## 3 Results and Discussion
### Antincociceptive Activity of Chrysin-Loaded Microemulsion  
The hyperalgesia induced by intraplantar injection of carrageenan (CG) is widely used to test the efficacy of new anti-inflammatory and antihyperalgesic compounds in preclinical models [43]. In particular, edema (swelling), hypersensitivity to a noxious stimulus (hyperalgesia), or sensitivity to a non-noxious stimulus (allodynia) can be used to assess inflammation in an animal model after CG administration. CG administration is in fact well known to induce the development of cardinal signs of inflammation as a consequence of the action of pro-inflammatory agents (mainly pro-inflammatory cytokines such as IL-1\(\beta\), IL-6, and TNF\(\alpha\)) as well as complement and reactive oxygen and nitrogen species. Inflammation manifests itself in a biphasic cutaneous reaction in the injected area, which consists of a first release of serotonin and histamine (up to 2 h later), followed by the release of bradykinin (3-6 h) occurring through the production of cytokines and prostaglandandins by both local cells and infiltered neutrophils [44; 45; 46]. The release of prostanoids and sympathomimetic amines stimulates peripheral A\(\delta\) and C fiber sensory nerve terminals, hence contributing to the consequent peripheral and central hyperalgesia [44]. Previous reports have demonstrated that chrysin inhibits COX-2 expression in both in vitro and in silico inflammation models [18,47]. The results of this study indicate that chrysin may be involved in other paths of the inflammatory cascade, thereby reducing mechanical hyperalgesia. However, after 1 h of CG induction, Blank-ME treated animals also showed a significant (\(p<0.05\)) antinociceptive effect, which suggests that at least one of the NE constituents may have interacted with some mediators involved in the inflammatory process. Additionally, it has recently been demonstrated that chrysin is able to effectively relieve oxidative insults and apoptosis in primary rat mesencephalic cultures and to exert a protective effect against brain damage induced by chronic cerebral hypoperfusion in rodents [48]. These neuroprotective and anti-inflammatory effects appear to be associated with the antioxidant profile of chrysin, along with its ability to reduce the levels of inflammatory mediators such as NF-kB p65 unit, TNF-\(\alpha\), IL-1\(\beta\), and IL-6 [49]. Since the observed antihyperalgesic effects of CS-ME and chrysin on the tested CG-induced inflammatory pain model may be associated with the regulation of inflammatory cytokine expression, levels of TNF-\(\alpha\) and IL-10 were measured to help understand the mechanism of chrysin activity. The levels of pro-inflammatory (TNF-\(\alpha\)) and anti-inflammatory (IL-10) cytokines in the control group were significantly (\(p<0.05\)) different from those in  
Figure 3: (**A**) Effects of chrysin (CS), chrysin-loaded microemulsion (CS-ME) (25 mg/kg) and blank-ME on the carrageenanan-induced mechanical hyperalgesia. All treatments were carried out 30 min before carrageenanan injection. The intensity of the force (g) needed for the mice to withdraw their hind paws or to “flinch” was determined using a digital analgesimeter 1 h before (baseline) as well as 1, 2, 3, and 4 h after carrageenanan injection. (**B**) Area under the curve (AUC) for antihyperalgesic effect of CS and CS-ME from 0 to 4 h. Results are expressed as means \(\pm\) standard error of the mean (n = 8): *** \(p\) < 0.001 vs. the control group; _*** \(p\) < 0.01 vs. the sham group; (one-way ANOVA followed by Tukey’s test). the sham group (Figure 4). All treatments significantly reduced (\(p<0.05\)) the TNF-\(\alpha\) level in the mouse paw compared with the control group after 4 h of CG injection. It is important to remember that TNF-\(\alpha\) is one of the key cytokines in the control of inflammation, being responsible for important effects of the repair process, and is the first cytokine released after an inflammatory stimulus such as carrageenan injection.