Anti-inflammatory Activity of Grains of Paradise Extract Notes

ANTI-INFLAMMATORY ACTIVITY OF GRAINS OF PARADISE

Authors and Affiliations

  • Nebojsa M. Ilic, Institute of Food Technology, University of Novi Sad, Serbia

  • Moul Dey, Biotech Center, Rutgers University, USA

  • Alexander A. Poulev, Biotech Center, Rutgers University, USA

  • Sithes Logendra, Biotech Center, Rutgers University, USA

  • Peter E. Kuhn, Biotech Center, Rutgers University, USA

  • Ilya Raskin, Biotech Center, Rutgers University, USA

ABSTRACT

  • Goal: Evaluate the anti-inflammatory activity of ethanolic extract of grains of paradise (Aframomum melegueta Schum, Zingiberaceae) on COX-2 enzyme, in vivo anti-inflammatory effects, and expression of pro-inflammatory genes.

  • Key Findings:

    • The anti-inflammatory compound identified is [6]-paradol.

    • [6]-shogaol is notable in inhibiting pro-inflammatory gene expression.

    • At 1000 mg/kg, whole extract reduces inflammation by 49% in rat paw edema model.

    • Individual gingerols [6]-paradol (20%), [6]-gingerol (25%), and [6]-shogaol (38%) showed variable efficacy when administered at 150 mg/kg.

    • The extract exhibits comparable effects to aspirin, the positive control.

KEYWORDS

  • anti-inflammatory, grains of paradise, Aframomum melegueta Schum, Zingiberaceae, gingerols, COX-2, paw edema

INTRODUCTION

  • Inflammation: Localized response to tissue injury (mechanical, biological, or autoimmune).

  • Arachidonic Acid Pathway: Main cellular mechanism in inflammation, including:

    • Cyclooxygenase pathway leading to prostaglandin synthesis.

    • COX Enzymes:

    • COX-1: Constitutive, “housekeeping”, protective functions (e.g., gastric lining).

    • COX-2: Inducible, primarily in inflamed tissues.

  • Importance of COX-2 inhibition in treating inflammatory responses for human health.

  • Aframomum melegueta: Traditional uses include treatment for stomachache, diarrhea, and as a spice in medieval Europe.

  • Previous studies indicated other properties, such as antiulcer, antimicrobial, and antioxidant activities.

OBJECTIVES

  • Assess anti-inflammatory effects through COX-2 inhibition and gene expression.

  • Identify compounds from the extract that may provide therapeutic benefits.

MATERIALS AND METHODS

Chemicals and Reagents

  • Source: Sigma-Aldrich Co. (unless otherwise stated).

  • Active compounds [6]-gingerol and [6]-shogaol from Dalton Chemical Laboratories.

  • Positive controls: Aspirin (>99% purity) and Vioxx (>98% purity).

Plant Material

  • Seeds of A. melegueta obtained from Abidjan, Ivory Coast. Verified by Dr. L. Struwe, Rutgers University.

  • Voucher specimen deposited at Chrysler Herbarium, Rutgers University.

Preparation of the Extract

  1. Grind seeds into powder.

  2. Extract 2 g seed powder with 20 mL 95% ethanol at room temperature for 24 hours.

  3. Filter and rotary-evaporate to recover 40.25 mg (2% yield).

COX-2 In Vitro Assay

  • Extract dissolved in 95% ethanol at 1 mg/mL.

  • Perform colorimetric COX (ovine) inhibitor assay (Cayman Chemical).

  • Measure peroxidase activity via TMPD absorbance at 590 nm.

Bioassay-Guided Purification

  • HPLC used for separation via preparatory HPLC and LC-MS analysis.

  • Conduct fractionation to isolate compounds responsible for COX-2 inhibition.

Gene Expression: Macrophage Cell Culture

  • Use RAW 264.7 (mouse monocyte/macrophage) in Dulbecco's modified Eagle medium (DMEM).

  • Maintain cells at 37°C with 5% CO2 and treat with extracts/DMSO before elicitation with LPS.

Gene Expression: Quantitative PCR

  • cDNAs diluted for quantitative analysis using SYBR green PCR master mix.

  • Use β-actin as the housekeeping gene for normalization via ΔΔCt methodology.

Carrageenan-Induced Rat Paw Edema

  • Male Long Evans rats, housed under standard conditions.

  • Fasting overnight before treatment with extracts and compounds, measuring inflammation via a plethysmometer.

STATISTICAL ANALYSIS

  • Utilization of ANOVA and Tukey’s multiple-comparison test for statistical significance across treatment groups.

RESULTS AND DISCUSSION

COX-2 Enzyme Inhibitory Activity

  • The extract inhibited COX-2 at 76%, inferior to Vioxx (87%).

  • Bioactivity-Guided Fractionation:

    • Identified [6]-paradol with 91% inhibition, making it the most potent compound.

    • Other identified compounds include [6]-gingerol and [6]-shogaol showing 7% and 68% inhibition respectively.

Inhibition of Pro-Inflammatory Gene Expression (In Vitro)

  • Extracts and [6]-shogaol inhibited IL-1β in a dose-dependent manner, but not others, indicating selective inhibition.

  • Cytotoxicity observed at concentrations >20 μg/mL, preventing further tests at higher doses.

Anti-Inflammatory Activity in Rat Paw Edema Assay (In Vivo)

  • Extract significantly inhibited edema:

    • 1000 mg/kg: 49% reduction

    • 500 mg/kg: 11% reduction

  • For isolated compounds at 150 mg/kg:

    • [6]-paradol: 20% reduction

    • [6]-shogaol: 38% reduction

    • [6]-gingerol: 25% reduction

    • Aspirin serves as the benchmark at 36% reduction.

Conclusion

  • The ethanolic extract of grains of paradise and its compounds exhibit the potential for in vitro and in vivo anti-inflammatory activity.

  • Future evaluation necessary to elucidate the mechanisms, especially regarding prostaglandin levels and detailed interactions of extracts and gingerols.

AUTHOR INFORMATION

  • Corresponding Author: Nebojsa M. Ilic, Email: nebojsa.ilic@fins.uns.ac.rs, Phone: +381 21 485 3824

  • Funding Acknowledgments: Research supported by Phytomedics Inc., NIH grants, and Rutgers University.

REFERENCES

  • Extensive citations from various journals and studies emphasizing prior research on COX-2, anti-inflammatory compounds, and traditional uses of Aframomum melegueta.