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Antioxidant, anti-inflammatory, and analgesic activities of Citrus reticulata Blanco leaves extracts: An in vivo and in vitro study

Activités antioxydantes, anti-inflammatoires et analgésiques des extraits des feuilles de Citrus reticulata Blanco: étude in vivo et in vitro

  • Aromathérapie Expérimentale
  • Published:
Phytothérapie

Abstract

Citrus species are cultivated and consumed widely. Citrus have been investigated for their pharmacological activity and human health. Their beneficial effects include antibacterial, analgesic, anti-inflammatory, and antitumoral effects. This studywas designed to evaluate the analgesic effect and the antioxidant and anti-inflammatory activities of Citrus reticulata Blanco leaves extracts (ECR) in cell and animal models. Antioxidant, anti-inflammatory, and antinociceptive activities were evaluated in mice using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical inhibition, xylene-induced ear edema, formalin assay and acetic acid-writhing response. Expression of antiinflammatory genes was measured in lipopolysaccharide (LPS)-treated Huh7 cells. ECR showed a significant DPPH radical scavenging activity. No behavioral changes or deaths were observed in mice at doses less than 2,000 mg/kg body weight. Different concentrations of methanolic and aqueous extracts (100–500 mg/kg body wt.) reduced the duration of linking behavior in the second phase of the formalin chemical nociception assay and decreased the number of acetic acidinduced writhing responses in mice, indicating significant analgesic activity. ECR also diminished xylene-induced ear swelling in mice, suggesting an in vivo anti-inflammatory action. No toxicity of ECR in the range of 0.1–10 μg/ml was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Cell treatment with LPS-induced oxidative/nitrosative stress as assessed by flow cytometry as the fluorescence of 2′,7′-dichlorofluorescein. This effect was significantly inhibited in a dose-dependent manner by ECR. Administration of ECR caused a dose-dependent inhibition of cytochrome P450 2E1, inducible nitric oxide synthase, tumor necrosis factor α, and interleukin-6 expression in LPS-treated cells. The present study demonstrates that extracts of Citrus reticulata leaves are safe, having antioxidant, anti-inflammatory, and analgesic effects both in vivo and in vitro.

Résumé

Les citrus sont cultivés et consommés largement. Les citrus ont été étudiés pour leur activité pharmacologique et leurs avantages sur la santé humaine. Ces bienfaits incluent l’activité antibactérienne, analgésique, antiinflammatoire et antitumorale. Cette étude a été conçue pour évaluer l’effet analgésique et l’acticité antioxydante et anti-inflammatoire des extraits des feuilles de Citrus reticulata Blanco (ECR) dans des modèles cellulaires et animaux. Les activités antioxydante, anti-inflammatoire et antinociceptive ont été évaluées chez des souris en utilisant l’inhibition du radical DPPH, l’oedème de l’oreille induit par le xylène, test de formaline et writhing reponse par l’acide acétique. L’expression des gènes anti-inflammatoires a été évaluée dans les cellules Huh7 traitées par le lipopolysaccharide (LPS). L’ECR a montré une activité antiradicalaire visà- vis du 1,1-diphenyl-2-picryl-hydrazyl (DPPH). Aucun changement de comportement ni de décès n’ont été observés chez les souris à des doses inférieures à 2 000 mg/kg de poids corporel. Les différentes concentrations des extraits aqueux et méthanoliques (100–500 mg/kg poids corporel) ont réduit la durée de léchage dans la deuxième phase de l’essai de la nociception chimique de formaline et ont diminué le nombre de torsions induites par l’acide acétique chez les souris, ce qui indique une activité antalgique significative. L’ECR a également diminué l’oedème de l’oreille induit par le xylène chez les souris, ce qui suggère une action anti-inflammatoire in vivo. Aucune toxicité de l’ECR dans la gamme de 0,1–10 μg/ml n’a été observée avec le bromure de 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT). Le traitement des cellules avec du LPS induit le stress oxydatif/nitrosatif tel qu’évalué par la cytométrie en flux en tant que fluorescence de 2′,7′-dichlorofluorescéine. Cet effet a été significativement inhibé par l’ECR de manière dose-dépendante. L’administration d’ECR a provoqué une inhibition dose-dépendante du cytochrome P450 (CYP) 2E1, de l’oxyde nitrique synthase inductible (iNOS), du facteur de nécrose tumorale (TNF)α et de l’expression de l’interleukine (IL)-6, dans les cellules traitées par le LPS. La présente étude démontre que les extraits des feuilles de Citrus reticulata ne sont pas dangereux, et possèdent des effets antioxydants, anti-inflammatoires et analgésiques in vivo et in vitro.

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Abbreviations

CYP:

cytochrome

DCF:

2′,7′-dichlorofluorescein

DCFH-DA:

2′,7′-dichlorodihydrofluorescein diacetate

DPPH:

1,1-diphenyl-2-picrylhydrazyl

ECR:

Citrus reticulate Blanco leaves extracts

GAE:

gallic acid equivalents

IL:

interleukin

iNOS:

inducible nitric oxide synthase

LPS:

lipopolysaccharide

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NF-κB:

nuclear factor kappa B

NSAIDs:

nonsteroidal anti-inflammatory drugs

RNS:

nitrogen species

ROS:

reactive oxygen

TNF:

tumor necrosis factor

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Authors and Affiliations

  1. Laboratoire de biotechnologies végétales et d’ethnobotanique, faculté des sciences de la nature et de la vie, université de Bejaia, Bejaia, 06000, Algérie

    M. Nasri & F. Bedjou

  2. Institute of Biomedicine (IBIOMED), University of León, León, Spain

    D. Porras & S. Martínez-Flórez

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  1. M. Nasri
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  2. F. Bedjou
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  4. S. Martínez-Flórez
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Nasri, M., Bedjou, F., Porras, D. et al. Antioxidant, anti-inflammatory, and analgesic activities of Citrus reticulata Blanco leaves extracts: An in vivo and in vitro study. Phytothérapie (2017). https://doi.org/10.1007/s10298-017-1094-8

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