The current study evaluated the anti-inflammatory and pain relieving effects of A. Seyal’s 80% methanol stem bark extract [21]. Considering the socioeconomic significance of pain and inflammation together with the awareness of potential herbal medicines used for this condition, it would make reasonable to explore medicinal plant derived analgesic and anti-inflammatory agents as plant-derived agents are associated with minimal side effects and comparable efficacy [25]. A. Seyal is a traditional herb used to treat various inflammatory and pain disorders [26]. Nevertheless, to date, no scientific data is found concerning its pain relieving and anti-inflammatory properties in the literature. Therefore, to support its claimed traditional use, it could be worthwhile to do scientific research on the pain relieving and anti-inflammatory properties of A. seyal stem bark extract in rodent models.

In the first model, the analgesic effects of 80% methanol stem bark extract of A. seyal was investigated using acetic acid-induced writhing reflex model. Administrations of various doses of A.seyal extract considerably decreased the stomach twisting reactions following acetic acid administration when compared to the negative control group (P < 0.01). The observed inhibition activity was 16%, 33.36%, and 48% from ASME100, ASME200 and AMSE400 respectively, demonstrating a dose-dependent effect. The mechanism related to acetic acid triggered writhing reflex is mediated through increased production of PGs [27]. Furthermore, when acetic acid is injected through intraperitoneal site, it irritates the peritoneal cavity, which causes the release of natural inflammatory molecules such as histamine, serotonin, bradykinin and, substance P [21, 28]. This may indicate that the extract would elicit its action on peripherally situated pain transduction pathways through suppression of these inflammatory mediators.

The hotplate model was the second experimental technique employed to examine the pain relieving efficacy of A. seyal extract. This approach is a valuable tool for assessing centrally acting analgesic agent, that is widely used to enhances mice’s sensation of the pain in response to heat [29]. It is sensitive to strong analgesics and has a time limit of 15 s to minimize the duration rodents are exposed to the hot plate set at 55 ± 1 °C [30]. According to these models, the duration between the injection and the first behavioral response such as jumping or paw lickining is a measure of the analgesic action [31]. In the current experiment, administrations of the extract tend to produce analgesic effect beginning at 30 min with all doses (p < 0.001). The maximum effect was achieved at 2 h, as evidenced by percentage of elongation time of 69.5%, 74.47% and 76.62% for doses of 100, 200 and 400 mg/kg respectively. In consistence with this, positive control group which took morphine indicates significant anti-nociceptive at all interval of observation.

The third model, carrageenan-caused paw swelling, is another approach employed to evaluate the anti-inflammatory effects of substances [32]. Carrageenan is a potent molecule that imitates the release of pro and inflammatory mediators such as PGs, leukotrienes, histamine, and, bradykinin [33]. 1% carrageenan subplantar injections into the hind paw causes two stages of inflammation [34]. During one to two hour since administration, carrageenan cause bradykinin, 5-hydroxytryptamine, and histamine release, which are expected to be the reason for swelling. Throughout the second stage of edema formation, which lasted one to four hours, PG level is elevated [35]. The finding of the current investigation illustrates that ASME exhibited a statistically significant reduction of carrageenan induced paw edema (p < 0.001) across all time points except in the first hour compared to the negative control group. Similarly, the standard drug, indomethacin, significantly reduced paw edema starting from the first hour, with the effect lasting up to the 5th hour (p < 0.001).The noted swelling reduction was most significant in the last stage of inflammation, which mimics the possible effects of NSAID drugs like indomethacin. This implies that one of the possible anti-inflammatory effects of the current experimental plant might be due to inhibitions of the PG synthesis through blocking cyclooxygenase enzyme.

The cotton pallet-induced granulation approach is the final and fourth model. It is a popular techniques used for evaluating long-term anti-inflammatory potential of investigational agents [36]. The subcutaneous applications of a cotton pellet into a rodent results in the formation of a granuloma at the site of the implant. The initial events include accumulation of fluid and proteinaceous material together with an infiltration of macrophages, neutrophils, and fibroblasts, and multiplication of small blood vessels [13]. An increase in the moisture content of the cotton pellet indicates transudative phase of inflammation, whereas elevation in the dry density within the cotton pellet indicates the proliferative phase of inflammation [31]. The present study revealed that ASME100 (P < 0.05) and the middle and the highest doses of plant extract witnessed a significant reduction in the development of granuloma mass formation compared to the negative control group (p < 0.001). Moreover, middle and highest doses of the ASME reduces the production of inflammatory exudate significantly (p < 0.001) in contrast to DW administered group. The appreciable effect of the plant extract in this model might suggest that the extract could act through inhibiting transudative, exudative, and proliferative phases of subacute inflammation [36].

Following extraction using various solvents, findings have illustrate that stem and root bark of A. seyal contains flavonoids, saponins, terpenoids, steroids, alkaloids, phenols, and tannins [18]. Thus, the analgesic and anti-inflammatory effects of A.seyal is due to these secondary metabolites. For instance, flavonoids, tannins, and saponins are well known for their ability to inhibit pain perception and anti-inflammatory properties due to inhibition of enzymes involved in inflammation, especially synthesis of prostaglandins [37]. Furthermore, saponins, phenolic compounds, and terpenoids demonstrated profound anti-inflammatory and analgesic activity through reductions of the expressions of proinflammatory cytokines, particularly interleukin 1 and interleukin 6 [38, 39]. Studies have shown that the current plant material contains flavonoid compounds such as catechin and epicatechin, a triterpene isolated active constituent lupeol and a steroid compounds stigmasterol [26]. Catechin and epicatechin possess anti-inflammatory effect through free radical scavenging activity [40]. Furthermore, catechins can exert their significant anti-inflammatory properties by regulating the activation or deactivation of inflammation-related oxidative stress-related cell signaling pathways, such as mitogen activated protein kinases, nuclear factor-kappa B and transcription factor nuclear factor (erythroid-derived 2)-like 2 [41]. On the other hand, lupeol’s analgesic and anti-inflammatory effect is mediated through inhibitions of Inducible nitric oxide synthase thus the pro-inflammatory productions of Nitric oxide [42] and steroid compounds including stigmasterol abolish inflammation through decreasing leukocyte infiltration [43].