Pain is a complex protective response that arises when tissue injury or dysfunction disrupts physiological homeostasis. It is transmitted through ascending nociceptive pathways involving the spinothalamic tract, thalamus, midbrain, and pons before reaching the somatosensory cortex, where the intensity, location, and severity of pain are perceived. Although pain serves as an essential defense mechanism, persistent or pathological pain significantly impairs quality of life and represents a major global clinical burden [1, 2, 3].

Despite advances in pharmacological management, effective treatment of pain remains a major clinical challenge. Opioid analgesics and non-steroidal anti-inflammatory drugs (NSAIDs) are the mainstay of pain therapy; however, their long-term use is frequently associated with serious adverse effects. Opioids are well known to cause tolerance, physical dependence, and addiction [4, 5]. Whereas NSAIDs are commonly linked to gastrointestinal irritation, renal dysfunction, and increased cardiovascular risk. Moreover, these conventional analgesics often provide only partial relief, with many patients experiencing inadequate pain control [6, 7, 8]. Collectively; these limitations underscore the urgent need to develop safer and more effective non-opioid analgesic strategies for the management of acute and chronic pain.

In earlier studies, natural bioactive compounds have gained increasing attention as potential non-opioid analgesics due to their multi-target mechanisms and favorable safety profiles [8]. Among these, flavonoids represent a large class of polyphenolic compounds widely distributed in fruits and medicinal plants, exhibiting antioxidant, anti-inflammatory, and neuroprotective properties [9, 10]. Furthermore, previous research has demonstrated that flavonoids can modulate pain and inflammation by inhibiting cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, suppressing oxidative stress, and interacting with key neurotransmitter systems, including $\gamma$-aminobutyric acid (GABA), serotonin, dopamine, and glycine [11, 12, 13]. Notably, flavonoids such as epigallocatechin-3-gallate (EGCG) have shown significant analgesic and anti-inflammatory effects in both preclinical and clinical studies, supporting their translational potential as non-opioid therapeutic agents [14, 15, 16].

7-Hydroxyflavone (7-HF), a naturally occurring flavonoid isolated from Oxytropis falcata Bunge, a traditional Tibetan medicinal plant, has been reported to exhibit a broad spectrum of pharmacological activities, including antioxidant, anti-inflammatory, antiplatelet, and organ-protective effects [17, 18, 19]. Mechanistic studies have demonstrated that 7-HF activates the Nrf2/NQO1/HO-1 signaling pathway, thereby enhancing endogenous antioxidant defenses, as evidenced by increased superoxide dismutase activity and reduced lipid peroxidation [20]. In addition, 7-HF has been shown to suppress pro-inflammatory cytokines such as tumor necrosis factor-$\alpha$ (TNF-$\alpha$), interleukin-1$\beta$ (IL-1$\beta$), and interleukin-6 (IL-6) through modulation of the MAPK/NF-$\kappa$B signaling pathway [21]. Importantly, experimental evidence indicates that 7-HF exerts significant analgesic effects by activating ATP-sensitive potassium channels and by dual inhibition of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes, resulting in attenuation of inflammatory edema and nociceptive pain [21, 22].

Safety evaluations further support the therapeutic potential of 7-HF. Developmental toxicity assessments using zebrafish embryo-larval models have shown no significant adverse effects on growth or behavior, indicating a favorable safety profile [23].

Despite such promising pharmacological activities, no study has systematically evaluated the analgesic efficacy of 7-HF, and its underlying mechanisms have not been comprehensively investigated using both centrally and peripherally mediated pain models. Therefore, the present study was undertaken to investigate the analgesic and antioxidant effects of 7-hydroxyflavone using established experimental pain models and to elucidate its possible mechanisms of action. This investigation aims to contribute to the development of safer, non-opioid therapeutic strategies for effective pain management.

The present study provides experimental evidence that 7-hydroxyflavone (7-HF) exerts dose-dependent analgesic activity through both central and peripheral mechanisms, with the higher dose (10 mg/kg) producing significant and sustained antinociceptive effects. 7-Hydroxyflavone (7-HF) effectively modulates analgesic activity through modulation of behavioral and oxidative parameters. These findings suggest that 7-HF possesses analgesic potential in experimental pain models and supports its involvement in both central and peripheral antinociceptive pathways.

Pain is a complex protective response that arises when tissue injury or dysfunction disrupts physiological homeostasis [1]. Analgesics are medications that target the peripheral or central nervous system to alleviate pain without causing substantial changes in consciousness [34]. Centrally acting analgesics raise the pain threshold and modify the physiological response. In contrast, peripherally acting analgesics block chemoreceptor impulses [35]. Analgesic activities were screened in pain-state models employing thermal stimuli like tail-flick and hot plate methods. Both approaches demonstrate centrally mediated antinociceptive responses, which focus generally on changes above the spinal cord level [36]. The tail-flick approach mediates a spinal reflex to a nociceptive stimulus, while the hot plate method is a supraspinal structured response [37].

The efficacy of 7-HF in central nociceptive models (hot plate, tail flick, and tail immersion tests) suggests possible involvement of supraspinal and spinal pathways, which are typically sensitive to centrally acting analgesics such as morphine. These models are sensitive to agents that modulate opioid receptors, ion channels, and descending inhibitory pathways.

7-hydroxyflavone was administered at low and high dose (5 and 10 mg/kg) and tested for antinociceptive potential utilizing hot plate, tail flick, and tail immersion models. The lower dose of 7-hydroxyflavone (5 mg/kg) did not show any analgesic effect. Steroids like calcitriol have a similar lack of analgesic effect at low dosages, suggesting that sub-therapeutic dosing may not modulate central nociceptive pathways [38]. However, 7-hydroxyflavone at 10 mg/kg increased reaction time, tail flick latency, and tail withdrawal time, indicating a significant improvement in pain threshold. Furthermore, the hot plate and tail flick experiments showed antinociceptive effects at 30 and 240 min, suggesting a prolonged central analgesic effect. The consistent increase of pain threshold in the tail immersion test up to 240 minutes supports spinal and supraspinal neural mechanisms associated with 7-hydroxyflavone’s higher dose analgesic profile; these results are correlate with earlier studies [1].

The acetic acid–induced writhing test was employed to evaluate the peripheral analgesic activity of the test compound. This model is widely regarded as a sensitive and reliable method for detecting antinociceptive effects, particularly for natural products and novel compounds that may remain inactive in other analgesic assays [39]. The peritoneal cavity gets irritated and stimulated by intraperitoneal acetic acid injection, which results in the production and release of endogenous inflammatory mediators, including histamine, serotonin, bradykinin, substance P, and PGs [40]. These different endogenous inflammatory mediators caused chemical-induced visceral pain, which is characterized by elongation of the body, extension of the forelimbs, and contraction of the abdominal muscles. The writhing test in response to acetic acid is therefore regarded as a model of visceral pain [41]. 7-hydroxyflavone dose-dependently showed significant peripheral analgesic activities by reducing the number of writhing compared to the control. The peripheral antinociceptive effect of 7-HF observed in acetic acid-induced writhing model may be associated with inhibition of inflammatory mediator release or reduced sensitivity of peripheral nociceptors in the peritoneal free nerve endings for chemically induced pain [41].

In this present investigation, the major strength lies in the integration of behavioral analgesic evaluation with oxidative stress assessment, allowing a mechanistic interpretation of the observed antinociceptive effects. It is well established that pain induction, particularly in chemically induced nociceptive models such as acetic acid–induced writhing, is associated with excessive generation of reactive oxygen species (ROS), leading to lipid peroxidation and depletion of endogenous antioxidant defenses [42, 43]. Elevated oxidative stresses have been shown to sensitize nociceptive pathways and exacerbate pain perception [44].

In the current study, administration of 7-hydroxyflavone (7-HF) significantly restored the levels of key endogenous antioxidants, including reduced glutathione (GSH) and superoxide dismutase (SOD), while concurrently reducing lipid peroxidation (LPO). These findings indicate a pronounced antioxidant effect of 7-HF under nociceptive conditions. The observed antioxidant restoration is consistent with previous reports demonstrating that flavonoids exert protective effects by enhancing cellular redox balance and suppressing oxidative damage [45, 46]. Notably, accumulating evidence suggests that flavonoids activate the nuclear factor erythroid 2–related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway, which plays a central role in regulating antioxidant enzyme expression and maintaining cellular homeostasis [47]. Activation of the Nrf2/HO-1 axis has been linked to reduced oxidative stress–mediated neuronal sensitization and attenuation of pain signalling [48]. Therefore, the antioxidant effects observed in the present study may contribute at least in part, to the antinociceptive activity of 7-HF potentially through modulation of oxidative stress–dependent nociceptive pathways.

Morphine was employed as a standard due to its well-characterized and robust analgesic efficacy across multiple nociceptive models. Morphine’s well-known side effects sedation, respiratory depression, constipation, tolerance, and dependence limit its clinical use. Flavonoid-based drugs like 7-HF may reduce opioid-related side effects by acting as non-opioid analgesics. The tested doses of 7-HF did not show behavioral abnormalities. The limitation of the current study is orally administered 7-HF and intraperitoneally administered morphine may differ in bioavailability and latency to peak effects. Morphine was chosen as a reference standard due to its well-characterized and substantial analgesic efficacy in multiple nociceptive models. Therefore, the comparison assessed relative pharmacodynamic efficacy, not pharmacokinetic equivalency. Oral conventional analgesics, such as aspirin or other non-steroidal anti-inflammatory drugs, might provide a more direct route-matched comparison in future investigations.

The antinociceptive effects of 7-HF at the tested dose showed measurable efficacy in several parameters, although direct equivalence with morphine can not be concluded due to differences in route of administration and pharmacokinetics. Overall, this study supports the potential of 7-HF as a promising analgesic candidate with antioxidant properties, warranting further pharmacological, mechanistic and translational stuudies to confirm its therapeutic relevance. The efficacy of 7-HF at 10 mg/kg doses further provides scope for its dose optimization. More study is needed to be required for the confirmation of the precise mechanism of the test drug as an analgesic potential and factors associated with pain and analgesia.

In conclusion, the 7-hydroxyflavone (7-HF), particularly at a dose of 10 mg/kg, demonstrated significant analgesic activity in the experimental rat model. The treatment with 7-HF effectively attenuated both central and peripheral nociception, as evidenced by the hot plate, tail flick, tail immersion, and acetic acid-induced writhing tests. In addition, 7-HF showed a favorable safety profile and was associated with improvement in antioxidant parameters, suggesting a potential link between its analgesic and antioxidant effects. The observed analgesic efficacy of 7-HF may be attributed to its multi-target mechanism of action. However, further studies are required to confirm its efficacy, safety, and therapeutic potential in the management of pain.

In this study, Rats were used to investigate the analgesic properties of 7-hydroxyflavone compared to morphine and a control group. The experimental animals were subjected to various tests to measure pain perception, including the hot plate, tail flick, and tail immersion tests for central mechanisms, and acetic acid-induced writhing for peripheral mechanisms. 7-hydroxyflavone were tested at low and high dose (5 mg/kg and 10 mg/kg), with the higher dose it showing significant analgesic effects compared to the lower dose and control groups. Moreover, the higher dose of 7-hydroxyflavone improved pain thresholds and reduced writhing responses in the experimental animals. Notably, in this study 7-hydroxyflavone treatment rats showed more significant restoration of antioxidant enzymes level like glutathione and superoxide dismutase and by reducing lipid peroxidation level that indicates inhibition of oxidative stress to pertaining its analgesic effect via central and peripheral mechanism.

7HF, 7 hydroxyflavone; SOD, Superoxide Dismutase; LPO, Lipid Peroxidation; GSH, Glutathione.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

ADT and SVK designed and conducted behavioural experiments. SVK performed statistical analysis. SPB, SBK, MRJ, and SPJ assisted in data compilation and manuscript drafting. ADT prepared the initial manuscript draft. SPJ supervised the work and revised the manuscript to its final version. All authors read and approved the final manuscript. All authors contributed to editorial changes in the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The study was done in accordance with the committee for the purpose of control and supervision of experimental animals (CPCSEA) guidelines after approval of the experimental protocols by the institutional animal ethics committee (IAEC) Rajarshi Shahu College of Pharmacy, Buldana. Project Proposal No. IAEC 1865/24-25/P-11.

The authors would like to acknowledge the Dr. Prakash N. Kendre vice principal of Rajarshi Shahu College of Pharmacy, Buldhana for the estimation of biomarkers of oxidative stress.

This research received no external funding.

The authors declare no conflict of interest.