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.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
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
References
Ghosh R, Alajvegovic A, Gomes AV (2015) NSAIDs and cardiovascular diseases: Role of reactive oxygen species. Oxid Med Cell Longev 2015:536962
González Gallego J, García-Mediavilla MV, Sánchez-Campos S, et al (2010) Fruit polyphenols, immunity and inflammation. Br J Nutr 104:S15–27
Wallace JL, Vong L (2008) NSAID-induced gastrointestinal damage and the design of GI-sparing NS. Curr Opin Investig Drugs 9:1151–6
Pisonero-Vaquero S, Martínez-Ferreras Á, García-Mediavilla MV, et al (2015) Quercetin ameliorates dysregulation of lipid metabolism genes via the PI3K/AKT pathway in a diet-induced mouse model of nonalcoholic fatty liver disease. Mol Nutr Food Res 59:879–93
Shah AS, Alagawadi KR (2011) Anti-inflammatory, analgesic and antipyretic properties of Thespesia populnea Soland ex. Correa seed extracts and its fractions in animal models. J Ethnopharmacol 137:1504–9
Lota ML, de Rocca Serra D, Tomi F, et al (2000) Chemical variability of peel and leaf essential oils of mandarins from Citrus reticulata Blanco. Biochem Syst Ecol 28:61–78
Álvarez B, Ramón-Laca L (2005) Pharmacological properties of citrus and their ancient and medieval uses in the Mediterranean region. J Ethnopharmacol 97:89–95
Perez YY, Jimenez-Ferrer E, Alonso D, et al (2010) Citrus limetta leaves extract antagonizes the hypertensive effect of angiotensin II. J Ethnopharmacol 128:611–4
Dallas C, Gerbi A, Elbez Y, et al (2014) Clinical study to assess the efficacy and safety of a citrus polyphenolic extract of red orange, grapefruit, and orange (Sinetrol-XPur) on weight management and metabolic parameters in healthy overweight individuals. Phytother Res 28:212–18
Graziano AC, Cardile V, Crascì L, et al (2012) Protective effects of an extract from Citrus bergamia against inflammatory injury in interferon-? and histamine exposed human keratinocytes. Life Sci 90:968–74
Singh J, Sood S, Muthuraman A (2014) In-vitro evaluation of bioactive compounds, anti-oxidant, lipid peroxidation and lipoxygenase inhibitory potential of Citrus karna L. peel extract. J Food Sci Technol 51:67–74
Zhou Z, Xi W, Hu Y, et al (2016) Antioxidant activity of Citrus fruits. Food Chem 196:885–96
García BF, Torres A, Macias FA (2015) Synergy and other interactions between polymethoxiflavones from Citrus byproducts. Molecules 20:20079–106
Chutia M, Deka Bhuyan P, Pathak MG, et al (2009) Antifungal activity and chemical composition of Citrus reticulata Blanco essential oil against phytopathogens from North East India. Food Sci Technol 42:777–80
Chen J, Zhuang D, Cai W, et al (2009) Inhibitory effects of four plants flavonoids extracts on fatty acid synthase. J Environ Sci 21:S131–S4
Kim MJ, Park HJ, Hong MS, et al (2005) Citrus reticulata Blanco induces apoptosis in human gastric cancer cells SNU-668. Nutr Cancer 51:78–82
Fan K, Kurihara N, Abe S, et al (2007) Chemopreventive effects of orange peel extract (OPE). I: OPE inhibits intestinal tumor growth in ApcMin/+ mice. J Med Food 10:11–7
Jung KH, Ha E, Kim MJ, et al (2007) Suppressive effects of nitirc oxide (NO) production and inducible nitric oxide synthase (iNOS) expression by Citrus reticulata extract in RAW 264.7 macrophage cells. Food Chem Toxicol 47:1545–59
Xiu LJ, Sun DZ, Jiao JP, et al (2015) Anticancer effects of traditional Chinese herbs with phlegm-eliminating properties — An overview. J Ethnopharmacol 172:155–61
Parhiz H, Roohbakhsh A, Soltani F, et al (2015) Antioxidant ansd anti-inflammatory properties of the citrus flavonoids hesperidin and hesperetin: an updated review of their molecular mechanisms and experimental models. Pytother Res 29:323–31
Zhang Y, Sun Y, Xi W, et al (2014) Phenolic compositions and antioxidant capacities of Chinese wild mandarin (Citrus reticulata Blanco) fruits. Food Chem 145:674–80
Lagha-Benamrouche S, Madani K (2013) Phenolic contents and antioxidant activity of orange varieties (Citrus sinensis L.and Citrus aurantium L.) cultivated in Algeria: peels and leaves. Ind Crops Prod 50:723–30
Manthey JA, Bendele P (2008) Anti-inflammatory activity of an orange peel polymethoxylated flavone, 3',4',3,5,6,7,8-heptamethoxyflavone, in the rat carrageenan/paw edema and mouse lipopolysaccharide-challenge assays. J Agric Food Chem 22: 9399–403
Gülçin Ì, Güngör SI, Beydemir S, et al (2004) Comparison of antioxidant activity of clove (Eugenia caryophylata Thunb) buds and lavender (Lavandula stoechas L.). Food Chem 87:393–400
Li BB, Smith B, Hossain MM (2006) Extraction of phenolics from citrus peels: I. Solvent extraction method. Sep Purif Technol 48:182–8
Bahorun T, Gressier B, Trotin F, et al (1966) Oxygen species scavenging activity of phenolic extracts from hawthorn fresh plant organs and pharmaceutical preparations. Arzneimittel Forschung 46:1086–9
Cuendet M, Dyatmiko W, Potterat O, et al (1997) Iridoid glucosides with free radical scavenging properties from Fagvaea blumei. Helv Chim Acta 80:1144–52
Mota AS, de Lima AB, Albuquerque TL, et al (2015) Antinociceptive activity and toxicity evaluation of the fatty oil from Plukenetia polyadenia Mull. Arg. (Euphorbiaceae). Molecules 20:7925–39
Xu Q, Wang Y, Guo S, et al (2014) Anti-inflammatory and analgesic activity of aqueous extract of Flos populi. J Ethnopharmacol 152:540–5
De la Puente B, Romero-Alejo E, Vela JM, et al (2015) Changes in saccharin preference behavior as a primary outcome to evaluate pain and analgesia in acetic acid-induced visceral pain in mice. J Pain Res 6:663–73
Crespo I, García-Mediavilla MV, Gutiérrez B, et al (2008) A comparison of the effects of kaempferol and quercetin on cytokine-induced pro-inflammatory status of cultured human endothelial cells. Br J Nutr 100:968–76
García-Mediavilla MV, Sánchez-Campos S, González-Pérez P, et al (2005) Differential contribution of hepatitis C virus NS5A and core proteins to the induction of oxidative and nitrosative stress in human hepatocyte-derived cells. J Hepatol 43:606–13
Pisonero-Vaquero S, García-Mediavilla MV, Jorquera F, et al (2014) Modulation of PI3K-LXRalpha-dependent lipogenesis mediated by oxidative/nitrosative stress contributes to inhibition of HCV replication by quercetin. Lab Invest 94:262–74
Miquilena-Colina ME, Lima-Cabello E, Sánchez-Campos S, et al (2011) Hepatic fatty acid translocase CD36 upregulation is associated with insulin resistance, hyperinsulinaemia and increased steatosis in non-alcoholic steatohepatitis and chronic hepatitis C. Gut 60:1394–402
Carbajo-Pescador S, Ordoñez R, Benet M, et al (2003) Inhibition of VEGF expression through blockade of Hif1a and STAT3 signalling mediates the anti-angiogenic effect of melatonin in HepG2 liver cancer cells. Br J Cancer 109:83–91
Karimi E, Oskoueian E, Hendra R, et al (2012) Phenolic compounds characterization and biological activities of Citrus aurantium bloom. Molecules 17:1203–18
Turkmen N, Sari F, Velioglu YS (2006) Effects of extraction solvents on concentration and antioxidant activity of black and black mate tea polyphenols determined by ferrous tartrate and Folin-Ciocalteu methods. Food Chem 99:835–41
Metrouh-Amir H, Duarte CMM, Maiza F (2015) Solvent effect on total phenolic contents, antioxidant, and antibacterial activities of Matricaria pubescens. Ind Crops Prod 67:249–56
Muthiah P, Asokkumar K (2012) In vitro antioxidant activities of leaves, fruits and peel extracts of citrus. Int J Phytopharmacy 2:13–20
Tripoli E, Guardia ML, Giammanco S, et al (2007) Citrus flavonoids: molecular structure, biological activity and nutritional properties: a review. Food Chem 104:466–79
Bourgou S, Ksouri R, Bellila A, et al (2005) Phenolic composition and biological activities of Tunisian Nigella sativa L. shoots and roots. C R Biol 331:48–55
Wei L, Dong L, Zhao T, et al (2011) Analgesic and antiinflammatory effects of the amphibian neurotoxin, anntoxin. Biochimie 93:995–1000
Khan S, Mehmood MH, Ali AN, et al (2011) Studies on antiinflammatory and analgesic activities of betel nut in rodents. J Ethnopharmacol 135:654–61
Saragusti AC, Bustos PS, Pierosan L, et al (2012) Involvement of the L-arginine–nitric oxide pathway in the antinociception caused by fruits of Prosopis strombulifera (Lam.) Benth. J Ethnopharmacol 140:117–22
Thun MJ, Henley SJ, Patrono C (2002) Nonsteroidal antiinflammatory drugs as anticancer agents: Mechanistic, pharmacologic, and clinical issues. J Natl Cancer Inst 94:252–66
Zakaria ZA, Ghani ZD, Nor RN, et al (2008) Antinociceptive, anti-inflammatory, and antipyretic properties of an aqueous extract of Dicranopteris linearis leaves in experimental animal models. J Nat Med 62:179–87
Ishola IO, Awodele O, Olusayero AM, et al (2014) Mechanisms of analgesis and anti-inflammatory properties of Annona muricata Linn. (annonaceae) fruit extract in rodents. J Med Food 17:1375–82
He XY, Liu QC, Peng W, et al (2013) Bioactivities and serum pharmacochemistry of Qi-Wei-Xiao-Yan-Tang. Pharm Biol 51:629–34
Molinett S, Nuñez F, Moya-León MA (2015) Chilean strawberry consumption protects against LPS-induced liver injury by antiinflammatory and antioxidant capability in Sprague-Dawley rats. Evid Based Complement Alternat Med 2015:320136
Hassan W, Rongyin G, Daoud A, et al (2014) Reduced oxidative stress contributes to the lipid lowering effects of isoquercitrin in free fatty acids induced hepatocytes. Oxid Med Cell Longev 2014:313602
Xing SQ, Zhang CG, Yuan JF, et al (2015) Adiponectin induces apoptosis in hepatocellular carcinoma through differential modulation of thioredoxin proteins. Biochem Pharmacol 93:221–31
Ho SC, Kuo CT (2014) Hesperidin, nobiletin, and tangeretin are collectively responsible for the anti-neuroinflammatory capacity of tangerine peel (Citrus reticulata epericarpium). Food Chem Toxicol 71:176–82
Saluk J, Bijak M, Posmyk MM, et al (2015) Red cabbage anthocyanins as inhibitors of lipopolysaccharide-induced oxidative stress in blood platelets. Int J Biol Macromol 80:702–9
Choi SY, Hwang JH, Ko HC, et al (2007) Nobiletin from citrus fruit peel inhibits DNA-binding activity of NF-kappaB abs ROS production in LPS-activated RAW 264.7 cells. J Ethnopharmacol 113:149–55
Tuñon MJ, García-Mediavilla MV, Sánchez-Campos S, et al (2009) Potential of flavonoids as anti-inflammatory agents: modulation of pro-inflammatory gene expression and signal transduction pathways. Curr Drug Metab 10:256–71
García-Mediavilla V, Crespo I, Collado PS, et al (2007) Antiinflammatory effect of the flavones quercetin and kaempferol in Chang Liver cells involves inhibition of inducible nitric oxide synthase, cyclooxygenase-2 and reactive C-protein, and downregulation of the nuclear factor kappaB pathway. Eur J Pharmacol 557:221–9
Marcolin E, San-Miguel B, Vallejo D, et al (2012) Quercetin treatment ameliorates inflammation and fibrosis in mice with nonalcoholic steatohepatitis. J Nutr 142:1821–8
Ihara I, Yamamoto H, Ida T, et al (2012) Inhibition of nitric oxide production and inducible nitric oxide synthase expression by a polymethoxy flavone from young fruits of Citrus unshiu in rat primary astrocytes. Biosci Biotechnol Biochem 76:1843–8
Kang SR, Han DY, Park KI, et al (2011) Suppressive effect on lpopolysaccharide-induced proinflammatory mediators by Citrus aurantium L. in macrophage RAW 264.7 cells via NF-kB signal pathway. Evid Based Complement Alternat Med 2011:248592
Lin N, Sato T, Takayama Y, et al (2003) Novel anti-inflammatory actions of nobiletin, a citrus polymethoxy flavonoid, on human synovial fibroblasts and mouse macrophages. Biochem Pharmacol 65:2065–71
Cui Y, Wu J, Jung SC, et al (2010) Anti-neuroinflammatory activity of nobiletin on suppression of microglial activation. Biol Pharm Bull 33:1814–21
Yoshigai E, Machida T, Okuyama T, et al (2013) Citrus nobiletin suppreßses inducible nitric oxide synthase gene expression in interleukin-1ß-treated hepatocytes. Biochem Biophys Res Commun 439:54–9
Vafei Adou K, Vauzour D, Lee HY, et al (2009) The citrus flavanone naringenin inhibits inflammatory signalling in glial cells and protects against neuroinflammatory injury. Arch Biochem Biophys 484:100–9
Yang HL, Chen SC, Senthil Kumar KJ, et al (2012) Antioxidant and anti-inflammatory potential of hesperetin metabolites obtained from hesperetin-administered rat serum: an ex vivo approach. J Agric Food Chem 60:522–32
Zhang S, Zheng L, Dong D, et al (2013) Effects of flavonoids from Rosa laevigata Michx fruit against high-fat diet-induced non-alcoholic fatty liver disease in rats. Food Chem 141:108–16
Cao Q, Mak K, Lieber C (2005) Cytochrome P4502E1 primes macrophages to increase TNF-alpha production in response to liposaccharide. Am J Physiol 289:G95–G107
Abdulla D, Goralski KB, Renton KW (2006) The regulation of cytochrome P450 2E1 during LPS-induced inflammation in the rat. Toxicol Appl Pharmacol 216:1–10
Gaudineau C, Beckerman R, Welbourn S, et al (2004) Inhbition of human P450 enzymes by multiple constituents of the Gingko biloba extract. Biochem Biophys Res Commun 318:1072–8
Kusirisin W, Jaikang C, Chaiyasut C, et al (2009) Effect of polyphenolic compounds from Solanum torvum on plasma lipid peroxidation, superoxide anion and cytochrome P450 2E1 in human liver microsomes. Med Chem 5:583–8
Author information
Authors and Affiliations
Corresponding author
About this article
Cite this article
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
Published:
DOI: https://doi.org/10.1007/s10298-017-1094-8