Anti-inflammatory Effects of Astaxanthin Extracted from Microalgae Hematococcus pluvialis and Combinations with Palm Tocotrienol Rich-Fraction in RAW 264.7 Macrophages

Astaxanthin and tocotrienols, known as antioxidants derived from natural compounds and shown to have anti-inflammatory properties. This study aims to investigate the effects of a combination of astaxanthin extracted from Hematococcus pluvialis microalga and palm tocotrienols rich-fraction (TRF) on inflammatory reaction in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages cells. MTT assay was used to test cell viability and nitrite oxide (NO) was determined using Griess assay. Isobologram confirmed that the combined treatment produced synergistic effect and measurement of inflammatory cytokines such as interleukin 6 (IL-6) and interleukin 12 (IL-12) through ELISA assay. Our results showed that the combination of astaxanthin and TRF exhibited inflammatory markers such as NO production. The concentration of astaxanthin ranging from 10 to 100 μg/mL and TRF at 4-25 μg/mL had no toxicity and achieved higher cell viability. The combination treatments led to more potent inhibition of NO production compared to single treatments. Combination Index (CI) was achieved from the combination treatments at IC80, resulting in synergism at a CI value of 0.81. Furthermore, ELISA showed that the combined treatment significantly further reduced the expression levels of pro-inflammatory cytokines IL-6 and IL-12. Our findings suggest that the combination of astaxanthin and TRF enhanced anti-inflammatory and antioxidant activities in stimulated macrophages and may act synergistically to produce health effects reducing inflammation.

Key Words: Inflammation, Tocotrienol-Rich Fraction, Astaxanthin, Antioxidant, Combination, Macrophages