Historically, natural products have been the main source of medicine used to treat various human ailments. In addition to offering a number of innovative and potent chemical entities for use in modern medicine, plants serve as a major source of natural products with a remarkable diversity of structural variations (David et al., 2015). Numerous novel medications have been discovered as a result of historical experiences using plants as medicinal tools. Natural products derived from plants have long been recognized for their diverse pharmacological activities. The intricate chemical structures found in natural products often contribute to their potent biological activities, making them promising candidates for therapeutic interventions (Mishra et al., 2016). The search for effective analgesics, antipyretics, and anti-inflammatory agents is a persistent challenge in medicine, driven by the need to alleviate pain, reduce fever, and mitigate inflammatory responses effectively. Natural products offer a compelling avenue in this regard as they often exhibit complex chemical profiles that interact with biological targets in unique ways. Advancements in analytical methods have significantly enhanced the isolation and characterization of bioactive compounds from plants, allowing researchers to identify specific molecules with analgesic, antipyretic, and anti-inflammatory activities (Porras et al., 2020).

Compounds isolated from plants play crucial roles in the development of treatments for various diseases. These compounds often act through diverse mechanisms, including inhibition of cyclooxygenase (COX) enzymes and modulation of inflammatory mediators such as prostaglandins, to mitigate inflammation (Süntar, 2020). Moreover, the global interest in sustainable and natural alternatives to synthetic drugs has amplified the importance of exploring natural products for pharmacological activities. Plants represent a renewable resource with immense chemical diversity, offering a promising avenue for discovering new analgesics, antipyretics, and anti-inflammatory agents that are effective yet potentially safer and more sustainable than their synthetic counterparts (Veeresham, 2012).

Hypericum, with 50 genera and 1,200 species, is a large genus of herbs and shrubs found widely in temperate climates worldwide. In Pakistan, nine species of this genus are known and are considered a rich source of flavones, lactones, and xanthones (Ravisankar et al., 2016). Hypericum oblongifolium Wall (Hypericaceae), locally known as “Pinli,” is an evergreen, widespread shrub in Himalayas and China, growing at elevations of five to six thousand feet. It is a flowering plant 6–12 m in height. Scientific interest in H. oblongifolium extends to its phytochemical composition, with researchers isolating bioactive compounds for their potential pharmaceutical applications, particularly in pain management and inflammation (V Simoben et al., 2015). The extract of this plant has traditionally been used to treat various ailments. Their antidepressant and antinociceptive effects have also been studied. According to the literature, the crude extract of this plant possesses potent analgesic, antipyretic, anti-inflammatory, anti-glycation, antioxidant, and anti-lipid peroxidation activities. Additionally, it shows relatively potent anti-proliferative activities (Ali et al., 2014). To date, several phytochemicals have been isolated from this plant, such as folecitin and folenolide. However, their biological potential remains unclear (Farooq et al., 2022).

Folenolide (3, 4-dihydroxy-6-oxabicyclo [3.2.1] oct-1-en-7-one) (Figure 1) is a lactone isolated from H. oblongifolium. It occurs as white crystals with a molecular formula of C₇H₈O₄. In the present study, folenolide was investigated for the first time because of its pharmacological and molecular docking properties, revealing its potential as a lead compound in the development of novel analgesic, anti-inflammatory, and antipyretic agents.

The assessment of acute toxicity is essential in evaluating the safety profile of pharmaceutical agents (Mondal et al., 2019). The results of the study (Table 1) revealed the safety of folenolide as no mortality was recorded. This indicates that folenolide exhibits promising safety characteristics in acute toxicity assessments.

Folenolide demonstrated strong analgesic properties in both peripheral and central pain models. In the acetic acid-induced writhing assay, folenolide at 3 and 6 mg/kg resulted in dose-dependent inhibition of writhing by 31.07% and 50.13%, respectively, indicating a peripheral analgesic effect comparable to that of mefenamic acid, as shown in Figure 2A. The efficacy of folenolide demonstrated enhanced potency compared to other well-known phytochemicals, including curcumin, as a dose of 50 mg/kg of it is required to produce similar analgesic effects (Mohammed and Sabry, 2020). These findings suggest its promising peripheral analgesic activity, like other bioactive compounds (Singsai et al., 2020). Similarly, significant reductions in paw-licking time were observed in both phases of the formalin test, indicating potential analgesic and anti-inflammatory effects of folenolide, as shown in Figure 2B. Formalin-induced pain is characterized by two distinct phases: the early neurogenic phase, involving direct activation of pain-sensing nerves, and the late inflammatory phase, characterized by the release of inflammatory mediators (Turnaturi et al., 2023). Finally, in the hot-plate test, folenolide significantly prolonged latency times at 90 and 120 min post-administration compared to the control, highlighting its sustained central analgesic activity, as shown in Figure 2C. These results align with the analgesic actions commonly observed in natural compounds, which frequently act by modulating central and peripheral system pain pathways (Liktor-Busa et al., 2021; Jabbari et al., 2024).

The test compound demonstrated strong anti-inflammatory effects across various established in vivo models (Matos et al., 2021; Oqal et al., 2023). Figure 3A demonstrates that significant reductions (p < 0.05) in paw edema were observed in both dose groups, where the group that was administered the 6 mg dose showed the highest suppression (94.98%) at the fifth hour interval, comparable to the standard treatment (89.3%) in the carrageenan-induced anti-inflammatory study. This showed the potent anti-inflammatory effects of folenolide compared to other bioactive natural compounds, including quercetin, which often requires doses above 25 mg/kg to achieve similar effects (Batiha et al., 2020). Formalin-induced paw edema serves as a critical tool in preclinical research (Horváth et al., 2021). The efficacy of folenolide against formalin-induced paw edema in a mouse model revealed significant anti-inflammatory effects. Over the 5-h observation period, the study showed reductions in paw edema volume, with percent suppressions of 78.26%, 69.17%, and 72.88% for the standard, 3, and 6 mg dose groups, respectively, compared to baseline measurements. These findings (Figure 3B) indicate the potential of folenolide to modulate inflammatory mediators involved in edema formation (Ferraz et al., 2020). Figure 3C shows that folenolide antagonized the inflammation induced by histamine. Significant reductions (p < 0.05) in paw edema were observed at both dose levels. However, animals that received a higher dose (6 mg) showed the highest suppression (88.6%) after 5 h, surpassing the standard treatment (83.12%). Folenolide may alleviate histamine-induced inflammation pathways (Almasarwah et al., 2023). In the xylene-induced ear edema model, the anti-inflammatory effect is calculated as the percentage weight gain of the ear. The low values of 6.19%–8.19% observed in the treatment groups indicate a very strong suppression of edema (approximately 92%–94% inhibition compared to the control), which is consistent with the high levels of efficacy observed in the paw edema models. Xylene induces localized inflammation, making it a suitable model for evaluating anti-inflammatory agents (Basit et al., 2022; Rousdy et al., 2022). The results from Figure 3D suggest a dose-dependent anti-inflammatory response of the test compound.

Brewer’s yeast is a useful test for the screening of isolated compounds for their antipyretic effect (Mohankumar et al., 2022; Negi et al., 2023). Yeast-induced pyrexia could be the production of prostaglandins, and it can be achieved by blocking the cyclo-oxygenase enzyme activity (Chansiw et al., 2024; Abd-Elhak et al., 2024; Veena et al., 2022). Folenolide significantly attenuated the rectal temperature of mice during the 5-hour observation period, and consistently lowered rectal temperatures indicate its potent antipyretic properties. In particular, the 6 mg/kg dose group showed pronounced temperature reductions from the second hour onward, achieving statistically significant differences compared to controls (Table 2). These results suggest that the test compound showed effective antipyretic activities by mediating the mechanisms that regulate fever response pathways (Ogbonna et al., 2025).

The test compound was docked against transcription regulators 1IK3 (15 lipoxygenase), 2R4R, 5KIR [COX (cyclooxygenase-2, COX-2)], 3UA9 (tankyrase-1 and tankyrase-2), 5C1M (opioid receptors), 3RZE (histaminic receptors), and 1IK3 (Tegegne and Alehegn, 2023). The highest free binding energy (ΔG) was observed with pose 1 (−5.9 ΔG (kcal/mol). More importantly, N H-bonding interaction was observed with any pose (1–9) that shows less affinity for binding interactions. Furthermore, hydrophobic interactions of folenolide were noticed with Gly720, Gln716, Ille770, Val372, Ser510, Thr728, Arg726, Phe576, and His513, which were mainly due to van der Waals forces and π–alkyl interactions, reflecting weak interactions with the target gene 1IK3. In the case of the transcription regulator 2R4R, no H-binding was observed in docking analysis. However, the highest free energy (ΔG) was recorded in the case of pose 1 [−5.4 ΔG (kcal/mol)]. In interaction analysis, weak interactions of the ligand were recorded with Ala57, Phe332, Val54, Leu64, Ile72, and Asn69 amino acid residues, which were due to van der Waals forces and π–alkyl interactions (Table 3; Figure 4). Nevertheless, the transcriptional regulator 5KIR did not participate in H-bonding interactions with the tested ligand (poses 1–9). In this case, the highest free binding energy (ΔG) was observed in the case of pose 1 (−4.4 ΔG kcal/mol), which was again less than that of other targets. The neighboring interacting amino acids included Phe187, Asp393, Gln429, Pro392, and Thr394. The π–sigma, van der Waals forces, and π–alkyl interactions contributed to these binding interactions (Table 3). In the molecular docking of the ligand molecule with the target gene 3UA9, only weak interactions were recorded with Asp147, Trp133, His54, Cys217, Ser55, Gln214, Val143, and Ile144 amino acids, with the highest binding free energy (ΔG) (−5.7ΔG (kcal/mol)) with pose 1 compared to that of all other poses (up to 9). In this case, no H-binding interactions were noticed (Table 3; Figure 4). In the docking of the ligand with 3RZE, no H-bond formation was observed, indicating no interaction with the target. Furthermore, weak interactions were recorded with Arg127, Tyr65, Asp124, Ile123, Thr147, Tyr138, and Arg143, with the highest free energy [−5.3 ΔG (kcal/mol)], which was due to π–sigma, van der Waals forces, and π–alkyl interactions (Table 3; Figure 5). Finally, no H-bond formation was recorded with 5C1M. However, weak interactions were recorded with Asp147, Trp133, His54, Cys217, Ser55, Gln124, Val143, and Ile144. In this case, interacting forces included weak alkyl and van der Waals forces, with the highest free energy (ΔG) [−5.0 ΔG (kcal/mol)] in pose 1 (Table 3; Figure 5). Weak intermolecular interactions, such as hydrogen bonding and hydrophobic interactions, are key players in stabilizing energetically favored ligands within the open conformational environment of protein structures (Beatrice, 2022). Weak intermolecular interactions, such as H-bonding and hydrophobic (π–π, π–amide, π–cation/anion, π–alkyl, and π–σ) interactions, are of great significance since they determine the stability and structure of proteins that are crucial for therapeutic effects (Siddique et al., 2022; Rafey et al., 2022). Hydrogen-bonding interaction governs the stability of the host–guest complex. However, the presence of multiple hydrophobic interactions competes with hydrogen bonding interaction in several conformers, resulting in a decrease in binding energy (Deivasigamani et al., 2024). In current investigations, the interactions were mainly ruled by hydrophobic forces and, therefore, play a key role in the therapeutic efficacy of folenolide. Furthermore, hydrophobic interactions play an important role in the folding of proteins, contributing to the stability and biological activity of the protein moiety (Berishvili et al., 2020). This allows the protein to reduce the surface area and decrease the unwanted interactions with water (Huang et al., 2015). It is thus concluded that folenolide has strong interactions with the tested molecule and the ability to interact with key targets involved in analgesia and inflammation, indicating that the potent in vivo effects observed may, therefore, be attributed to a polypharmacological mode of action.

Pharmacological evaluation and molecular docking studies have demonstrated the significant potential of folenolide as a drug. It showed marked analgesic effects through both peripheral and central mechanisms, suggesting its efficacy in modulating the pain pathways. It also exhibits significant anti-inflammatory properties in various models, indicating its ability to attenuate inflammatory responses through different pathways. Moreover, its antipyretic activity indicated its potential for fever management. Molecular docking studies have suggested that folenolide exerts its effects by modulating protein structures involved in the pain, inflammation, and fever pathways. In conclusion, the findings from this study collectively support the promising pharmacological profile of folenolide and its potential as a natural-product-based therapeutic option for pain and inflammatory disorders.