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. 2019 Aug 14;56(12):5492–5499. doi: 10.1007/s13197-019-04021-z

Pro-oxidant versus anti-oxidant effects of seeds aglycone extracts of Lepidium sativum and Eruca vesicaria Linn., in vitro, and on neutrophil nitro-oxidative functions

Nabila Tounsi 1, Bahia Djerdjouri 1,✉, Ouahiba Ait Yahia 2, Aicha Belkebir 2
PMCID: PMC6838261  PMID: 31749497

Abstract

This study evaluated the anti-inflammatory and antioxidant properties of seeds aglycone extracts from Lepidium sativum (LS) and Eruca vesicaria (EV) Linn., on oxidative damages in vitro and on neutrophil nitro-oxidative functions. The results showed that LS and EV aglycone extracts attenuated liver microsomal lipids and proteins oxidation through a potent antioxidant effect as attested by the dose dependent quenching of DPPH radical scavenging activity. LS and EV aglycone extracts inhibited dose dependently the production of superoxide anion by BALB/c mice-derived peritoneal neutrophils, whereas they slightly enhanced exocytosis of myeloperoxidase (MPO), a marker of azurophilic granules. Interestingly, only LS replenished glutathione (GSH) and nitric oxide levels, indicating a fine differential effect. This study highlighted the subtle oxidative and antioxidant capacity of LS and EV seeds aglycone extracts. These health promoting compounds could be used to finely modulate critical events involved in microbial infection, inflammation and nitro-oxidative stress.

Keywords: Eruca vesicaria, Lepidium sativum, Glutathione, Lipid peroxides, Myeloperoxidase, Nitro-oxidative stress

Introduction

Plants used as food supply and in traditional herbal medicine, have beneficial potential with determinant impacts on human health.

Plants secondary metabolites were extensively exploited as antioxidants to treat nitro-oxidative stress-associated human diseases such as rheumatisms, asthma, cardiopathies, aging and neuropathies, diabetes and cancer (Podsędek 2007; Pandey and Rizvi 2009). This common pharmacological feature of vegetables and medicinal plants is supported by various naturally occurring aglycone compounds including polyphenols, flavonoids, terpens and alkaloids (Lee et al. 2010; Impellizzeri et al. 2011).

Inflammatory syndromes develop on chronic inflammation and acute activation of inflammatory cells, namely neutrophils (Itou et al. 2006).

Neutrophils (PN) are the first line of defence against infection. Their intracellular killing functions result from synergistic release of reactive oxygen species (ROS) and granular proteolytic and oxidative enzymes into the phagolysosome. They can also eliminate extracellular pathogens trapped into a network of proteins-studded chromatin known as neutrophil extracellular traps (NETs) (Rigby and DeLeo 2012).

Neutrophils are the major source of ROS initiated by the most powerful phagocyte NADPH oxidase2 (NOX2) which reduces oxygen to superoxide anion (O·−2). NOX2 crosstalk with superoxide dismutase, catalase and granular myeloperoxidase (MPO) generates various oxygen metabolites including hydrogen peroxide (H2O2), hydroxyl radical (·OH), singlet oxygen (1O2), and peroxyl (ROO·) (Rigby and DeLeo 2012). Persistent nitro-oxidative stress results from mixed reactive species with strong oxidative capacity such as peroxynitrite (ONOO−) and nitrogen dioxide (NO2−), or reactive chlorine species generated upon reaction between ROS and nitric oxide (NO) produced by NO synthase (NOS) activity.

Modulation of neutrophil functions was shown to be beneficial in inflammatory, infectious and tumor conditions, and in prevention of tissue oxidative injury (Kruger et al. 2015).

The ROS dependent scission of polyunsaturated fatty acids, releases oxidized volatile lipids such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA) (Gaweł et al. 2004). Similarly, the carbonylation of proteins irreversibly alters amino acid side-chains into aldehyde or ketone groups, and thereby proteins structure and function (Suzuki et al. 2010).

Lepidium sativum (LS) and Eruca vesicaria (EV) belong to the large family of Brassicaceae. These two eatable cruciferous plants are enriched in various bioactive components, namely the sulforaphane, a potent inducer of the NRF2-HO-1 antioxidant pathway (Fahley et al. 2001; Bell et al. 2015). Usefully, a high intake of brassicaceous vegetables has been associated with a reduced risk of diabetes, cardiovascular diseases and cancer (Podsędek 2007).

Aglycones such as flavonoids have recognized antioxidant, anti-bacterial, anti-inflammatory, anti-mutagenic and anti-tumoral potential (Impellizzeri et al. 2011; Al-Sheddi et al. 2016). The antioxidant effect of flavonoids encompasses inhibition of ROS production, ROS scavenging and neutralising capacity, prevention of GSH depletion and oxidative damage of biomolecules. Flavonoids finely modulate the expression of antioxidant enzymes including glutathione S-transferase (GST), NAD(P)H quinone dehydrogenase-1 (NQO-1) and heme oxygenase-1 (HO-1) through activation of intracellular redox sensor, nuclear factor like Nuclear factor (erythroid derived 2)-like 2 (Nrf2), and its recruitment to the antioxidant response element (ARE), a cis-acting DNA sequence (Prestera et al. 1993; Hayes and Dinkova-Kostova 2014).

This study investigated the anti-inflammatory and antioxidant potential of seeds aglycone extracts from LS and EV on DPPH radical scavenging activity, oxidative damages in vitro, and on neutrophil nitro-oxidative functions.

Materials and methods

Animals and chemicals

Reagents were obtained from Sigma Aldrich except otherwise indicated. BALB/c mice (6–8 weeks old) purchased from Institut Pasteur (Algiers, Algeria), were acclimated to standard laboratory conditions before use.

All experiments were approved by the Ethical Committee for animal handling of the of the “Université des Sciences et de la Technologie Houari Boumediene” (Algiers, Algeria).

Extraction of aglycones and determination of flavonoids content

The flavonoid aglycones were extracted from seeds of two domestic plants Eruca vesicaria and Lepidium sativum Linn., according to Chang et al. (2002) with slight modifications. In brief, after ethyl ether extraction, crude extracts were dried and solubilized in methanol. The flavonoid content were quantified by differential spectrophotometry at 420 nm in the presence of AlCl3, and expressed as quercetin equivalent.

Based on preliminary assays, seeds aglycone extracts of Lepidium sativum (LS) were used at 0.016 mg/ml (LS1) and 0.16 mg/ml (LS2) and those of Eruca vesicaria (EV) at 0.16 mg/ml (EV1) 0.72 mg/ml (EV2).

DPPH radical scavenging capacity assay

Free radical scavenging capacity of EV and LS aglycone extracts was determined using the DPPH (2,2-diphenyl-2-picrylhydrazyl radical) assay (Vivot et al. 2001). In brief, the reaction medium containing 150 μM DPPH was incubated in the absence (control) or presence of aglycone extracts, 30 min at 37 °C. Their antioxidant activity was measured at 515 nm, and expressed as % inhibition of control. Ascorbic acid (500 µM) was used as a standard antioxidant.

Evaluation of seeds aglycone extracts effects on oxidative stress, in vitro

Proteins carbonylation assay

BSA (1 mg/ml) was oxidized in 10 mM phosphate buffer (pH 7.4), 30 min at 37 °C, and treated with diphenylhydrazine (DNPH, 10 mM) at room temperature. Proteins extracts were pelleted by centrifugation at 10,000×g for 10 min, washed three times with ethanol/ethyl acetate (1:1, v/v) and dissolved in phosphate buffer. Carbonyls content was determined from absorbance at 360 nm (Yan et al. 1996). The preventing effect of aglycone extracts on BSA-carbonyl group’s formation was expressed as percentage % of control inhibition.

Evaluation of aglycone extracts on microsomal oxidative stress

Isolation of liver microsomal fraction and treatment

Fresh livers isolated from BALB/c mice were homogenized in ice cold isotonic sucrose solution 10% (w/v) containing 50 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 25 mM KC1 and 8 mM CaCl2, and centrifuged at 10,000×g, 15 min at 4 °C. The post-mitochondrial supernatant was diluted with 12.5 mM sucrose solution containing 8 mM CaCl2 and 5 mM MgCl2 and centrifuged at 1500×g for 10 min. The pellet was diluted in sucrose solution and centrifuged twice at 1500×g for 10 min, and finally homogenized in Tris–HCl buffer containing 5 mM MgCl2 and 25 mM KCl (Schenkman and Cinti 1978). The liver fraction oxidized with 1 mM ferric chloride and 1 mM ascorbic acid was incubated 1 h, at 37 °C, treated or not (control) with aglycone extracts and the levels of lipid peroxides and gluthatione (GSH) were measured.

Lipid peroxidation assay

Thiobarbituric acid reactive substances (TBARS) were determined as described by Ohkawa et al. (1979). The liver fraction was added to a reaction mixture containing 8.1% (w/v) SDS, 20% (v/v) acetic acid (pH 3.5) and 0.8% (w/v) thiobarbituric acid, boiled 1 h at 95 °C, and then centrifuged at 3000×g for 10 min. Absorbance of supernatants was measured at 532 nm. N-Acetylcysteine (NAC) at 500 µM was used as a standard antioxidant.

The percentage of inhibition of lipid peroxidation (LPI) was calculated as follows

A=absorbance532nm=ALPI%=1-[Asample/Ablank]×100

Reduced glutathione assay

Reduced glutathione (GSH) assay was performed according to Sedlak and Lindsay (1968). Liver fractions were treated with 5-sulfosalicylic acid (5%) for 30 min, and centrifuged at 10,000×g, 20 min at 4 °C. The pellets suspended in 0.2 M Tris–EDTA (pH 8.9) were treated with 10 mM Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid)) (DTNB). Absorbance was read at 412 nm, and the amount of GSH expressed in µM, using GSH standard curve.

Evaluation of aglycone extracts on neutrophil functions

Isolation of BALB/c mice derived-peritoneal neutrophils

Exudates of peritoneal neutrophils were elicited by intra-peritoneal injection of thioglycollate broth (4%) to BALB/c mice. Neutrophils (PN) were collected in sterile PBS (pH 7.35) and pelleted by centrifugation at 350×g, 10 min at 4 °C (Itou et al. 2006). Crude PN were washed 2 times with erythrocyte lysis buffer, and suspended in sterile PBS at 106 PN/ml.

Effects of aglycone extracts on superoxide anion production

PN were suspended at 106 cells/ml in PBS pH 7.35 supplemented with 7.5 μM glucose, 1.26 mM CaCl2, 0.5 mM MgCl2, and 80 μM cytochrome c and incubated, 30 min at 37 °C in the absence (control) or presence of aglycone extracts, prior to stimulation with phorbol myristate acetate (PMA, 160 nM). NAC (500 µM) was used as a standard scavenger of superoxide anion. The production of superoxide anion (O·−2) was continuously recorded for 30 min by monitoring superoxide dismutase-inhibitable reduction of cytochrome at 550 nm. Results were expressed as nM of reduced cytochrome c/min/106 cells (Djerdjouri et al. 1995).

Effect of aglycone extracts on myeloperoxidase exocytosis

PN (106 cells/ml) in sterile PBS (pH 7.35) were assayed for azurophilic granules exocytosis according to Kanashiro et al. (2007).

Aliquots of 106 cells treated with aglycone extracts (LS1, 0.016 mg/ml, LS2, 0.16 mg/ml, EV1, 0.16 mg/ml and EV2, 0.72 mg/ml) for 30 min, and then with 5 µg/ml cytochalasin B, 5 min at 37 °C. After stimulation with bacterial peptide formyl methionyl leucyl phenylalanine (fMLP, 10−6 M) for an additional 15 min, the reaction was stopped in ice and the supernatants were collected by centrifugation at 300×g, 5 min at 4 °C.

Myeloperoxidase (MPO) activity was assayed in PBS (pH 6.0) at 25 °C, containing 0.167 mg/ml ortho-dianisidine and 0.0006% H2O2. Absorbance was measured at 460 nm for 2 min, and results were expressed as µM H2O2/min/mg protein (Bradley et al. 1982). The protein concentration was quantified by Bradford assay (Bradford 1976).

Effects of aglycone extracts on nitric oxide production

Nitric oxide (NO) level was determined as a marker of NO synthase (NOS) activity by measuring nitrites (NO2−), the stable end products of NO (Green et al. 1982).

Equal volumes of supernatant of phagocytes culture and Griess reagent (1% sulphanilamide and 0.1% N-(1-napthyl)ethylenediamine dihydrochloride (in 2.5% H3PO4) were incubated, 10 min at room temperature. Absorbance was read at 540 nm and NO2− expressed in µM, using sodium nitrite curve as standard.

Statistical analysis

Data were analyzed using GraphPad Prism software (Version 5.02; San Diego, CA, USA). Results were expressed as mean ± SD (standard deviation of the mean). Statistical evaluation was performed by a two-tailed unpaired one way ANOVA, followed by Newman–Keuls multiple comparison test with p < 0.05 considered statistically significant.

Results

Dose dependent ROS scavenging effect of LS and EV aglycone extracts

Aglycone extracts inhibited dose dependently the DPPH radical scavenging activity.

The inhibitory effect reached 12% (LS1, p < 0.001) and 78% (LS2, p < 0.01), and 28% (EV1) and 81% (EV2) (p < 0.001) of control, respectively (Fig. 1).

Fig. 1.

Fig. 1

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Lepidium sativum and Eruca vesicaria effect on DPPH scavenging capacity in vitro. Data are mean ± SD of three independent experiments, two-tailed unpaired one way ANOVA followed by Newman–Keuls multiple comparison test

At 0.16 mg/ml, the quenching activity of LS was found to be 2.78-fold higher than EV (p < 0.001). Comparatively, ascorbic acid (500 µM) shifted by 80% the DPPH radical scavenging activity.

LS aglycone extracts selectively enhanced microsomal GSH content

EV aglycone extract had no impact on microsomal GSH content, whereas LS aglycone extract enhanced it by 117% (LS1 p > 0.05) and 283% (LS2, p < 0.001) of control. The antioxidant NAC, a precursor of GSH synthesis, enhanced GSH levels by fivefold, compared to LS2 (Fig. 2a).

Fig. 2.

Fig. 2

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In vitro effects of Lepidium sativum and Eruca vesicaria seeds aglycone extracts on glutathione levels in vitro (a), on protein carbonylation in vitro (b), and on lipid peroxidation inhibition  % in vitro (c). Data are mean ± SD of three independent experiments, two-tailed unpaired one way ANOVA followed by Newman–Keuls multiple comparison test

Protective effects of aglycone extracts on protein oxidation

EV aglycone extract displayed a dose dependent protection on BSA oxidation that reached 28% (LS1, p < 0.001) and 41% (LS2, p < 0.001), respectively, of control (Fig. 2b).

The protective effect of EV1 on oxidative damage of BSA was 1.75-fold (p < 0.05) higher than that of LS2.

LS and EV aglycone extracts inhibited equipotently liver lipids peroxidation

Aglycone extracts were equipotent (LS2 vs. EV1) in reducing lipid peroxidation in liver microsomal fraction (Fig. 2c). The LPI reached 43% (LS1, p < 0.01), and 44% (LS2, p < 0.01), and 48% (EV1, p < 0.01) and 59% (EV2, p < 0.01) of control.

The highest concentration of EV aglycone extract was as efficient as 500 µM NAC, used as standard antioxidant (57%, p < 0.01).

Aglycone extracts inhibited superoxide production by PMA-stimulated neutrophils

LS and EV aglycone extracts exerted a potent anti-inflammatory effect. They depressed superoxide anion production by PMA-stimulated neutrophils by 36% (LS1) and 72% (LS2), and by 71% (EV1) and 87% (EV2) (p < 0.001) (Fig. 3a). Moreover, 500 µM NAC, a standard ROS scavenger, displayed an inhibitory effect reaching 54% of control.

Fig. 3.

Fig. 3

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Lepidium sativum and Eruca vesicaria aglycone extracts effects on superoxide anions production by PMA-stimulated neutrophils (a), on nitrites release by fMLP-stimulated neutrophils (b), and on myeloperoxidase exocytosis by fMLP-stimulated neutrophils (c). Data are mean ± SD of three independent experiments, two-tailed unpaired one way ANOVA followed by Newman–Keuls multiple comparison test

Differential effect of aglycone extracts on nitric oxide production

EV aglycone extracts attenuated nitric oxide (NO) production by fMLP-stimulated neutrophils by approximately 16% (EV1, p<0.05) and 18% (LS1, p < 0.01).

However, LS aglycone extract had antioxidant effect at low concentration (LS1, p < 0.05), but at higher concentration enhanced NO release by 36% (LS2, p < 0.001), through a pro-oxidant effect (Fig. 3b).

Potentiating effects of aglycone extracts on myeloperoxidase exocytosis

Aglycone extracts enhanced dose dependently MPO release by fMLP-stimulated neutrophils. The levels reached 12% (LS1, p < 0.001) and 24% (LS2, p < 0.01), and 22% (EV1, p < 0.001) and 33% (EV2, p < 0.001) of control (Fig. 3c).

Discussion

Plants are an inexhaustible source of bioactive secondary metabolites with beneficial potential on many human inflammatory syndromes. This study evaluated the anti-inflammatory and antioxidant effect of seeds aglycone extracts of two cruciferous vegetables, Lepidium sativum (LS) and Eruca vesicaria (EV) on neutrophils oxidative functions and oxidative damages, in vitro.

Our results showed that aglycone extracts exert a direct and dose dependent inhibition of DPPH radical scavenging activity, reflecting a strong antioxidant potential.

Cell redox homeostasis is the result of a fine balance between the production of reactive oxygen and nitrogen species (ROS) and their clearance by nitro-antioxidant systems.

Physiological ROS levels navigate between various metabolic enzymes, primarily the phagocyte NOX2, MPO, NO synthase, xanthine oxidase, cytochromes P450, cyclooxygenases and lipooxygenases, and between biochemical processes namely mitochondrial respiration and oxidative killing of pathogens (Zhang et al. 2016). Under eustress conditions, ROS act as signaling molecules that control cell survival and proliferation (Liou and Storz 2010). When accumulated, ROS sustain lipids and proteins oxidation that subsequently decrease antioxidant capacity of tissues and cells. In the in vitro model of lipid peroxidation, Fe2+ catalyzed the conversion of superoxide anion (O·−2) and hydrogen peroxide (H2O2) into highly damaging hydroxyl radical (·OH) through the Fenton reaction or by the Fe2+ catalyzed Haber–Weiss reaction. In turn, Fe3+ can be reduced either by O·−2, or by ascorbate to further propagate self oxidative cycles (Fig. 4) (Jomova and Valko 2011). Low-dose of LS and EV aglycones, equipotently reduce microsomal lipid oxidation.

Fig. 4.

Fig. 4

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Diagram showing the differential anti-inflammatory and antioxidant potential of Lepidium sativum and Eruca vesicaria seeds aglycone extracts

Moreover, EV and LS aglycones inhibit dose dependently protein carbonylation in the model of BSA-oxidation, a result supported by a previous study showing that aglycones of some antioxidants (namely quercetin and genistein) were effective in reducing the carbonyl and sulfhydryl group formation of BSA (Utrera and Estévez 2013).

Nitric oxide (NO) is a key modulator of inflammatory responses and achieved both protective and toxic effect. This dual role of NO depends on NO concentration, the NO synthase isoform involved and the cells in which it is synthesized (Thippeswamy et al. 2006).

At similar dose, EV aglycone exerts anti-inflammatory effect, whereas LS aglycone stimulates NO release as it was shown with glycyrrhizin aglycone which can increase NO production and up-regulates iNOS expression through NF-κB pathway (Jeong and Kim 2002). Indeed, enhanced nitrites levels were associated with higher concentration of GSH production (Fig. 1b). This enhanced nitrites levels may explain the inefficiency of LS aglycone at higher concentration to further inhibit LPI (Fig. 2a). Low O·−2 level can interact with NO to produce strong oxidants such as peroxynitrite (ONOO−) and thereby hydroxyl radical, that actively contribute to lipid peroxidation and cell membranes alteration (Szabó et al. 2007).

Both EV and LS aglycone dramatically inhibits O·−2 release by PMA-stimulated neutrophils. Previous studies reported O·−2 scavenging by polyphenol aglycones (Lee et al. 2010).

Neutrophils have multifaceted functions, and ROS derived from oxygen by NOX2 and nitrogen by iNOS can synergize with the release of granular enzymes inducing beneficial inflammatory response, but could also amplify pathological nitro-oxidative reactions chains (Fig. 4) (Mayadas et al. 2014).

Our results show that aglycones induce an unexpected increase on MPO degranulation of fMLP-stimulated neutrophils. In a mouse model of carrageenan-induced pleurisy, oleuropein aglycone caused significant reduction of neutrophils infiltration and MPO activity (Impellizzeri et al. 2011). Moreover, 7-hydroxycoumarin and their derivatives and quercitin might act as co-substrates of MPO resulting in the formation of the oxidized coumarin and quercetin (Shiba et al. 2008; Kabeya et al. 2013). The catechol group is required for the inhibition of MPO activity. MPO catalyzes the oxidation of etoposide phenolic ring into phenoxyl radical, whose reactivity may determine their pro-oxidant effects in cells (Kagan et al. 2001).This prooxidant effect of oxidized antioxidant is only observed in a cell system. In vivo, aglycones with a catechol group are mostly converted into inactive metabolites by conjugation. The ratio of active aglycone to inactive metabolites is regulated by the balance between conjugation and deconjugation reaction (Shiba et al. 2008).

Conclusion

As a result, aglycone free radicals scavenging capacity, prevention of lipid peroxidation and protein carbonylation, and restoration of intracellular GSH may account for the beneficial of LS and EV aglycone extracts. The antioxidant activity might be helpful against various oxidative stress-related diseases, whereas the pro-oxidant effect through enhanced MPO activity shared by LS and EV and the selective enhanced release of NO by LS could account for potent killing process as well as antimicrobial and anti-tumoral potential.

The balance between the anti- and pro-oxidant properties of aglycones is essential to exert beneficial pharmacological effects without suppressing the oxidative microbial killing ability of neutrophils. Thus, it is relevant to understand the molecular basis namely NOS and MPO regulation and GSH turnover by aglycones on effector functions of neutrophils, a key cells involved in the regulation of inflammatory processes.

Acknowledgements

The authors are grateful to Dr Haj-Arab. H. for the identification of the plant

Abbreviations

AA

Ascorbic acid

EV

Eruca vesicaria

GSH

Reduced glutathione

HNE

4-Hydroxynonenal

LPI

Lipid peroxidation inhibition

LS

Lepidium sativum

MDA

Malondialdehyde

MPO

Myeloperoxidase

NAC

N-Acetylcysteine

NO

Nitric oxide

NOS

Nitric oxide synthase

NOX2

NADPH oxidase2

ProtCO

Protein carbonyls

PN

Neutrophils

ROS

Reactive oxygen species

O·−2

Superoxide anion

Compliance with ethical standards

Conflict of interest

All authors declare that there is no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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