CBD, THC and the combination of both cannabinoids have been reported to exert a wide range of analgesic effects including nociceptive pain and neuropathic pain with distinct mechanism, which are potentially applicable for the treatment of severe chronic pain [32,33]. Despite the promising therapeutic applications, cannabis products and cannabinoids remain a controlled substance in many countries due to its psychoactive and addictive properties [34]. Meanwhile, CR, which contains only trace amounts of THC and CBD but is rich in bioactive compounds such as friedelin and epifriedelin [35], have been reported to exert multifaceted effects through various active ingredients [36]. Demonstrating the analgesic effect and mechanism of CR extract, in relation to the predicted bioactive compound, is necessary for its potential industrial or pharmaceutical application as a regulation-free alternative to conventional cannabis products.

In the present study, CSREA reduced Phase I pain-related behavior, which predominantly reflects direct peripheral nociceptor activation in the formalin test [37]. This early effect is compatible with a peripheral analgesic mechanism. Our neuronal cell data showed modulation of SCN9A (Nav1.7), ASIC1A, and TACR1, indicating reduced excitability and neuropeptide signaling (Figure 9). Concomitant alterations in FAAH/MAGL (Figure 8) and the CB1/CB2 receptor balance (Figure 10) further support enhanced endocannabinoid tone. Elevated AEA/2-AG would be expected to rapidly suppress nociceptor activity through presynaptic CB1-mediated inhibition of transmitter release and TRPV1 desensitization [38,39]. Taken together, these findings suggest that the attenuation of Phase I nocifensive behavior by CSREA may be mediated through modulation of the endocannabinoid system, leading to suppression of peripheral nociceptor excitability [40,41].

CSREA exhibited significant anti-inflammatory effects across various in vivo models. In the formalin-induced inflammation model, systemic IL-6 and IL-1β levels were markedly elevated in the formalin group but were significantly reduced by CSREA treatment, particularly at the higher dose (Figure 3). These results are consistent with behavioral outcomes observed during the late phase of the formalin test, where CSREA attenuated inflammatory pain (Figure 2). Similarly, in the carrageenan-induced inflammation model in rats, CSREA significantly reduced serum pro-inflammatory cytokines and paw edema, supporting its systemic anti-inflammatory efficacy (Figure 5 and Figure 6).

Histopathological analysis of H&E-stained tissue sections revealed severe dermal edema in the carrageenan-treated group, which was significantly reduced in the CSREA-treated group (Figure 7). Notably, the high-dose CSREA group exhibited only minimal interstitial swelling, indicating near-complete suppression of local inflammation. These findings suggest that CSREA provides local protection against tissue damage caused by the carrageenan-induced inflammatory response, as evidenced by the reduction in serum inflammatory markers.

Pro-inflammatory mediators produced by activated immune cells in damaged tissue or following nerve injury increase the resting membrane potential of neurons, lowering the threshold for activation and leading to nociceptor sensitization [42]. Therefore, the anti-inflammatory properties of CSREA demonstrated in our study may contribute to its pain-relieving effects. Inflammation and pain are closely linked through overlapping pathophysiological processes.

Pain is triggered by the recognition of harmful stimuli via nociceptors, which are specialized sensory neurons located as free nerve endings on Aδ and C fibers. These fibers possess distinct properties and transmit noxious signals to the cerebral cortex, where they are perceived as pain [43]. Although inflammatory and neuropathic pain have distinct etiologies, they share key mechanisms including altered ion channel expression, glial activation, and spinal sensitization. Understanding these overlapping pathways may help explain the transition from acute to chronic pain states [44].

Cannabinoids have been shown to inhibit C fiber-induced responses in dorsal horn neurons under normal conditions as well as in models of inflammation and nerve injury [45]. This effect is consistent with evidence demonstrating that cannabinoids reduce the expression of Fos protein-a marker of prolonged neuronal activation through CB1 and CB2 receptor-mediated pathways in various models of chronic pain [46,47,48]. However, as we noted, CSREA contains trace amounts of cannabinoids, which may not sufficiently account for explaining the analgesic properties of CSREA.

In our previous study, we hypothesized that the endocannabinoid system might be modulated by bioactive compounds derived from CR, which are not classified as cannabinoids (data not published). Unique phytochemicals of CR such as friedelin, epifriedelinol, and stigmasterol, which are known for their potential anti-inflammatory and analgesic effects [35,49,50], and may influence downstream signaling pathways. In addition, these non-cannabinoid compounds may affect the cannabinoid system directly (by interacting with traditional cannabinoid receptors (CB1 and CB2) or non-classical cannabinoid receptor- transient receptor potential cation channel subfamily V member 1 (TRPV1) and G protein-coupled receptor 55 (GPR55) [51] or indirectly (by modulating the endocannabinoid synthesis/degradation to control endocannabinoid levels, or even may affect other unknown pathways) to exhibit analgesic efficacy [45]. For instance, β-caryophyllene, a non-cannabinoid compound, is known to act as a CB2 receptor agonist, directly interacting with CB2R [52]. Moreover, non-cannabinoid agents, including certain non-steroidal anti-inflammatory drugs (NSAIDs), have been reported to exert indirect analgesic effects by inhibiting the metabolic degradation of endocannabinoids, thereby enhancing local endocannabinoid tone [53].

In our previous study, we demonstrated that CSREA regulates the balance of CB1 and CB2 receptors and reduces inflammatory cytokine levels at the in vitro level (Not published). The delicate changes in expression pattern of CB1 and CB2 receptors have been observed in various conditions, and are considered crucial in the pain regulation [54]. To control the balance of the receptor activity, various agonists and antagonists targeting cannabinoid receptors have been tested across different types of pain and pathological conditions [55]. Moreover, CB2-linked anti-inflammatory effects may contribute more prominently to Phase II.

2-AG, a key endocannabinoid, plays a significant role in modulating inflammation and nociception via CB1 and CB2 receptors [56,57]. 2-AG is synthesized on demand and rapidly degraded by hydrolytic enzymes, and its levels can be influenced by signaling lipids in central nervous systems which are critical regulators of neurotransmitter release. This biological process is involved in both physiological modulation of pain and pathological condition of neuroinflammation [39,58,59]. In this context, the enzymatic degradation of 2-AG by enzymes such as FAAH and MAGL is equally important in regulating its levels and, consequently, its effects on pain and inflammation [60,61]. Therefore, inhibition of these enzymes may increase 2-AG concentrations, enhancing anti-nociceptive and anti-inflammatory responses [60,62,63].

CSREA significantly reduced lipopolysaccharide (LPS)-induced elevations in 2-AG levels, accompanied by a marked decrease in pro-inflammatory cytokine expression. Conversely, in the absence of LPS stimulation, CSREA increased 2-AG levels, indicating a potential role in modulating endocannabinoid synthesis or degradation. This homeostatic effect of CSREA on 2-AG level may contribute to both its analgesic and anti-inflammatory potential. As demonstrated by the carrageenan-induced in vivo model, CSREA has the potential to inhibit local inflammation and reduce hind paw edema and serum cytokine levels, which may contribute to the attenuation of inflammatory pain responses. Furthermore, the therapeutic potential of CSREA may not be limited to acute pain response; it may attenuate chronic pain caused by neuronal inflammation or injury-associated neuropathic pain.

In addition to the receptor-based mechanisms, we have conducted cell-based experiments focusing on inflammation-related cytokines and nociceptive effect of CSREA. Our results indicate that treatment with CSREA led to a reduction in the levels of key pro-inflammatory cytokines, including IL-6 and IL-1β (Figure 3 and Figure 5). This suggests that the anti-inflammatory effects of the extract may extend to modulating cellular responses, particularly in immune cells, and reducing inflammation at the molecular level.

CSREA effectively counteracted RA-induced receptor imbalance by restoring the CB1R/CB2R ratio (Figure 10a,b). The transcript expression pattern of CB1R and CB2R is highly tissue-specific, and the ratio reflects the differentiation or developmental stages in neurons or immune cells [64,65]. This rebalancing suggests that CSREA modulates endocannabinoid receptor dynamics, potentially enhancing CB1R-mediated neuroprotective signaling and regulating CB2R-mediated immune responses. Such modulation may underlie the antinociceptive and anti-inflammatory effects observed in this study.

However, this study has several limitations. First, it does not identify the specific endocannabinoid receptors through which CSREA may exert its effects. The hypothesis that CSREA exhibits anti-nociceptive effects by modulating endocannabinoid receptors requires validation using rigorous experimental models, including receptor-specific knockout in vivo studies. Furthermore, in vivo measurements of cannabinoid levels—both local and systemic—are needed to confirm the correlation between altered expression of degradation enzymes and the observed analgesic effects. While diclofenac was used as a reference drug, a direct comparison using equipotent doses was not performed in this study. Future investigations will aim to establish pharmacological equivalence between CSREA and diclofenac by incorporating additional dose-ranging groups to better elucidate the relative efficacy and optimal dosing strategy of CSREA.

It is important to acknowledge several limitations in the present study that should be considered when interpreting its translational potential. Our experimental design employed a preventative analgesic model, wherein CSREA was administered prior to the induction of pain. While this approach is widely used for the initial screening of a compound’s intrinsic antinociceptive properties [66]; however, this design does not fully replicate the typical clinical scenario in humans, where analgesic treatments are administered after pain is already established. Future studies should therefore incorporate therapeutic models (i.e., administration of CSREA after the pain stimulus) to assess its efficacy in a more clinically relevant context.

This study presents evidence that CSREA possesses analgesic effects via modulating the endocannabinoid system in vivo. However, to fully elucidate the mechanisms underlying these effects, future studies should focus on investigating the specific signaling pathways involved in the antinociceptive effect of CSREA, their interaction with CB1 and CB2 receptors, and the modulation of 2-AG levels through the inhibition of FAAH and MAGL. Additionally, further in vitro and in vivo studies are needed to explore the long-term effects of CSREA on chronic pain and neuroinflammatory diseases. Additional positive controls, such as opioids, will be essential to better distinguish whether the antinociceptive effects of CSREA are mediated predominantly through neurogenic or inflammatory mechanisms. A comprehensive understanding of the pharmacological profile of the CSREA, including the role of FAAH and MAGL in the 2-AG pathway, will be essential in advancing its clinical applications.