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Phenolic rich Cocos nucifera inflorescence extract ameliorates inflammatory responses in LPS-stimulated RAW264.7 macrophages and toxin-induced murine models

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Abstract

Anti-inflammatory and antinociceptive effects of the acetone extract of Cocos nucifera (CnAE), an important ingredient in several traditional drugs, have been studied using different in vitro and in vivo models. CnAE did not show any observable toxicity in RAW264.7 macrophages by MTT assay. The calorimetric analysis (total COX, 5-LOX, MPO, iNOS and NO), ELISA (IL-1β, IL-6, TNF-α and PGE2) and qRT-PCR (IL-1β, IL-6, TNF-α and NF-κB) were performed in LPS-induced RAW264.7 macrophages. Phosphorylation of NF-κBp65 and IκB was determined by western blotting. CnAE (100 µg/mL) remarkably inhibited total COX (68.67%) and 5-LOX (63.67%) activities, and subsequent release of iNOS, NO and PGE2 (p ≤ 0.05) in RAW264.7 cells treated with LPS. ELISA showed CnAE markedly decreased the level of pro-inflammatory cytokines IL-1β (p ≤ 0.001), IL-6 (p ≤ 0.001) and TNF-α (p ≤ 0.001) in LPS treated RAW264.7 cells. CnAE (100 µg/mL) also significantly down-regulated the mRNA expressions of pro-inflammatory cytokines (IL-1β, p ≤ 0.05; IL-6, p ≤ 0.01 and TNF-α, p ≤ 0.001) and NF-κB (p ≤ 0.001) against LPS-induction. Moreover, LPS-induced phosphorylation of IκB-α and NF-κB p65 was significantly inhibited by CnAE (100 µg/mL). In vivo anti-inflammatory studies showed that CnAE (400 mg/kg) significantly inhibited carrageenan-induced acute paw oedema (59.81%, p ≤ 0.001) and formalin-induced chronic paw oedema (52.90%, p ≤ 0.001) in mice. CnAE at a dose of 400 mg/kg also showed a significant anti-nociceptive effect on acetic acid-induced writhing (48.21%, p ≤ 0.001) and Eddy’s hot plate methods. These findings suggest that CnAE has significant anti-inflammatory and anti-nociceptive properties, mainly attributed to the inhibition of NF-κB/IκB signalling cascade.

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Abbreviations

ANOVA:

Analysis of variance

CD14:

Cluster of differentiation 14

CnAE:

Cocos nucifera acetone extract

COX:

Cyclooxygenase

DCFDA:

2′,7′-Dichlorofluorescin diacetate

DMSO:

Dimethyl sulfoxide

ELISA:

Enzyme-linked immunosorbent assay

IL:

Interleukin

IкB:

Inhibitor kappa B

iNOS:

Inducible nitric oxide synthase

IRAK:

Interleukin receptor-associated kinase

LOX:

Lipoxygenase

LPS:

Lipopolysaccharide

MD2:

Myeloid differentiation 2

MPO:

Myeloperoxide

mRNA:

Messenger ribonucleic acid

MyD88:

Myeloid differentiation primary response 88

NF-кB:

Nuclear factor kappa B

NO:

Nitric oxide

NOX:

Nicotinamide adenine dinucleotide phosphate oxidase

OECD:

Organisation for economic co-operation and development

PCR:

Polymerase chain reaction

PGE2:

Prostaglandin 2

qRT:

Quantitative real time

SD:

Standard deviation

TLR4:

Toll-like receptor 4

TNF:

Tumor necrosis factor

TRAF6:

TNF receptor associated factor 6

UV:

Ultra violet

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Acknowledgements

The authors are grateful to Dr. Ashok K. Chauhan, Founder President, Ritnand Balved Education Foundation (RBEF) and Amity Group of Institutions, and to Dr. Atul Chauhan, Chancellor, Amity University Uttar Pradesh (AUUP) for facilitating this work. Also we acknowledge our sincere gratitude to all those from AIPP, AUUP, Amala Cancer Research Centre, Thrissur, India and Biogenix Research Center, Thiruvananthapuram, India who extended their kind help and support.

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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Authors and Affiliations

  1. Amity Institute of Phytochemistry and Phytomedicine, 3 Ravi Nagar, Peroorkada P.O, Thiruvananthapuram, Kerala, 695005, India

    Manikantan Ambika Chithra, Thadiyan Parambil Ijinu, Palpu Pushpangadan & Varughese George

  2. Amity University Uttar Pradesh, Noida, Uttar Pradesh, 201303, India

    Harsha Kharkwal

  3. Pharmacopoeial Laboratory for Indian Medicine, Department of AYUSH, Ghaziabad, Uttar Pradesh, 201002, India

    Rajeev Kumar Sharma

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  1. Manikantan Ambika Chithra
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Contributions

MAC, TPI, HK, RKS, VG, PP designed study; MAC, TPI performed research; TPI, MAC, HK, RKS, VG analyzed data; TPI, MAC, VG wrote the paper.

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Correspondence to Varughese George.

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All procedures performed in studies involving animals were in accordance with the ethical standards of the Institution. Experiments were conducted after getting the approval of the Institute’s Animal Ethics Committee (IAEC; Reg. No. 149/199/CPCSEA), Amala Cancer Research Centre, Thrissur, Kerala, India.

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Chithra, M.A., Ijinu, T.P., Kharkwal, H. et al. Phenolic rich Cocos nucifera inflorescence extract ameliorates inflammatory responses in LPS-stimulated RAW264.7 macrophages and toxin-induced murine models. Inflammopharmacol 28, 1073–1089 (2020). https://doi.org/10.1007/s10787-019-00620-6

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