Nutriceutical pharmacology
Anti-oxidant and anti-inflammatory effects of cinnamaldehyde and eugenol on mononuclear cells of rheumatoid arthritis patients

https://doi.org/10.1016/j.ejphar.2019.02.031Get rights and content

Abstract

Rheumatoid arthritis (RA) is an autoimmune disorder affecting joints and frequently characterized by initial local and later systemic inflammation. The present study was conducted with the aim to determine the anti-inflammatory and antioxidant effects of cinnamaldehyde and eugenol in the peripheral blood mononuclear cells (PBMC) of RA patients. PBMCs obtained from RA patients were treated with varying concentrations of cinnamaldehyde and eugenol. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were monitored in the 24-h culture supernatant of PBMCs. Reactive oxygen species formation, biomolecular oxidation and the activities of antioxidant enzymes were also determined. FTIR analysis was done to determine structural alterations in the PBMCs. Molecular docking was performed to gain an insight into the binding mechanism of eugenol and cinnamaldehyde with pro-inflammatory cytokines. The levels of pro-inflammatory cytokines and markers of oxidative stress were found to be elevated in the PBMC culture of RA patients as compared to the healthy controls. Cinnamaldehyde and eugenol have significantly reduced the levels of cytokines. Reactive oxygen species formation, biomolecular oxidation and antioxidant defense response were also ameliorated by treating PBMCs with both the compounds. FTIR results further confirms cinnamaldehyde and eugenol mediated protection to biomolecules of PBMCs of RA patients. Molecular docking results indicates interaction of cinnamaldehyde and eugenol with key residues of TNF-α and IL-6. Cinnamaldehyde and eugenol were found to exert potent anti-inflammatory and anti-oxidant effects on the PBMC culture of RA patients. So, these compounds may be used as an adjunct in the management of RA.

Introduction

Rheumatoid arthritis (RA), a severe disabling arthritis is characterized by synovial infiltration and hyperplasia, thus leading to progressive destruction of articular tissues (Vivar and Van Vollenhoven, 2014). It affects around 1–2% of the world population, with women being 3 times more susceptible than man, probably due to the effect of female hormones on the activity of immune cells (Ngo et al., 2014). Although the etiology of RA is still not completely understood but some reports confirm the role of macrophages, T cells and B cells in the destruction of bone and cartilage (Hu et al., 2013). This autoimmune disorder is characterized by the presence of rheumatoid factor (antibody against IgG/IgM) and anti-citullinated peptide antibodies (ACPA) (Rantapää‐Dahlqvist et al., 2003). Activation of CD4+T cells by the arthritogenic agent (microbial or self-antigen) drives the autoimmune attack in the joints (Ahmed et al., 2017). Pro-inflammatory cytokines produced by activated T cells cause macrophages to release inflammatory mediators which in turn leads to secondary inflammatory injuries in arthritis (Chun et al., 2016).

The inflammatory cascade is also associated with the activation of oxidant generating enzymes (NADPH oxidase, xanthine oxidase, myeloperoxidase) thus promoting the production of reactive oxygen (ROS) and nitrogen (RNS) species (Fay et al., 2006). These reactive species are involved in cellular damage in joint tissues via various independent mechanisms such as depletion of antioxidants, lipid peroxidation, protein oxidation and DNA damage (Mahajan and Tandon, 2004). Thus inflammatory mediators and reactive species have direct or indirect effects on the pathophysiology of RA.

Non-steroidal anti-inflammatory drugs (NSAIDs) and disease modifying anti-rheumatic drugs (DMARDs) used for the treatment of RA are associated with various adverse drugs reaction, for example gastrointestinal ulcers, cardiovascular complications, and emergence of opportunistic infections due to immunosuppressants (Desai et al., 2014). Moreover, biologics such as TNF-α inhibitors, IL-1ß inhibitors, IL-6 inhibitors are also used in the treatment of RA. However their cost, side effects and interference in immune defense response limit their use (Li et al., 2013). Therefore, there is a need to evaluate anti-arthritic efficacy of natural compounds which are relatively safe, tolerable and easily accessible.

Cinnamaldehyde is a bioactive compound isolated from the bark of Cinnamomum cassia (Hancı et al., 2016). It has been used in the manufacturing industry as a spice and flavoring agent in beverages, sweets, ice-creams and chewing gums (Roth-Walter et al., 2014). Eugenol is a phenylpropanoid compound present in honey and essential oil of spices such as Syzgium aromaticum, Cinnamomum verum and Pimenta racemosa (Jaganathan et al., 2011). Both these compounds have been used in traditional medical practices (local analgesic and for the treatment of disturbances in blood circulation, dyspepsia, inflammation, gastritis) due to their anti-fungal, anti-bacterial, anti-inflammatory, anti-mutagenic and anti-oxidant effects (Jaganathan et al., 2011, Roth-Walter et al., 2014).

In our previous publication we have shown that cinnamaldehyde and eugenol have ameliorated oxidative stress and inflammation in collagen induced arthritis in rats (Mateen et al., 2019). In the present study keeping into consideration that RA is a systemic disorder, PBMCs obtained from RA patients were treated with various concentrations of cinnamaldehyde and eugenol in order to determine their effect on oxidant-antioxidant status and secretion of pro-inflammatory cytokines.

Section snippets

Human subjects

The study protocol was approved from the Institutional Ethical Committee of Jawaharlal Nehru Medical College, A.M.U. Informed consent was obtained from all the participants of the study. Blood was obtained from healthy controls (n = 10) and RA patients (n = 20) who attended the Orthopaedics Out Patient Department (OPD) of Jawaharlal Nehru Medical College and Hospital, Aligarh, India. Patients were diagnosed according to the European League Against Rheumatism (EULAR) 2010 classification criteria

Effect of cinnamaldehyde and eugenol on cell viability

To examine the effect of cinnamaldehyde and eugenol on the viability of PBMCs, MTT assay was performed. As shown in Fig. 1[A]. 10 20, 40 µM of cinnamaldehyde and eugenol caused no significant change in the viability of PBMCs. Therefore, for further experiments 10, 20 and 40 µM of cinnamaldehyde and eugenol were used.

Effect of cinnamaldehyde and eugenol on cytokine level

The level of pro-inflammatory cytokines TNF-α and IL-6 [Fig. 1B and C] were monitored in the 24-h culture supernatant of PBMCs. RA patients showed significantly higher levels of

Discussion

Rheumatoid arthritis is an inflammatory autoimmune disorder. Reactive oxygen species and pro-inflammatory cytokines have been shown to play an important role in the pathophysiology of RA (Mateen et al., 2016, Mateen et al., 2017). Various natural compounds have been shown to impart their antioxidant and anti-inflammatory effects in the management of many diseases (Umar et al., 2013, Umar et al., 2012, Wang et al., 2017). The results of the present study showed heightened inflammatory state in

Conclusion

Cinnamaldehyde and eugenol were effective in suppressing the secretion of pro-inflammatory cytokines from the cultured PBMCs of RA patients. These compounds have also reduced the neutralizing reactive oxygen/nitrogen species formation which in turn has ameliorated biomolecular oxidation and antioxidant defence response in the PBMC culture of RA patients. Thus these compounds have potential to be used as an adjunct in the management of RA by virtue of their free radical scavenging and

Acknowledgement

Assistance from the Institution (AMU) as well as infrastructural support from DST-FIST to the department is duly acknowledged. The authors extend their appreciation to the Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia for funding this work through research group project number RGP-215. The authors are grateful to Chairman, Dept. of Physiology for allowing us to avail various lab facilities. The authors are also thankful to Dr Saba Tufail, Dr. Asif Zaman, Mr. Mohd.

Author contributions

Conceived and designed the experiments: SMateen, SMoin. Performed the experiments: SMateen, MTR, SS, SK, FMH. Analyzed the data: SMateen, MTR, SMoin. Contributed samples/reagents/materials/analysis tools: SMoin, SSN, AQK, MTR, SK, FMH. Wrote the paper: SMateen, MTR, SSN.

Conflict of interest

The authors declare that they have no conflict of interest.

Author agreement

The manuscript entitled ‘Anti-oxidant and Anti-inflammatory effects of Cinnamaldehyde and Eugenol on Mononuclear Cells of Rheumatoid Arthritis Patients’ was reviewed by all authors, agreed with its content and agreed to its submission in European journal of pharmacology.

References (62)

  • S. Islam et al.

    Activation of Caspase-3 in human chondrosarcoma cells by Tumor Necrosis Factor-α and epigallocatechin-3-gallate in vitro

    Biochem. Biophys. Res. Commun.

    (2000)
  • S. Islam et al.

    Structural and immunological characterization of hydroxyl radical modified human IgG: clinical correlation in rheumatoid arthritis

    Spectrochim. Acta Part A: Mol. Biomol. Spectrosc.

    (2018)
  • H.M. Khojah et al.

    Reactive oxygen and nitrogen species in patients with rheumatoid arthritis as potential biomarkers for disease activity and the role of antioxidants

    Free Radic. Biol. Med.

    (2016)
  • R.L. Levine et al.

    [49] Determination of carbonyl content in oxidatively modified proteins

    Methods in Enzymology

    (1990)
  • X. Li et al.

    E)-3-(3, 4-Dimethoxyphenyl)-1-(5-hydroxy-2, 2-dimethyl-2H-chromen-6-yl) prop-2-en-1-one ameliorates the collagen-arthritis via blocking ERK/JNK and NF-κB signaling pathway

    Int. Immunopharmacol.

    (2013)
  • E. Lipiec et al.

    Synchrotron FTIR shows evidence of DNA damage and lipid accumulation in prostate adenocarcinoma PC-3 cells following proton irradiation

    J. Mol. Struct.

    (2014)
  • O.H. Lowry

    Protein measurement with folin phenol regent

    J. Biol. Chem.

    (1951)
  • S. Mateen et al.

    Cinnamaldehyde and eugenol attenuates collagen induced arthritis via reduction of free radicals and pro-inflammatory cytokines

    Phytomedicine

    (2019)
  • K.M. Miranda et al.

    A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite

    Nitric Oxide

    (2001)
  • S. Ngo et al.

    Gender differences in autoimmune disease

    Front. Neuroendocrinol.

    (2014)
  • A.T. Paul et al.

    Modulating TNF-alpha signaling with natural products

    Drug Discov. Today

    (2006)
  • A. Seven et al.

    Lipid, protein, DNA oxidation and antioxidant status in rheumatoid arthritis

    Clin. Biochem.

    (2008)
  • N.P. Singh et al.

    A simple technique for quantitation of low levels of DNA damage in individual cells

    Exp. Cell Res.

    (1988)
  • S. Umar et al.

    Modulation of the oxidative stress and inflammatory cytokine response by thymoquinone in the collagen induced arthritis in Wistar rats

    Chem.-Biol. Interact.

    (2012)
  • X. Wang et al.

    Anti-arthritic effect of berberine on adjuvant-induced rheumatoid arthritis in rats

    Biomed. Pharmacother.

    (2017)
  • X. Yao et al.

    Targeting interleukin-6 in inflammatory autoimmune diseases and cancers

    Pharmacol. Ther.

    (2014)
  • H. Yuan et al.

    Therapeutic role of a vaccine targeting RANKL and TNF-α on collagen-induced arthritis

    Biomaterials

    (2012)
  • Y.M. Ahmed et al.

    Granisetron and carvedilol can protect experimental rats against adjuvant-induced arthritis

    Immunopharmacol. Immunotoxicol.

    (2017)
  • D. Bezerra et al.

    The dual antioxidant/prooxidant effect of Eugenol and its action in cancer development and treatment

    Nutrients

    (2017)
  • R.J. Desai et al.

    Tumor necrosis factor-α inhibitor treatment and the risk of incident cardiovascular events in patients with early rheumatoid arthritis: a nested case-control study

    J. Rheumatol.

    (2014)
  • H. Fabian et al.

    A comparative infrared spectroscopic study of human breast tumors and breast tumor cell xenografts

    Biospectroscopy

    (1995)
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