For centuries, humans have leveraged the health-promoting properties of plants for our well-being. While research has been conducted on numerous medicinal plants, the specific benefits of many species remain underexplored. Eupatorium Japonicum (EJ), a member of the Asteraceae family, has historically been consumed in Japan, South Korea, China, and Vietnam for its traditional use in soothing digestive issues. This study aimed to explore the radical scavenging and antiinflammatory efficacy of EJ extract using RAW 264.7 cells. The radical-scavenging effects were assessed using the DPPH and ABTS assays, where an anti-oxidative molecule in the test sample will react with a stable free radical in DPPH and ABTS causing discoloration. The anti-inflammatory efficacy was assessed using the nitric oxide (NO) assay in LPS-induced RAW 264.7 cells, where the amount of NO produced in response to infection was measured using Griess reagent. Reversetranscriptase polymerase chain reaction (RT-PCR) and real-time PCR were executed to confirm the anti-inflammatory activity by measuring the RNA levels of pro-inflammatory cytokines. The DPPH and ABTS assays revealed that EJ extract decreased oxidation in a concentration-dependent manner (7.8-1,000 μg/mL) compared to ascorbic acid and Trolox respectively. EJ extract significantly reduced NO production concentration independently. Furthermore, EJ extract showed no cytotoxic effects as determined through the MTT assay. RT-PCR and real-time PCR analyses revealed inhibition of mRNA expression of pro-inflammatory cytokines (iNOS, COX-2, TNF-α, and IL-6). Western blotting demonstrated EJ’s anti-inflammatory activity by reducing protein levels of iNOS, COX-2, TNF-α, and IL-6. These findings suggest that EJ extract exhibits anti-inflammatory activities and can be further evaluated in the future.

Abstract
INTRODUCTION
MATERIALS AND METHODS
    Materials and reagents
    Sample preparation
    2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay
    2,2'-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid(ABTS) assay
    Cell culture
    Nitric oxide (NO) assay
    Cell viability assays
    RNA extraction, reverse-transcriptase polymerasechain reaction (RT-PCR), and real-time polymerasechain reaction (real-time PCR)
    Western blot analysis
    Statistical analysis
RESULTS
    Anti-oxidant activity of EJ extract via DPPH andABTS assays
    Anti-inflammatory activity of EJ extract via NO assay
    Cell viability of EJ extract via MTT assay
    Reduction of pro-inflammatory mediators and cytokinesby EJ extract
    EJ alleviates iNOS, COX-2 phosphorylation
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES

• Uyanga Batmunkh(Department of Veterinary Medicine, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea)
• Abdul Wahab Akram(Department of Veterinary Medicine, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea)
• Sung Dae Kim(Department of Veterinary Medicine, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea)
• Tae-Wan Kim(Department of Veterinary Medicine, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea)
• Man Hee Rhee(Department of Veterinary Medicine, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea, Institute for Veterinary Biomedical Science, Kyungpook National University, Daegu 41566, Korea) Corresponding author