Uncovering the anti-inflammatory potential of *Lactiplantibacillus Plantarum* fermented *Cannabis Sativa* L seeds

Inflammation, a crucial immune response to harmful stimuli, plays a dual role in disease initiation and progression. The overproduction of inflammatory factors such as TNF-α, IL-6, IL-1β, IL-18, and nitric oxide (NO) contributes to various inflammatory diseases posing a significant global health burden. Current treatments often involve biological agents like anti-TNF-α, anti-IL-1, and anti-IL-6, but these can have complicated application conditions. Therefore, exploring safer and more effective natural anti-inflammatory compounds is crucial. *Cannabis sativa* L. (hemp) seeds, rich in protein and oil and containing low levels of psychoactive compounds, are emerging as a promising food source. They also contain various phytochemicals, including cannabinoic acids (CBDA, CBGA) and their decarboxylated forms (CBD, CBG), phenolics, organic acids, unsaturated fatty acids, and protein hydrolysates, which have demonstrated anti-inflammatory, antioxidant, neuroprotective, and anxiolytic properties. However, research on hemp seeds' physiological benefits is limited. Fermentation, a well-established food processing and medicinal technique, efficiently releases bioactive compounds from food matrices. Lactic acid bacteria (LABs), known for their safety and diverse bioactivities (immunomodulatory, anti-inflammatory, anti-diabetic, and antioxidant), are commonly used in fermentation. This study aims to investigate the anti-inflammatory effects of hemp seeds fermented with LABs, focusing on *Lactiplantibacillus plantarum*, and to identify the responsible metabolites using untargeted metabolomics.

Existing research highlights the anti-inflammatory properties of various components in hemp seeds, including CBD and other cannabinoids. Studies have also shown the benefits of fermentation in enhancing the bioactivity of food products by releasing bioactive compounds. However, research specifically exploring the anti-inflammatory potential of *Lactiplantibacillus plantarum* fermented hemp seeds is limited. Untargeted metabolomics, a powerful tool for identifying metabolic changes, has not been extensively applied to analyze the metabolic profiles of raw and fermented hemp seeds. The study aims to address this gap by employing this technique to identify key anti-inflammatory metabolites produced during fermentation.

This study employed several methods to investigate the anti-inflammatory properties of *Lactiplantibacillus plantarum* fermented hemp seeds (FHS) and to identify the underlying mechanisms. **Fermentation:** Raw hemp seeds were fermented with ten different probiotic strains, including *L. plantarum*, under controlled conditions. The fermented samples were then freeze-dried for further analysis. **Cytotoxicity assay:** The cytotoxicity of FHS on RAW 264.7 cells was assessed using the EZ-Cytox assay to determine the safe concentration range for subsequent experiments. **Anti-inflammatory assays:** The anti-inflammatory effects of FHS were evaluated using LPS-induced RAW 264.7 cells, measuring the levels of TNF-α, IL-1β, IL-6, IL-18, and NO using ELISA kits. A dose-response curve was generated to determine the optimal concentration of FHS for further studies. **Immunoblotting analysis:** Western blotting was used to analyze the protein expression levels of TLR4, NF-κB p65, and iNOS in LPS-stimulated RAW 264.7 cells treated with FHS, to investigate the mechanism by which FHS exerts its anti-inflammatory effects. **UHPLC-QTOF-MS-based untargeted metabolomics:** This technique was used to compare the metabolic profiles of raw hemp seeds (RHS) and FHS. Multivariate (PCA), univariate, and correlation analyses were performed to identify metabolites associated with anti-inflammatory activity. **HPLC-DAD analysis:** This was conducted to validate and quantify the concentrations of key anti-inflammatory metabolites identified through metabolomics. **In vitro simulated digestion and bioaccessibility assay:** This involved exposing FHS and key identified metabolites to simulated oral, gastric, and intestinal conditions to assess the stability and bioaccessibility of these compounds using a Caco-2 cell monolayer model.

The study found that *L. plantarum* fermented hemp seeds (FHS) exhibited significantly enhanced anti-inflammatory activity compared to raw hemp seeds (RHS). FHS demonstrated a dose-dependent inhibition of TNF-α, IL-6, IL-1β, and NO within a concentration range of 50-500 µg/mL. The anti-inflammatory effect was also accompanied by a reduction in the expression of TLR4, NF-κB p65, and iNOS, suggesting an impact on the TLR4/NF-κB signaling pathway. Untargeted metabolomics analysis revealed significant metabolic alterations in FHS compared to RHS. Indolelactic acid (IA) and homovanillic acid (HVA) emerged as the main anti-inflammatory metabolites. HPLC analysis confirmed the increased concentrations of IA and HVA in FHS compared to RHS. Both IA and HVA exhibited potent anti-inflammatory abilities with low IC₅₀ values for TNF-α, IL-1β, IL-6, IL-18, and NO inhibition. In vitro gastrointestinal digestion coupled with the Caco-2 cell monolayer model indicated that IA and HVA in FHS maintain stability and have high bioaccessibility (approximately 60% for both IA and HVA).

The findings of this study clearly demonstrate the enhanced anti-inflammatory potential of hemp seeds after fermentation with *L. plantarum*. The significant reduction in inflammatory markers and cytokines, coupled with the identification of IA and HVA as key bioactive compounds, supports the development of FHS as a novel functional food ingredient. The in vitro digestion and bioaccessibility studies further substantiate the potential of FHS for human consumption and absorption of the beneficial compounds. The mechanism of action appears to involve the modulation of the TLR4/NF-κB signaling pathway, a critical player in inflammation. The identification of IA and HVA as key bioactive compounds offers valuable insights into the potential health benefits of FHS and provides potential biomarkers for future studies. The results align with previous research showcasing the anti-inflammatory properties of individual compounds like IA and HVA, strengthening the validity of the findings. The study contributes to the growing body of research on the potential of hemp seeds as a functional food and emphasizes the role of fermentation technology in enhancing their bioactivity.

This study successfully demonstrated the significant anti-inflammatory properties of *L. plantarum* fermented hemp seeds (FHS). Indolelactic acid (IA) and homovanillic acid (HVA) were identified as key anti-inflammatory metabolites with high bioaccessibility. These findings suggest FHS holds significant potential for developing novel functional foods targeting inflammation-associated disorders. Future research could focus on in vivo studies to confirm these findings in animal models and eventually human clinical trials to validate the efficacy and safety of FHS for various inflammatory conditions. Further exploration of other bioactive compounds present in FHS and their mechanisms of action is also warranted.

This study was primarily conducted at the cellular level using in vitro models. While the in vitro digestion model provides valuable insights into bioaccessibility, it does not fully represent the complexity of human digestion. In vivo studies are necessary to confirm the anti-inflammatory effects and bioavailability of FHS in animal models and ultimately in humans. The study also focused on a limited number of inflammatory markers and cytokines; investigation of additional markers might provide a more comprehensive understanding of the anti-inflammatory effects of FHS.