Rice, a vital staple crop cultivated worldwide, is the primary dietary component for a substantial segment of the global population. In 2021, the Food and Agriculture Organization estimated global rice production at approximately 520.3 million tonnes (milled basis), with India contributing significantly, producing around 122 million tonnes (Statista) (Kumar et al., 2023). Throughout history, rice has held a place of esteem in traditional medicine across Asian countries, offering various therapeutic applications (Kowsalya et al., 2022). In the Philippines, rice bran has been employed as a rich source of Vitamin B for treating beriberi. At the same time, Malayan Medicine's Medicinal Book recommends using boiled rice greens as an eye lotion for acute inflammation and applying rice powder to specific skin ailments. In Cambodia, rice plant hulls are considered diuretic and used to treat dysentery. Chinese medicine attributes rice with the capacity to strengthen the spleen, stimulate appetite, and alleviate indigestion.

Consequently, sprouted rice grain powder is an external medicine to aid digestion, muscle toning, and gas release from the digestive tract (Umadevi et al., 2012). In India, traditional medicinal systems such as Ayurveda and Unani extensively use various parts of rice plants to address a wide range of health conditions, including high blood pressure, diabetes, gastrointestinal disorders, diarrhea, skin aging, anemia, lactation, and ulcers (Kowsalya et al., 2022). This traditional knowledge shows that, apart from rice grain, other parts of the rice plants also have significant medicinal value.

Rice bran, constituting approximately 8% of the whole grain, is the outer layer of the rice grain and is obtained as a by-product during rice milling. Rice bran is known for its rich content of bioactive compounds, including phenolic acids (e.g., ferulic acid, p-coumaric acid, p-hydroxybenzoic, protocatechuic acid, and caffeic acid), flavonoids (e.g., catechin, myricetin, quercetin, apigenin, and luteolin), and steroidal compounds (e.g., tocotrienol, tocopherol, β-sitosterol ferulate, γ-oryzanol, and campesterol ferulate). These bioactive compounds have been associated with rice bran's various pharmaceutical and therapeutic properties, including preventing diabetes, cancer, hypertension, fungal and bacterial infections, cholesterol reduction, radical scavenging, and inflammation (Kumar et al., 2023).

Inflammation, a fundamental component of the immune response, is pivotal in immune modulation to prevent diseases (Zhang and Mosser, 2008). Two primary types of inflammation exist-acute inflammation, caused by microbes and toxic compounds over a short period, and chronic inflammation, resulting from long-term exposure to foreign materials, autoimmune disorders, and immune cell dysregulation (Pahwa et al., 2023). Inflammatory stimuli activate macrophages in inflamed tissues, leading to the production of oxidative stress markers such as reactive oxygen species (ROS), nitric oxide (NO), prostaglandins, and inflammatory markers like various cytokines, interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-α) (Zhang and Mosser, 2008). While these inflammatory markers are critical players in local and systemic inflammation, their overproduction can damage tissue and contribute to various chronic illnesses, including cardiovascular disease, atherosclerosis, psoriasis, rheumatoid arthritis, and cancer (Chen et al., 2018). Consequently, the modulation of uncontrolled inflammatory responses, especially with dietary components possessing antioxidant and anti-inflammatory properties, is of paramount importance. Nutraceuticals such as phenolics, flavonoids, tocopherols, tocotrienols, and γ-oryzanol have been reported to influence inflammatory pathways and exhibit antioxidant and anti-inflammatory properties (Callcott et al., 2018). Recent studies have specifically highlighted the anti-inflammatory activities of major bioactive components found in rice bran, including γ-oryzanol, cycloartenyl ferulate, ferulic acid, catechin, vitamin E and rice bran extract, by inhibiting pro-inflammatory markers (Kumar et al., 2023; Lyu and Park, 2005; Ronchetti et al., 2009; Mizushina et al., 2013; Ng and Ko, 2012). Nevertheless, there is a need to explore the anti-inflammatory properties of pigmented rice bran.

The pigmented rice brans-purple, black, and red, are widely acknowledged as a potent source of bioactive components, including phenolics, flavonoids, vitamin E, and γ-oryzanol fractions. Moreover, pigmented rice brans are reported to be a potent source of antioxidants and possess ROS scavenging and quenching activity, thereby demonstrating antioxidant activities (Laokuldilok et al., 2011). Few available studies have shown that red and black rice and red rice bran extract have exerted anti-inflammatory properties by inhibiting the production of inflammatory markers and improving the expression of anti-inflammatory cytokines IL-10 (Kitisin et al., 2015; Limtrakul et al., 2016; Junmarkho and Hansakul, 2019; Onsa-Ard et al., 2022).

However, despite the promising evidence of the anti-inflammatory potential of rice bran, the bioactive components responsible for these properties are often trapped within the complex bran matrix, limiting their bioavailability (Guido and Moreira, 2017). Thus, there is a compelling need to explore effective processing techniques that release these bioactive components from the bran matrix while enhancing their anti-inflammatory properties. Recent studies have demonstrated that enzymatic processing can effectively break down the cell matrix and release these bioactive compounds (Guido and Moreira, 2017). Intriguingly, earlier research from our laboratory has shown that enzymatic treatment of red rice bran alters the profile of bioactive compounds and enhances its antioxidant activities (Sapna & Jayadeep, 2021a, 2021b). Nevertheless, such treatremainimpact on red rice bran's anti-inflammatory properties remains unexplored. Therefore, investigating bioactive-rich enzyme-treated red rice bran's anti-inflammatory efficacy is worthwhile.

Considering the abovementioned reports, this study explored the anti-inflammatory properties of enzyme-treated red rice bran extracts. It seeks to evaluate their efficacy in inhibiting pro-inflammatory cytokines (TNF-α, IL-6, IL-10, IL-1β) and mediators (ROS, NO, PGE2, COX2, iNOS) in LPS-induced RAW 264.7 macrophage cells. Additionally, the study intended to uncover the molecular mechanisms underlying these anti-inflammatory properties through mRNA expression studies. This research also endeavors to compare the anti-inflammatory efficacy of enzyme-treated bran extracts with the major bioactive components of rice bran with known anti-inflammatory properties, including ferulic acid, catechin, γ-tocopherol, and γ-oryzanol.