Inflammation is one of the mechanisms of pain. Cytokines and other pro-inflammatory mediators, such as prostaglandins, nitric oxide, serotonin, and histamine, are released from cells after the tissue damage, resulting in pain and hyperalgesia [1]. The inflammatory cascade involves major targets, including cyclooxygenase (COX), interleukins (IL), and tumor necrosis factor-α [2]. Recent interest in chromones as potential candidates for drug development is attributed to their anti-inflammatory effects [2,3].

Saposhnikovia divaricata and Peucedanum japonicum belonging to family Umbelliferae, known as ‘Bangpung’ in Korea, have been used as traditional herbal medicine to treat fever, rheumatism, neuralgia, and headache [4]. Bioactive substances derived from ‘Bangpung’ showed antioxidant [5] and anti-inflammatory effects [6,7]. These properties of ‘Bangpung’ suggested potential pharmaceutical candidates [2]. Compounds isolated from S. divaricata, including sec-O-glucosylhamaudol (SOG), hamaudol, ledabouriellol, divaricatol, isofraxidin,and cimifugin showed an analgesic effect [8]. We previously demonstrated the strong antinociceptive effect of intrathecal SOG, which is one of the constituents of P. japonicum Thunb. via opioid receptors [9]. Recent studies showed anti-inflammatory effects of cimifugin, a chromone (1,4-benzopyran, Fig. 1), which is one of the bioactive substances derived from S. divaricata [10,11]. Thus, we hypothesized that the anti-inflammatory effect of cimifugin may result in an analgesic effect. Thus, we conducted a preliminary study to determine the antinociceptive effect of intrathecal cimifugin using a formalin test to confirm the analgesic effect of cimifugin mediated via anti-inflammatory effect. The primary outcome of the study was to the concentration-dependent analgesic effect of cimifugin based on a formalin test. The secondary outcome was calculating the effective dose (ED) of cimifugin required for the analgesia. 

Typical biphasic flinch responses to the formalin test were observed in all rats following intrathecal administration of vehicle (DMSO 70%) (Fig. 3).

Pre-treatment with intrathecal cimifugin decreased the flinch response triggered by formalin injection significantly in both phases (Figs. 3, 4). During phase 1, a statistically significant reduction in flinch response was found using the dose of 300 μg, and the maximal reduction in flinch response was observed at a dose of 1,000 μg (41.0% of control, P < 0.001, Figs. 4, 5, and Table 1). During phase 2, a statistically significant reduction of flinch response was found at a dose of 100 μg (65.5% of control, P = 0.011), and the maximal reduction in flinch response occurred at a dose of 1,000 μg (50.6% of control, P = 0.003, Figs. 4, 5, and Table 1).

The ED₅₀ (95% CIs) values of intrathecal cimifugin were 696.1 (360.8-1,342.8) μg during phase 1 and 1,242.8 (42.0-48,292.5) μg during phase 2.

As mentioned above, ‘Bangpung’ was traditionally used to treat fever and pain. The anti-inflammatory and antioxidant activity of the bioactive substances derived from S. divaricata may correspond to those effects [5-7]. According to the phytochemical studies, Prim-O-glucosylcimifugin is the main chromone present in S. divaricata, with antipyretic, anti-inflammatory, and analgesic properties [4]. Cimifugin is an aglycone of prim-O-glucosylcimifugin, synthesized during blood absorption [4]. According to Li et al. [16] cimifugin showed the highest concentration in plasma of rats treated with the extracts of S. divaricata orally. They concluded that cimifugin is the potential pharmacodynamic component of S. divaricata formed via biotransformation in vivo even though prim-O-glucosylcimifugin is the major constituent [4,16].

As far as we know, only one study reported the analgesic effect of cimifugin. Okuyama et al. [8] reported that oral administration of 80 mg/kg of cimifugin showed significant analgesia in mice during acetic acid-induced writhing test. The results suggested that the analgesic potency of chromones may be attributed to the non-glycosylated dihydropyran-type C-ring. Chromones are an important class of natural products that exhibit antibacterial, anticoagulant, anti-inflammatory, antioxidant, and anticancer activities [3]. Several types of compounds in chromones targeting multiple inflammatory pathways have been reported. The anti-inflammatory effects of chromones are mediated not only via inhibition of the COX pathway but also by neutrophil-dependent superoxide anion generation [3]. A recent study involving RAW264.7 cells showed that cimifugin inhibits the activities of mitogen-activated protein kinase and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathways and suppresses the release of inflammatory factors [11]. Another study of reported that cimifugin significantly attenuated allergic inflammation by reducing thymic stromal lymphopoietin and IL-33 synthesis [10].

These studies suggest that cimifugin has an anti-inflammatory role resulting in an analgesic effect via inhibition of the inflammatory process. Neuroinflammation plays an important role in the generation and modulation of pain [17], so the anti-inflammatory effect of cimifugin was thought to decrease the induction of inflammatory pain. Thus, we conducted the formalin test as a preliminary study to evaluate the analgesic effect of intrathecal cimifugin and its relation to inflammation. In the current study, we confirmed that intrathecal administration of cimifugin decreased the flinch response to formalin injection in both phases. The two phases in the formalin test indicate different nociceptive mechanisms [18]. The flinch response in phase 1 is due to a direct effect on the nociceptors, while the phase 2 response represents both inflammatory pain in the peripheral tissue and functional changes in the dorsal horn mediated via inflammatory reaction in the spinal cord. Thus, the inflammatory pathway mediated by prostaglandins does not play an important role during the early phase but the latent phase. However, some nonsteroidal anti-inflammatory drugs such as paracetamol show analgesic effects in both phases, suggesting effects against non-inflammatory pain. In the current study, cimifugin reduced the flinch response in both phases; however, significant antinociceptive effects were observed upon treatment with 300 μg in phase 1 (70.8% of control) and 100 μg in phase 2 (65.5% of control). However, its maximal antinociceptive effect with the 1,000 μg dose was more effective in phase 1 (phase 1: 41.0% of control vs. phase 2: 50.6% of control).These results are consistent with a previous study reporting the anti-inflammatory effects of isolated compounds from P. japonicum Thunb., which showed a relatively weak inhibitory activity of cimifugin against both COX-1 and COX-2 [7]. Therefore, it is suggested that cimifugin has an antinociceptive effect mediated via anti-inflammatory effect at low concentrations and via other non-inflammatory analgesic effects at high concentration.

Other hypotheses have been proposed to explain the mechanisms of action. First, derivatives of natural products display analgesic effect via multiple mechanisms. For example, eugenol, the bioactive compound derived from clove, carries not only anti-inflammatory and antioxidant potential but also analgesic effects mediated via α₂ -adrenergic and opioid receptors [19]. Our previous study investigating SOG, and other extracts derived from S. divaricata, showed that SOG exhibits analgesic effect against incisional pain via the μ-opioid receptor [20]. Thus, other targets may modulate acute peripheral nociception. Second, the antioxidant effect may mediate the formalin-induced pain response. According to the study evaluating the analgesic effect of antioxidants in the formalin test, the pro-oxidant may play an important role in the pain induced by tissue injury and intrathecal administration of an antioxidant (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy) effectively reduced the response in both phases [21]. Hong et al. [22] suggested that antioxidants such as vitamin E induce antinociceptive effects by not only decreasing the central sensitization but also scavenging the reactive oxygen species in the peripheral tissue.

Although the current study showed an analgesic effect of cimifugin in both phases of the formalin test, there were several limitations. First, we merely evaluated the pharmacological effect of different doses of cimifugin in the formalin test in this preliminary study, without elucidating the mechanisms of action. Further studies using different routes of administration using antagonists of specific receptors and methods other than formalin test, as well as other molecular studies are needed to determine the exact site of action of cimifugin. Second, a higher dose of cimifugin (> 1,000 μg) may yield a more potent analgesic effect, but the maximal dose of the current study was 1,000 μg. Higher concentrations of cimifugin could not be administered in this study because of technical difficulties and inability to dissolve higher concentrations of cimifugin (> 1,000 μg/10 μl of 70% DMSO). Thus, we used 1,000 μg of cimifugin as the maximal dose for the intrathecal administration. Moreover, cimifugin powder was only dissolved in DMSO at a concentration greater than 70%. Even though the control dose of DMSO alone had no analgesic effect, DMSO showedsome reducing effects in the formalin test [23]. The high concentration of DMSO used for drug preparation may affect the study results. Third, the CI of the ED₅₀ values in phase 2 was large according to the linear regression (42.0 to 48,292.5 μg), which was considered as computational limitation due to the inability to use higher-concentrations of cimifugin. Such a large CI limits further evaluation because of difficulty associated with the intrathecal administration. Further studies investigating the side effects of treatment with large doses are needed. Fourth, further investigations using biological markers are needed for objective results.

In conclusion, intrathecal cimifugin had antinociceptive effects in both phases of the formalin test, suggesting that cimifugin has an anti-inflammatory effect at low concentrations and non-inflammatory analgesic effects at higher concentrations. Further studies are required to investigate the actual mechanisms of cimifugin.