a density of 5 × 10⁴ cells/well for 24 h, then the cells were treated with different concentrations of P. argentea extract (0.1–100 μg/mL) for another 24 h. 5% Triton X-100 was used as a negative control; finally 2 mg/ml MTT was added and the plate was incubated for 1 h at 37 °C. Then, the formazan crystals formed were dissolved by adding DMSO and the absorbance was measured at 550 nm using Digiscan 340 microplate reader (ASYA Hitech GmbH, Eugendorf, Austria). 2.10. Intracellular ROS production assay ROS production was evaluated by the DCFH-DA assay (Le Bel et al., 1992) with some modification. This assay is based on the oxidation of the nonfluorescent compound 2′,7′-dichlorohydrofluorescein (DCFH) into the fluorescent compound dichlorofluorescein (DCF) in presence of ROS. Cells were incubated in 96-well multiwells and treated with different concentrations of P. argentea extract for 24 h. Then, 50 μL of DCFH-DA at a concentration of 10 μM were added for 30 min; then, the unabsorbed probe was removed and fluorescence in a microplate fluorescence reader (FLx800, Bio-Tek Instrumentation) at 480/510 nm
Fig. 1. Total polyphenol content in Paronychia argentea Lam. extracts. Values are means ± SD, n = 3. Different letters indicate statistically significant differences (p < 0.05) among groups. 3
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Table 1 ORAC values and free radical scavenging activity (DPPH) for P. argentea extracts. Values are means ± SD, n = 3. Different letters indicate statistically significant differences (p < 0.05) among groups. Extract
ORAC value (μmol Trolox equivalent/ mextract)
DPPH (IC50)
Ethanol Chloroform Aqueous-chloroform Trolox
1.452b ± 0.167 0.466a ± 0.072 2.587c ± 0.102 –
169.34b ± 2.19 601.72c ± 3.93 178.53b ± 4.43 102.59a ± 2.77
argentea extracts was first determined by the ORAC assay. Results show ORAC values ranging from 0.466 to 2.587 μmol TE/mg (Table 1), this indicating a moderate to strong antioxidant capacity (Dávalos et al., 2004; Zheng and Wang, 2001), the aqueous-chloroform extract being the most active. Results for the free radical scavenging activity of P. argentea extracts are expressed as the concentration of each extract which is capable of scavenge 50% of DPPH radicals (Table 1). The highest this concentration is, the lowest the oxygen scavenging activity is, as a higher quantity is needed to reach the same effect (Brand-Williams et al., 1995; Atoui et al., 2005). Our results show a high scavenging activity of both ethanol and aqueous-chloroform extracts from P. argentea, although lower than that of the positive control, Trolox. In fact, IC50 values for ethanol and aqueous-chloroform extracts (169.34 and 178.53 μg/mL, respectively) are significantly lower than those previously reported by other authors for the ethanolic extract (IC50 value of 208 μg/mL), but similar to that of the infusion (IC50 = 144.92 μg/mL) (Sait et al., 2015). This difference can be attributed to the variable polyphenol content, as shown in other vegetal species (Kang et al., 2003). When analyzing the results obtained for the antioxidant ability by the ORAC assay in relation to the phenolic composition, a positive (r = 0.987) and highly significant correlation (p < 0.0001) between both variables was found (Fig. 2a). These results strongly suggest that the total polyphenolic content remarkably contributes to the antioxidant capacity of P. argentea extracts. Thus, the higher total polyphenolic content obtained in the aqueous-chloroform extract resulted in higher total antioxidant capacity. These data are in line with previous studies which demonstrate the direct relationship between the antioxidant capacity and the polyphenol content (Dudonné et al., 2009; Hayase and Kato, 1984; Maisuthisakul et al., 2007;; Velioglu et al., 1998) and are consistent with our results revealing the aqueouschloroform extract from P. argentea with the highest content in total polyphenols. Moreover, a statistically significant (p = 0.01) negative (r = −0.784) correlation between total polyphenol content and IC50 values by DPPH scavenging assay was obtained (Fig. 2b), this indicating a moderate relationship between both variables. Collectively, the polyphenol content seems to be responsible for 78.4% of the antioxidant capacity of P. argentea extracts, whereas 21.6% of the antioxidant activity may be due to the presence of other antioxidant compounds which might be capable of inducing a synergistic effect. Also, the presence of non-phenolic compounds such as peptides, proteins and sugars, among others, may have greater impact on the DPPH results. The aqueous-chloroform extract showed the lowest value of IC50 value and the highest phenolic content, as they are inversely proportional, also being the most active extract in scavenging DPPH radicals. These results were reflected in the r value (r = 0.843; p = 0.004) obtained for the correlation between DPPH and ORAC values (Fig. 2c), which confirmed the higher antioxidant capacity of the aqueous extract. Taken together, the results of the antioxidant activity of P. argentea by different assays gave comparable results and confirm the relationship between phenolic compounds concentration and the oxygen radical scavenging ability. Therefore, the aqueous-chloroform extract was
Fig. 2. Correlation coefficient R between assays and total polyphenol content in Paronychia argentea Lam. extracts. a) ORAC assay versus total polyphenols. b) DPPH assay versus total polyphenols. c) DPPH assay versus ORAC assay.
then selected among the tested extracts for further evaluation in cells. 3.3. HPLC analysis In this study, the HPLC analysis allows the identification and quantification of the flavonoids in the aqueous-chloroform extract of P. argentea, the main components being quercetin derivatives (Table 2). Representative chromatograms at 352 and 372 nm are shown in Fig. 3. Our results are in accordance with those previously reported by other authors (Sait et al., 2015) who described the ethanolic extract of aerial parts of this species with a higher content in flavonoids than other preparations such as the decoction or infusion of the plant. 3.4. Cell viability The effect of the aqueous-chloroform extract of P. argentea on cell viability was assessed in NRK-52E renal cells, a useful cell model for the study of mechanisms and signal transduction pathways in physiological and pathological conditions (Álvarez-Cilleros et al., 2018), with the aim to select non-toxic concentrations of the plant extract. P. argentea extracts are not toxic at any of the assayed concentrations. The results of MTT assay indicated no-statistically significant 4
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Table 2 Flavonoids identified in P. argentea Lam. ethanolic extract by HPLC. Peak number
Compound
mg/g dried planta
t < SUB > R < /SUB > (min)
1 2 3
Quercetin-3-O-(glucosyl) galactoside Quercetin-3-O-galactoside Quercetinmethylether-O-hexoside
1.3 ± 0.1 3.7 ± 0.3 1.1 ± 0.1
15.33 18.57 22.21
a
Values are mean ± SEM, n = 3.
realistic concentration within the physiological range for the further ROS assays.
differences between the tested concentrations of P. argentea extract and the control group (untreated cells). On the contrary, renal cell viability was significantly reduced when rnal cells were treated with the negative and cytotoxic control, triton-X 100 (data not shown). In the MTT experiment we evaluated the effect of a concentration range of the plant extract on the renal cell line with the aim to select the
3.5. ROS production To test the effect of P. argentea on intracellular ROS levels,
Fig. 3. Chromatogram of the aqueous-chloroform extract of P. argentea (10 mg/mL) at a) 352 nm and b) 372 nm. Experimental conditions are described in the text. For peak identification, see Table 2. 5
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Evaluation of the probe 2′-7′Dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol. 5, 227–231. Luczkiewicz, M., Cisowski, W., Kaiser, P., Ochocki, R., Piotrowski, A., 2001. Comparative analysis of phenolic acids in mistletoe plants from various hosts. Acta Pol. Pharm. 58, 373–379. Maisuthisakul, P., Suttajit, M., Pongsawatmanit, R., 2007. Assessment of phenolic content and free radical scavenging capacity of some Thai indigenous plants. Food Chem. 100, 1409–1418. Montreau, F.R., 1972. Sur le dosage des composes phenoliques totaux dans les vins par la methode Folin-Ciocalteau. Connaiss. Vigne Vin 24, 397–404. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application
Fig. 4. Effect of treatment with P. argentea on intracellular ROS production on NRK-52E cells. For the Direct effect, NRK-52E cells were treated with different concentrations of the extract for 24 h. For the Protective effect, NRK-52E cells were treated with different concentrations of the extract for 24 h. Then, cells were washed and 1 mM H2O2 was added to all the cultures except for controls for 3 h. Values are expressed as a percent relative to control condition ± SD (n = 3, 3 replicates). Different letters indicate statistically significant differences (p < 0.05) among groups.
concentrations of 1.0, 10, 25 and 50 μg/mL of aqueous-chloroform extract were added and evaluated by the DCFH-DA assay (Fig. 4). Treatment with 1 mM H2O2 caused an increase of about 10% in ROS levels, when compared to control cells. P. argentea alone did not significantly increase ROS concentration, this indicating no cellular stress or oxidative damage which could affect the functional conditions of cells. Pretreatment of the cells with P. argentea extracts at 1 and 10 μg/ mL, led to a ROS production similar to that of untreated cells or those treated with the positive control, Trolox. Differences were statistically significant with respect to the negative control, H2O2 (Fig. 4). Our results agree with those from other authors who assessed the protective role of the butanolic extract of P. argentea against oxidative stress (Zama et al., 2007) and those who related the antioxidant and freeradical scavenging activities of the plant to the reduction of urinary concentrations of stone-forming constituents (Bouanani et al., 2010).
4. Conclusion The study of P. argentea aerial parts by two different assays based on both hydrogen atom transference and electron transference (ORAC, DPPH, respectively) revealed the antioxidant activity of this specie. The total phenolic content determination, together with the chromatographic analysis showed a high content in phenolic compounds, mainly derivatives from the flavonol quercetin, which are the main responsible for the reported antioxidant capacity, although other antioxidant compounds might induce a synergistic effect. MTT assay showed that P. argentea is not toxic for cultured renal cells at concentrations ranging from 0.1 to 100 μg/mL, whereas ROS assays supported the chemoprotective effect of realistic concentrations of P. argentea extract, especially the lowest concentrations (1 and 10 μg/ mL), against oxidative damage induced with 1 mM H2O2 on the NRK52E renal endothelial cell line. This effect allows endothelial renal cells to be in a favourable condition to face an oxidative challenge. These results may contribute to the knowledge of the biochemical mechanisms involved in the physiological chemoprotection, although further studies regarding the bioavailability and the effects of gastrointestinal digestion, hepatic metabolism, distribution of the metabolites in the organism and urinary pH are needed previous to assess the efficacy of P. argentea on renal diseases in humans. 6
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from various host trees. Bulletin UASVM, Agriculture Cluj-Napoca 65, 327–332. Williamson, E.M., Okpako, D.T., Evans, F.J., 1996. Pharmacological Methods in Phytotherapy Research. Selection, Preparation and Pharmacological Evaluation of Plant Material, vol. 1. Wiley & Sons, New York, pp. 155–168. Wojcikowski, K., Wohlmuth, H., Johnson, D.W., Rolfe, M., Gobe, G., 2009. An in vitro investigation of herbs traditionally used for kidney and urinary system disorders: potential therapeutic and toxic effects. Nephrology 14 (1), 70–79. https://doi:10. 1111/j.1440-1797.2008.01017.x. Yadav, R., Alok, S., Jain, S., Verma, A., Mahor, A., Bharti, J., Jaiswal, M., 2011. Herbal plants used in the treatment of urolithiasis: a review. Int. J. Pharm. Sci. Res. 2 (6), 1412–1420. Yaniv, Z., Friedman, J., Palevitch, D., Dafni, A., 2002. Plants used for the treatment of diabetes in Israel. J. Ethnopharmacol. 19 (2), 145–151. Zama, D., Meraihi, Z., Tebibel, S., Benayssa, W., Benayache, F., Benayache, S., Vlietinck, A.J., 2007. Chlorpyrifos-induced oxidative stress and tissue damage in the liver,kidney, brain and foetus in pregnant rats: the protective role of the butanolic extract of Paronychia argentea L. Indian J. Pharmacol. 39, 145–150. Zheng, W., Wang, S.Y., 2001. Antioxidant activity and phenolic compounds in selected herbs. J. Agric. Food Chem. 49, 5165–5170.
to proliferation and cytotoxicity assays. J. Immunol. Methods 65 (1–2), 55–63. Sait, S., Hamri-Zeghichi, S., Boulekbache-Makhlouf, L., Madani, K., Rigou, P., Brighenti, V., Pio Prencipe, F., Benvenuti, S., Pellati, F., 2015. HPLC-UV/DAD and ESI-MSn analysis of flavonoids and antioxidant activity of an Algerian medicinal plant: Paronychia argentea Lam. J. Pharm. Biomed. Anal. 111, 231–240. Schoeneberger, H., Belz, K., Schenk, B., Fulda, S., 2014. Impairment of antioxidant defense via glutathione depletion sensitizes acute lymphoblastic leukemia cells for Smac mimetic-induced cell death. Oncogene. https://doi.org/10.1038/onc.2014.338. Sharma, O.P., Bhat, T.K., 2009. DPPH antioxidant assay revisited. Food Chem. 113, 1202–1205. Stypinski, P., 1997. Biology and Ecology of Mistletoe (Viscum album, Viscaceae) in Poland. Institute of Botanic. W. Szafera PAN, Kraków, Poland, pp. 1–117. Tandon, C., Aggarwal, A., Tandon, S., Singla, S., 2010. Reduction of oxalate-induced renal tubular epithelial (NRK-52E) cell injury and inhibition of calcium oxalate crystallisation in vitro by aqueous extract of Achyranthes aspera. Int. J. Green Pharm. 4 (3), 159. Velioglu, Y.S., Mazza, G., Gao, L., Oomah, B.D., 1998. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. J. Agric. Food Chem. 46, 4113–4117. Vicas, S., Rugina, D., Sosaciu, C., 2008. Antioxidant activities of Viscum album's leaves
7
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Paronychia argentea Lam. protects renal endothelial cells against oxidative injury
T
L. Arkoub-Hamitouchea, V. González-del-Campob, M.E. López-Olivac, B. Fatihaa, O.M. Palominob,∗ a
Laboratoire de Biothechnologie Végétale et Ethnobotanique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, 06000, Bejaia, Algeria Department of Pharmacology, Pharmacognosy and Botany, Faculty of Pharmacy, University Complutense of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain c Departmental Section of Physiology, Faculty of Pharmacy, University Complutense of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain b
A R T I C LE I N FO
A B S T R A C T
Keywords: Renal endothelial cells Radical scavenging ability Polyphenols Quercetin derivatives HPLC
Ethnopharmacological relevance: Paronychia argentea Lam. (Arabic tea), a species spontaneously growing in the Mediterranean area, has been used in folk medicine for renal diseases. Aim of the study: To assess the antioxidant and protective potentials of different extracts from P. argentea in the renal endothelial NRK-52E cell line by several in vitro models, including a H2O2-induced oxidative stress model. Material and methods: Aerial parts of P. argentea were collected in Algeria and ethanolic, chloroform and aqueous-chloroform extracts were obtained from dried plant. The antioxidant capacity was first evaluated by the Oxygen Radical Absorbance Capacity (ORAC) and the free radical scavenging activity (DPPH) methods. Cellular viability was assessed by MTT method assay after 24 h pretreatment with each extract concentration in order to measure protection from H2O2 in NRK-52E cells. Furthermore, the intracellular ROS formation (DCFH-DA method), was determined. Results: P. argentea showed in vitro antioxidant activity as evidenced by the ORAC and DPPH assays. No cell toxicity was observed for concentrations ranging from 0.1 to 100 μg/mL of each extract. These extracts also exerted a protective effect on renal endothelial cells simultaneously treated with 1 mM H2O2. Chemical composition for the aqueous-chloroform extract was assessed by HPLC, as it showed the strongest antioxidant ability, revealing three quercetin derivatives as the main phenolic compounds. Conclusion: P. argentea is endorsed with antioxidant activity and protects renal endothelial cells against oxidative damage which indicate this plant constitutes a potential treatment for renal diseases.
1. Introduction Paronychia argentea Lam., a plant belonging to the Caryophyllaceae family, is widely distributed in the Mediterranean area and commonly known as Arabic tea. Extracts from its aerial parts have been frequently used in folk Algerian medicine as diuretic, to treat kidney stones or prostate discomfort, stomach ulcer, anorexia and as an antimicrobial agent (Abu-Irmaileh and Afifi, 2000; Al-Bakri and Afifi, 2007; Beloued, 1998; Bouanani et al., 2010; Ferreira et al., 2006). P. argentea has also been largely used in Portugal as a carminative remedy and in Israel, Jordan or Morocco to treat diabetes (Afifi and Al-Khalidi, 2005; Bellakhdar, 1997; Dafni et al., 1984; Ferreira et al., 2006; Hudaib et al.,
2008; Kim et al., 2004; Yaniv et al., 2002). Its reported hypoglycemic effect has been related to its content in glycosides and flavonoids aglycones which shown a marked inhibitory effect on alpha-amylase activity (Williamson et al., 1996). In vivo and in vitro antioxidant activities have also been reported for the aqueous and alcoholic extracts of P. argentea. They have shown potent inhibition of lipid peroxidation and a chemoprotective effect by quenching free radicals. This antioxidant efficiency is mostly attributed to their phenolic components (phenolic acids, tannins and flavonoids) which seem to be crucial considering the key role of free radicals in the origin and pathogenesis of degenerative diseases like arthritis, cancer or Alzheimer disease, among others (Sait et al., 2015; Yadav et al., 2011).
Abbreviations: DCF, Dichlorofluorescein; DCFH, 2′,7′-dichlorohydrofluorescein; DCFH-DA, 2ʹ, 7ʹ-dichlorohydrofluorescin diacetate; DMEM, Dulbecco's modified eagle's medium; DPPH, 2, 2-diphenyl-1-picrylhydrazyl; FBS, Fetal bovine serum; GAE, Gallic acid equivalents; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide; ORAC, Oxygen radical absorbance capacity; ROS, Reactive oxygen species ∗ Corresponding author. Pza Ramón y Cajal s/n, 28040, Madrid, Spain. E-mail addresses: [email protected], [email protected] (O.M. Palomino). https://doi.org/10.1016/j.jep.2019.112314 Received 24 June 2019; Received in revised form 14 October 2019; Accepted 15 October 2019 Available online 17 October 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
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evaporated under reduced pressure by means of rotary evaporation at 40 °C until dryness. The ethanolic extract was splitted by using a Chloroform:Water (3:1, v/v) mixture to obtain three different extracts: ethanolic, chloroform and aqueous-chloroform. Dried extracts were stored at 5 °C and protected from light until use.
The chemical composition of P. argentea aerial parts has been studied by Sait et al. (2015), who used HPLC coupled with diode array detection and electrospray ionization mass spectrometry to report the flavonoids content. Eleven compounds were identified; among them, isorhamnetin-3-O-dihexoside, quercetin-3-O-glucoside, quercetin-methyl-ether-O-hexoside, quercetin, jaceosidin and isorhamnetin, were described for the first time in this species. These flavonoids, along with some phenolic acids and tannins, are accountable of the observed antioxidant and chemoprotective effect (Cartea et al., 2010). One of the most frequent renal pathologies for which there is no fully satisfactory therapy yet is kidney stones. Cell models are currently utilized to investigate regular renal anatomy and physiology, as well as morphometric changes derived from age and the development of kidney diseases. Cell lines such as NRK-52E (rat), LLC-PK1 (pig), MDCK (dog), VERO (monkey), BHK (hamster) and OK (opossum) are frequently used for toxicological studies (Heussner and Dietrich, 2013). In particular, NRK cells from kidneys of Rattus norvegicus show epithelial morphology and their main function is storage and subsequent secretion of excretory materials. Biological effects of several bioactive compounds on renal system have been studied in NRK-52E cells (Heussner and Dietrich, 2013). Tandon et al. (2010) showed that an extract from Achyranthes aspera was able to prevent the oxalate-induced cell damage on NRK-52E cells, suggesting a chemoprotective effect on the renal epithelial cells. The effect of several herbs traditionally used for kidney and urinary system disorders has been tested on normal renal mammalian fibroblasts (NRK49F) and NRK-52E cells, where Dioscorea villosa showed renotoxicity, while Angelica sinensis, Centella asiatica, Glycyrrhiza glabra, Scutellaria lateriflora, and Olea europaea extracts exerted potent antifibrotic and antioxidant activity (Wojcikowski et al., 2009). Previous studies have shown the beneficial effects of P. argentea butanolic extract in the prevention of kidney stones growth in experimental calcium oxalate nephrolithiasis in Wistar rats (Bouanani et al., 2010). The results suggest that butanolic extract might prevent and probably remove the pre-exisiting kidney stones, although the precise mechanism of action remains to be described. When butanolic and aqueous extract effects were compared, the former showed a more pronounced anti-litiasic effect which could be related to the differences in their chemical composition, mainly flavonoids and saponins derivatives (Braca et al., 2008; Lakshminarasimhan et al., 2002). In this study, different extracts from aerial parts of spontaneous populations of P. argentea collected in Algeria were assayed for the in vitro antioxidant potential and biological properties in renal endothelial NRK-52E cell line.
2.3. Reagents Fluorescein (3′, 6′-dihydroxyspiro [isobenzofuran-1[3H], 9′ [9H]xanthen]-3-one), 2, 2′-azobis (2-amidinopropane) dihydrochloride (AAPH), 2, 2-Diphenyl-1-picrylhydrazyl (DPPH), Folin – Ciocalteu's phenol reagent, gentamicin, penicillin G and streptomycin were purchased from Sigma-Aldrich (Spain). Dulbecco's modified Eagle's medium (DMEM), RPMI1640 medium, foetal bovine serum (FBS), PBS were purchased from Gibco (Invitrogen, Paisley, UK). Dimethyl sulphoxide (DMSO), Hydrogen peroxide solution (30% w/w), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,7dichloro-dihydrofluorescein diacetate (DCFH-DA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Standards of Quercetin-3-O-(glucosyl) galactoside, Quercetin-3-Ogalactoside and Quercetin-methylether-O-hexoside of HPLC grade were purchased from Extrasynthese (Genay, France). 2.4. Polyphenol quantification Total polyphenol content was determined by the spectrophotometric method of Folin-Ciocalteu (Montreau, 1972) using gallic acid (GA) as standard with small modifications. A UVIKON 930 Kontron instruments spectrophotometer was used. Briefly, 0.5 mL solution of each sample (1 mg/mL concentration) was mixed with 0.5 mL FolinCiocalteu reagent; after 3 min, a saturated sodium carbonate reagent (75 g/L) was added to the solution. The mixture was then incubated for 1 h at room temperature protected from light, and the absorbance was measured at 760 nm. A standard curve was obtained for GA and results were expressed as mg of gallic acid equivalents (GAE)/mg dry weight (DW). 2.5. Antioxidant capacity and oxygen scavenging activity The antioxidant capacity of the three extracts of P. argentea (ethanol, chloroform and aqueous-chloroform extracts) was evaluated by the oxygen radical absorbance capacity (ORAC) method (Dávalos et al., 2004). Briefly, 20 μL of sample (at different concentrations), blank (phosphate saline buffer) and Trolox calibration solutions (12.5–200 μM) were placed on black 96-well untreated microplate and mixed with 200 μl of working fluorescein solution (10 nM, in 10 mM phosphate buffer, pH 7.4). Samples, blank and Trolox were tempered at 37 °C during 10min; then, 60 μl of peroxyl generator AAPH was added to initiate the oxidation reaction., causing a decrease in fluorescence (excitation wavelength 485 nm and emission wavelength 528 nm), which is measured every 60 s for 90 min at 37 °C in a multiwell plate reader (FLUOstar OPTIMA fluorimeter, BMG LABTECH). Results calculate the relationship of the areas under the curve between blank and samples and are expressed as micromoles of Trolox equivalents per gram. The free radicals scavenging ability of the samples was determined by the DPPH assay according to Sharma and Bhat (2009) in a multiwell plate reader (FLUOstar OPTIMA fluorimeter, BMG LABTECH). Briefly, a stock solution of DPPH of 2.3 mg/1 mL MeOH was kept at 5 °C until use; then increasing concentrations of each sample were added and absorbance was recorded at 517 nm. Assays were done in triplicate. The free radical-scavenging activity of each solution was then calculated as the percentage of inhibition and results are expressed as IC50 value, which is defined as the concentration of extract (lg/mL) required to scavenge 50% DPPH radicals, according the following expression:
2. Materials and methods 2.1. Plant material Aerial parts of P. argentea spontaneously growing in Chemini (SidiAich, Bejaia, Algeria; geographic coordinates 36° 36′ 30″ North, 4° 41′ 2″ East) were harvested in April 2015. The plant was identified by a botanist from the University of Abderramane Mira and a voucher specimen was kept for internal control. The plant name has been checked with http://www.theplantlist.org June 2019. Samples were dried at room temperature, protected from light, grind down and sieved to obtain a powder of 250 μm diameter. The powder was kept at room temperature protected from light and moisture until use. 2.2. Extraction process Extracts were obtained according to Chiang et al. (1994) with small modifications. Briefly, 1 g of the powder was macerated in 4 mL of ethanol for 24 h under shaking, at room temperature. The suspension was then poured and centrifuged at 3000g for 10min. Then, it was 2
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was determined. In order to evaluate the protective effect against an oxidative insult, cells were treated with different concentrations of P. argentea extract for 24 h. Cells were then washed and 1 mM H2O2 (diluted in DMEM) was added to all the cultures except for controls for 3 h. DCFH-DA was added and the generation of ROS was determined as previously described.
% Scavenging = [(AC-AS)/AC]*100 AC: Absorbance shown by control (Trolox) AS: Absorbance of the tested sample 2.6. HPLC analysis P. argentea samples (10 mg of dry extract in 1 ml DMSO and then filtered through a 0.45 μm cellulose acetate filter) were analyzed by HPLC according to previous studies with small modifications (Sait et al., 2015) within a Jasco modular 2065 system, equipped with a vacuum degasser, quaternary pump PU-2080, thermostated autosampler, column oven and UV/VIS detector (UV-2070 Plus). The analyses were carried out on a Mediterranean Sea column (250 nm × 4.6 nm, 5 μm) (Teknokroma, Spain) with a mobile phase consisting in A: water/formic acid (0.1%) and B: ACN. The separation was achieved by gradient elution as follows: 0–15 min from 10% to 20%B; 15–35 min from 20% to 30% B; 35–45min from 30% to 50% B, and kept for 5 min. Flow-rate was set at 1 mL/min. Sample injection volume was 10 μl and column temperature was 25 °C. Phenolic compounds were identified according to their retention time and their purity checked throughout their absorption spectra at 352 nm and 372 nm. Every sample was analyzed in triplicate.
2.11. Statistical analysis Each experiment was carried out in triplicate. Data are expressed as Mean ± SD values of at least three experiments. For multiple comparisons, one-way ANOVA was followed by a Bonferroni test when variances were homogeneous or by Tamhane test when variances were not homogeneous. The level of significance was p < 0.05. In order to study the correlationship between the total polyphenol content and the antioxidant activity assessed by ORAC and DPPH assays, Pearson's correlation coefficient (p < 0.05) was applied. The statistical analyses were conducted using the statistical package SPSS version 22.0 for Windows (SPSS, Chicago; IL, USA). 3. Results and discussion 3.1. Polyphenol content
2.7. Cell culture
The highest total polyphenol content was found in the aqueouschloroform extract (278.76 mg GAE/g), followed by the ethanol and chloroform extracts (120.35 mg GAE/g and 29.72 mg GAE/g, respectively (Fig. 1), these results being probably related to the differences in the solvent polarity. Moreover, total content is much higher than that reported for the methanolic extract of 15.5 mg GAE/g (Alali et al., 2007). These differences may be attributed to the harvesting season and related environmental conditions (Stypinski, 1997; Luczkiewicz et al., 2001; Vicas et al., 2008).
NRK-52E renal endothelial line was a kind gift from Dr Sonia Pascual and Dr Luis Goya, ICTAN, CSIC (Madrid, Spain). Cells were grown in a humidified incubator at 5% CO2 and 95% air at 37 °C in Dulbecco's Modified Eagle's medium (DMEM) pyruvate free, from Invitrogen (Madrid, Spain), supplemented with 10% foetal bovine serum (FBS) (Biowhittaker) and 50 mg/L of each one of the following antibiotics: gentamicin, penicillin and streptomycin. 2.8. Cell treatment
3.2. Antioxidant capacity and oxygen scavenging activity
For all the experiments, different concentrations of P. argentea extract were dissolved in DMEM and added to the 96 multi-well cell plates for 24 h. Every sample was analyzed in triplicate, with 3 different experiments.
Different methods can be used to evaluate the antioxidant capacity of phenolic compounds. In this work, total polyphenol content, antioxidant capacity (ORAC) and free radical scavenging activity (DPPH) assays were used to assess to the ethanol, chloroform and aqueouschloroform extracts from aerial parts of P. argentea. Trolox, as a water-soluble analogue of vitamin E, was selected as a positive control in all the assays performed in this study. Trolox has been shown to decrease ROS production, to prevent cytotoxicity in human cell lines and to rescue cells from apoptotic death (Kello et al., 2014; Schoeneberger et al., 2014). The antioxidant capacity in P.
2.9. MTT assay Cell viability (cell growth inhibition) was determined by MTT assay (Mosmann, 1983; Carmichael et al., 1987) with some modifications. Cells were incubated in 96-well plates, at a density of 5 × 10⁴ cells/well for 24 h, then the cells were treated with different concentrations of P. argentea extract (0.1–100 μg/mL) for another 24 h. 5% Triton X-100 was used as a negative control; finally 2 mg/ml MTT was added and the plate was incubated for 1 h at 37 °C. Then, the formazan crystals formed were dissolved by adding DMSO and the absorbance was measured at 550 nm using Digiscan 340 microplate reader (ASYA Hitech GmbH, Eugendorf, Austria). 2.10. Intracellular ROS production assay ROS production was evaluated by the DCFH-DA assay (Le Bel et al., 1992) with some modification. This assay is based on the oxidation of the nonfluorescent compound 2′,7′-dichlorohydrofluorescein (DCFH) into the fluorescent compound dichlorofluorescein (DCF) in presence of ROS. Cells were incubated in 96-well multiwells and treated with different concentrations of P. argentea extract for 24 h. Then, 50 μL of DCFH-DA at a concentration of 10 μM were added for 30 min; then, the unabsorbed probe was removed and fluorescence in a microplate fluorescence reader (FLx800, Bio-Tek Instrumentation) at 480/510 nm
Fig. 1. Total polyphenol content in Paronychia argentea Lam. extracts. Values are means ± SD, n = 3. Different letters indicate statistically significant differences (p < 0.05) among groups. 3
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Table 1 ORAC values and free radical scavenging activity (DPPH) for P. argentea extracts. Values are means ± SD, n = 3. Different letters indicate statistically significant differences (p < 0.05) among groups. Extract
ORAC value (μmol Trolox equivalent/ mextract)
DPPH (IC50)
Ethanol Chloroform Aqueous-chloroform Trolox
1.452b ± 0.167 0.466a ± 0.072 2.587c ± 0.102 –
169.34b ± 2.19 601.72c ± 3.93 178.53b ± 4.43 102.59a ± 2.77
argentea extracts was first determined by the ORAC assay. Results show ORAC values ranging from 0.466 to 2.587 μmol TE/mg (Table 1), this indicating a moderate to strong antioxidant capacity (Dávalos et al., 2004; Zheng and Wang, 2001), the aqueous-chloroform extract being the most active. Results for the free radical scavenging activity of P. argentea extracts are expressed as the concentration of each extract which is capable of scavenge 50% of DPPH radicals (Table 1). The highest this concentration is, the lowest the oxygen scavenging activity is, as a higher quantity is needed to reach the same effect (Brand-Williams et al., 1995; Atoui et al., 2005). Our results show a high scavenging activity of both ethanol and aqueous-chloroform extracts from P. argentea, although lower than that of the positive control, Trolox. In fact, IC50 values for ethanol and aqueous-chloroform extracts (169.34 and 178.53 μg/mL, respectively) are significantly lower than those previously reported by other authors for the ethanolic extract (IC50 value of 208 μg/mL), but similar to that of the infusion (IC50 = 144.92 μg/mL) (Sait et al., 2015). This difference can be attributed to the variable polyphenol content, as shown in other vegetal species (Kang et al., 2003). When analyzing the results obtained for the antioxidant ability by the ORAC assay in relation to the phenolic composition, a positive (r = 0.987) and highly significant correlation (p < 0.0001) between both variables was found (Fig. 2a). These results strongly suggest that the total polyphenolic content remarkably contributes to the antioxidant capacity of P. argentea extracts. Thus, the higher total polyphenolic content obtained in the aqueous-chloroform extract resulted in higher total antioxidant capacity. These data are in line with previous studies which demonstrate the direct relationship between the antioxidant capacity and the polyphenol content (Dudonné et al., 2009; Hayase and Kato, 1984; Maisuthisakul et al., 2007;; Velioglu et al., 1998) and are consistent with our results revealing the aqueouschloroform extract from P. argentea with the highest content in total polyphenols. Moreover, a statistically significant (p = 0.01) negative (r = −0.784) correlation between total polyphenol content and IC50 values by DPPH scavenging assay was obtained (Fig. 2b), this indicating a moderate relationship between both variables. Collectively, the polyphenol content seems to be responsible for 78.4% of the antioxidant capacity of P. argentea extracts, whereas 21.6% of the antioxidant activity may be due to the presence of other antioxidant compounds which might be capable of inducing a synergistic effect. Also, the presence of non-phenolic compounds such as peptides, proteins and sugars, among others, may have greater impact on the DPPH results. The aqueous-chloroform extract showed the lowest value of IC50 value and the highest phenolic content, as they are inversely proportional, also being the most active extract in scavenging DPPH radicals. These results were reflected in the r value (r = 0.843; p = 0.004) obtained for the correlation between DPPH and ORAC values (Fig. 2c), which confirmed the higher antioxidant capacity of the aqueous extract. Taken together, the results of the antioxidant activity of P. argentea by different assays gave comparable results and confirm the relationship between phenolic compounds concentration and the oxygen radical scavenging ability. Therefore, the aqueous-chloroform extract was
Fig. 2. Correlation coefficient R between assays and total polyphenol content in Paronychia argentea Lam. extracts. a) ORAC assay versus total polyphenols. b) DPPH assay versus total polyphenols. c) DPPH assay versus ORAC assay.
then selected among the tested extracts for further evaluation in cells. 3.3. HPLC analysis In this study, the HPLC analysis allows the identification and quantification of the flavonoids in the aqueous-chloroform extract of P. argentea, the main components being quercetin derivatives (Table 2). Representative chromatograms at 352 and 372 nm are shown in Fig. 3. Our results are in accordance with those previously reported by other authors (Sait et al., 2015) who described the ethanolic extract of aerial parts of this species with a higher content in flavonoids than other preparations such as the decoction or infusion of the plant. 3.4. Cell viability The effect of the aqueous-chloroform extract of P. argentea on cell viability was assessed in NRK-52E renal cells, a useful cell model for the study of mechanisms and signal transduction pathways in physiological and pathological conditions (Álvarez-Cilleros et al., 2018), with the aim to select non-toxic concentrations of the plant extract. P. argentea extracts are not toxic at any of the assayed concentrations. The results of MTT assay indicated no-statistically significant 4
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Table 2 Flavonoids identified in P. argentea Lam. ethanolic extract by HPLC. Peak number
Compound
mg/g dried planta
t < SUB > R < /SUB > (min)
1 2 3
Quercetin-3-O-(glucosyl) galactoside Quercetin-3-O-galactoside Quercetinmethylether-O-hexoside
1.3 ± 0.1 3.7 ± 0.3 1.1 ± 0.1
15.33 18.57 22.21
a
Values are mean ± SEM, n = 3.
realistic concentration within the physiological range for the further ROS assays.
differences between the tested concentrations of P. argentea extract and the control group (untreated cells). On the contrary, renal cell viability was significantly reduced when rnal cells were treated with the negative and cytotoxic control, triton-X 100 (data not shown). In the MTT experiment we evaluated the effect of a concentration range of the plant extract on the renal cell line with the aim to select the
3.5. ROS production To test the effect of P. argentea on intracellular ROS levels,
Fig. 3. Chromatogram of the aqueous-chloroform extract of P. argentea (10 mg/mL) at a) 352 nm and b) 372 nm. Experimental conditions are described in the text. For peak identification, see Table 2. 5
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Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Abu-Irmaileh, B., Afifi, F., 2000. Treatment with medicinal plants in Jordan. Dirasat Med. Biol. Sci. 27 (1), 53–74. Afifi, F.U.B., Al-Khalidi, E.K., 2005. Studies on the in vivo hypoglycemic activities of two medicinal plants used in the treatment of diabetes in Jordanian traditional medicine following intranasal administration. J. Ethnopharmacol. 100, 314–318. Al-Bakri, A.G., Afifi, F.U., 2007. Evaluation of antimicrobial activity of selected plant extracts by rapid XTT colorimetry and bacterial enumeration. J. Microbiol. Methods 68, 19–25. Alali, F.Q., Tawaha, K., El-Elimat, T., Syouf, M., El-Fayad, M., Abulaila, K., Nielsen, S.J., Wheaton, W.D., Falkinham 3rd, J.O., Oberlies, N.H., 2007. Antioxidant activity and total phenolic content of aqueous and methanolic extracts of Jordanian plants: an ICBG project. Nat. Prod. 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Fig. 4. Effect of treatment with P. argentea on intracellular ROS production on NRK-52E cells. For the Direct effect, NRK-52E cells were treated with different concentrations of the extract for 24 h. For the Protective effect, NRK-52E cells were treated with different concentrations of the extract for 24 h. Then, cells were washed and 1 mM H2O2 was added to all the cultures except for controls for 3 h. Values are expressed as a percent relative to control condition ± SD (n = 3, 3 replicates). Different letters indicate statistically significant differences (p < 0.05) among groups.
concentrations of 1.0, 10, 25 and 50 μg/mL of aqueous-chloroform extract were added and evaluated by the DCFH-DA assay (Fig. 4). Treatment with 1 mM H2O2 caused an increase of about 10% in ROS levels, when compared to control cells. P. argentea alone did not significantly increase ROS concentration, this indicating no cellular stress or oxidative damage which could affect the functional conditions of cells. Pretreatment of the cells with P. argentea extracts at 1 and 10 μg/ mL, led to a ROS production similar to that of untreated cells or those treated with the positive control, Trolox. Differences were statistically significant with respect to the negative control, H2O2 (Fig. 4). Our results agree with those from other authors who assessed the protective role of the butanolic extract of P. argentea against oxidative stress (Zama et al., 2007) and those who related the antioxidant and freeradical scavenging activities of the plant to the reduction of urinary concentrations of stone-forming constituents (Bouanani et al., 2010).
4. Conclusion The study of P. argentea aerial parts by two different assays based on both hydrogen atom transference and electron transference (ORAC, DPPH, respectively) revealed the antioxidant activity of this specie. The total phenolic content determination, together with the chromatographic analysis showed a high content in phenolic compounds, mainly derivatives from the flavonol quercetin, which are the main responsible for the reported antioxidant capacity, although other antioxidant compounds might induce a synergistic effect. MTT assay showed that P. argentea is not toxic for cultured renal cells at concentrations ranging from 0.1 to 100 μg/mL, whereas ROS assays supported the chemoprotective effect of realistic concentrations of P. argentea extract, especially the lowest concentrations (1 and 10 μg/ mL), against oxidative damage induced with 1 mM H2O2 on the NRK52E renal endothelial cell line. This effect allows endothelial renal cells to be in a favourable condition to face an oxidative challenge. These results may contribute to the knowledge of the biochemical mechanisms involved in the physiological chemoprotection, although further studies regarding the bioavailability and the effects of gastrointestinal digestion, hepatic metabolism, distribution of the metabolites in the organism and urinary pH are needed previous to assess the efficacy of P. argentea on renal diseases in humans. 6
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