## 2 Results and Discussion  
### In Silico ADMET Prediction  
Early determination of the ADMET properties of a therapeutic candidate is crucial, as they significantly reduce the attrition rates in drug discovery. Conventional approaches in the determination of ADMET parameters are often time-consuming, expensive, and involve ethical concerns [21]. Hence, computer-aided ADMET prediction has captured significant popularity owing to its feasibility and reliability. In the current study, we utilized the pkCSM web tool and the SwissADME web tool to determine pharmacokinetic descriptors and drug-likeness, respectively. In terms of pharmacokinetics, spilanthol has gained a favorable result in various pharmacokinetic descriptors. Importantly, spilanthol has demonstrated substantial blood-brain barrier (BBB) permeability, favoring its potential therapeutic application in NDDs. Additionally, spilanthol has accounted for high intestinal absorption, thereby favoring oral administration (Supplementary Table S1). Drug-likeness entails a qualitative assessment aimed at evaluating the likelihood of a compound to serve as an oral drug through scrutiny of its structural and physicochemical attributes. [PERSON]'s rule of five is a widespread guideline to evaluate drug-likeness compounds, and it consists of five descriptors including molecular weight (MW) \(\leq\) 500 g/mol, Log \(p\)\(\leq\) 5 (n-octanol/water distribution coefficient), nHA \(\leq\) 10 (number of hydrogen bond acceptors), and nHD \(\leq\) 5 (number of hydrogen bond donors) [22]. Interestingly, spilanthol exhibited promising drug-likeness with zero violation in the descriptors coming under [PERSON]'s rule. Additionally, spilanthol did not violate any descriptors of the [PERSON], [PERSON], [PERSON], and [PERSON] rules, which are also frequently utilized to evaluate the drug-likeness of compounds. Notably, spilanthol exhibited substantial oral bioavailability indicated by an Abbot bioavailability score of 0.55, predicting the probability of spilanthol achieving at least 10% oral bioavailability in rats (Supplementary Table S2 and Supplementary Figure S1) [23]. Protox-II is a widespread web server used to predict the rodent oral toxicity of compounds, and we leveraged Protox-II to evaluate the toxicity of spilanthol. Interestingly, spilanthol demonstrated relatively low toxicity with predicted LD50: 4378 mg/kg, and it was classified under predicted toxicity class 5 with both average similarity and average accuracy of 100%, indicating the safe therapeutic applicability of spilanthol (Supplementary Table S3). Despite the extensive ethopharmaceutical uses of spilanthol-rich herbal extracts and herbal preparations, a very limited number of safety studies have been conducted regarding the safety and toxicity of spilanthol. [PERSON] et al. evaluated the impact of ethanolic extracts of _A. oleracea_ flowers and leaves, as well as pure spilanthol in male Wistar rats. Intraperitoneal administration of crude extracts (10-100 mg/kg) and spilanthol (6.2 mg/kg) have not induced nephrotoxicity and hepatotoxicity [24]. Similarly, intraperitoneal treatment of _A. oleracea_ floral extract for 7 days up to 500 mg/kg has not produced significant toxic outcomes [25]. Chronic toxicity evaluation in male Wistar mice using 100 mg/kg/day of _A. oleracea_ floral extract orally for 60 days showed no signs of toxicity [26]. However, oral and immersion treatment with spilanthol and spilanthol-rich extracts has induced identifiable toxicity characterized by histopathological changes in vital organs in zebrafish models [27]. Similarly, substantial maternal toxicity and teratogenicity have been observed in zebrafish preclinical models upon treatment with spilanthol and spilanthol-rich crude extracts. Additionally, few metabolites of spilanthol with mutagenic potential have been identified during in silico evaluations [28; 29].