Abstract Clitoria ternatea L. (CT) is a perennial herbaceous plant with deep blue flowers native to tropical Asia. This work explores the endometrial pain (EP) regulation of CT flower through a multifaceted approach. Phytochemical screening unveiled the presence of alkaloids, steroids, flavonoids, glycosides, and tannins in CT flower methanolic extract (ME). In the in vitro membrane stabilizing experiment, the ME demonstrated 91.47% suppression of heat-induced hemolysis. Upon carrageenan-induced paw edema assay conducted on male Swiss albino mice at doses of 200 mg/kg and 400 mg/kg, 65.28% and 81.89% inhibition rates, respectively, of paw edema were reported. For the same doses, upon acetic acid-induced-writhing assay, 75.6% and 76.78% inhibition rates, respectively, were observed. For network pharmacology analyses, a protein–protein interaction network was constructed for 92 overlapping gene targets of CT and EP, followed by GO and KEGG pathway enrichment analyses. Network pharmacology-based investigation identified the anti-EP activity of CT to be mostly regulated by the proteins SRC homology, ESR1, and PI3KR1. Physicochemical, pharmacokinetic, and toxicity property predictions for the compounds with stable ligand–target interactions and a molecular dynamics simulation for the highest interacting complex further validated these findings. This work affirmed the anti-EP role of CT flower against EP, suggesting a probable molecular mechanism involved. Keywords: Clitoria ternatea, GO pathway, KEGG pathway, network pharmacology, molecular docking, molecular dynamics simulation, ADMET, anti-inflammatory, analgesic, endometriosis 1. Introduction Endometriosis is a debilitating gynecological disease affecting 10–15% of women worldwide [[44]1]. It is often termed as an estrogen-dependent disorder that is associated with the growth of endometrial glands and stroma in regions other than the uterus [[45]2]. Processes like proinflammatory, proangiogenic, endocrine, and immune activities play key roles in the overall prognosis of the disease [[46]3]. Recently, the homeobox (HOX) gene, which plays a key role in the development of the female reproductive system and is responsible for the regulation of several transcription factors involved in the differentiation and positioning of cells during embryogenesis, has been considered the key player behind the development of the disease [[47]4]. The chronic nature of endometrial pain and other symptoms can lead to crucial psychological suffering, including anxiety, depression, and a sense of isolation, adversely affecting quality of life. In addition, endometrial pain and symptoms frequently result in decreased productivity and absenteeism from work, and impacted women may lose 10.8 h of work per week on average [[48]5]. As a result, endometriosis may have long-term financial effects on women, such as decreased lifetime wages and retirement savings, which may exacerbate gender differences in financial well-being [[49]6]. Currently, medications such as progestogen, NSAIDS, GnRH antagonists (GnRH-As), selective estrogen receptor modulators (SERMs), combined oral contraceptives, aromatase inhibitors (AIs), novel products like resveratrol, IL-1 antagonists, prostaglandin E2 (PGE2) receptor antagonists, and dopamine receptor antagonists [[50]7] are used against endometriosis. GnRH-As appear to be the most promising medication in endometriosis treatment [[51]8]. The treatment plan may include surgical alternatives such as laparoscopy, laparotomy, or hysterectomy, which involves removal of the ovaries. The existing medicinal and surgical therapies for endometriosis have a fairly high recurrence rate. Additionally, approved endometriosis medications might cause melancholy, insomnia, and bone loss [[52]9]. Due to problems with the existing therapeutic approaches, individuals are beginning to favor herbal medications because of their mild nature and health benefits [[53]10]. Clitorea ternatea is a member of plant family Fabaceae and is a herbaceous perennial flowering plant commonly known as Butterfly pea ([54]Figure 1). Although its origins are unknown, this plant is endemic to tropical Asia and Africa. It is currently found in Madagascar, South America, India, Southern and Eastern Africa, and the Western Indian Ocean [[55]11]. The Butterfly pea flower is unique for its blue-hued petals, which have been widely utilized in Southeast Asian cuisine as a natural food-coloring agent [[56]12]. Butterfly pea flower tea has been popularized as blue tea for its easy availability, affordability, and anti-inflammatory and antioxidant qualities, which can help relieve inflammation and chronic pain [[57]13]. Owing to these diverse pharmacological effects, Clitorea ternatea has been traditionally used as a neuroprotective agent, an antimicrobial agent, an anti-inflammatory agent, and an anti-pyretic agent, making it a well-accepted remedy in treating anxiety, infection, pain, and inflammation. Moreover, Butterfly pea also promotes digestion, weight loss, diuresis, glaucoma alleviation, and blood glucose control. Butterfly pea flower is rich in polyphenolic constituents such as kaempferol, anthocyanins, phenolic acid, myricetin, and quercetin. Moreover, lipophilic constituents, including fatty acids such as palmitic acid, linolic acid, and arachidic acid, phytosterols such as campesterol, sitostanol, and sigmasterol, and various tocols like α-tocopherol and γ-tocopherol, are also present in the flowers of Butterfly pea [[58]14]. These constituents can provide the medicinal effects of blue pea flowers, establishing their status as functional foods [[59]15]. Due to the presence of promising phytochemicals, blue tea has the potential to alleviate pain, inflammation, and menstruation difficulties [[60]16]. Figure 1. [61]Figure 1 [62]Open in a new tab Dried flower of C. ternetea L. Traditional herbal medicines have been utilized for their therapeutic benefits for centuries, yet understanding their pharmacological effects remains challenging due to the complex interactions among multiple bioactive compounds. In response to this challenge, network pharmacology, a relatively newer computational field, has transpired as a propitious methodology to develop an understanding of the holistic mechanisms underlying herbal remedies [[63]17]. First introduced by Hopkins in 2008, network pharmacology offers a paradigm shift by emphasizing the “multi-compounds, multi-target, and multi-disease” model of drug action, in contrast to the traditional “a-drug, a-gene, a-disease” framework [[64]18]. Through the construction of network models integrating molecular, cellular, and organism-level interactions, an overall understanding of the intricate correlations between drugs, targets, and diseases can be achieved using network pharmacology [[65]19]. The molecular docking approach can further validate bioactive compound and hub-gene-expressed critical protein interactions [[66]20]. Previously, Pueraria lobata, known by the native name ‘kudzu’, has been found to have aromatase inhibitory activity, which has been proven to be very effective in the treatment of endometriosis [[67]21]. Similarly, the hexane fraction of black aged garlic (Allium sativum) has been found to have inhibitory effects on tumor necrosis factor (TNF)-activated endometriotic stromal cells [[68]22]. Uncaria tementosa extract, Silybum marianum extract, and Calligonum comosum extracts have been reported to be effective in the treatment of endometriosis [[69]23]. An extensive literature survey indicates that prostaglandin E2, cyclooxygenase-2, NFκB, and estrogen cytokines are key players in the prognosis of the disease; thus, their receptors are considered potential targets for relieving endometriosis [[70]24,[71]25,[72]26]. This study explored the prospective effects and molecular mechanism of an 80% methanolic crude extract of C. ternatea flower extract in alleviating endometriosis and related pain through network pharmacology and molecular docking-based analysis of the target proteins and active phytoconstituents followed by a molecular dynamics simulation analysis for the ligan–protein complexes [[73]27]. We also investigated the anti-inflammatory and analgesic activities of the crude extract of C. ternatea flower through a range of in vitro and in vivo experiments. 2. Materials and Methods 2.1. Plant Material and Reagents The reagents and chemicals used in the study included methanol (Sigma Aldrich Ltd., Tokyo, Japan), sodium chloride (50 mM), disodium hydrogen phosphate, and sodium dihydrogen phosphate to prepare phosphate buffer and freshly drawn human blood for erythrocyte (RBC) suspension. Analytical-grade reagents and solvents were purchased from a local chemical supplier (Active Fine Chemicals Ltd., Dhaka, Bangladesh) and used in this study. Aceclofenac was used as a standard for evaluating the anti-inflammatory and antinociceptive effects of the crude methanolic extract. For this purpose, raw Aceclofenac was obtained from Incepta Pharmaceuticals Ltd. (Dhaka, Bangladesh). Carrageenan and acetic acid were used to induce paw edema and analgesia, respectively. They were procured from Sigma Aldrich (St. Louis, MO, USA). The plant was obtained from the indoor facilities of Bailey Road, Bangladesh (latitude N 23°44.4524′, longitude-E 90°24.1467′). Khondokar Kamrul Islam, scientific officer, National Herbarium Bangladesh, Mirpur, Dhaka, identified and authenticated the plant sample, which was retained at the National Herbarium and Department of Pharmacy, East West University, Bangladesh, under the accession number DACB-94816 for future reference. The sample was sent to a herbarium directly after plucking and prior to collecting on a large scale. Upon confirmation, it was obtained in higher quantities and subjected to shade drying. 2.2. C. ternatea Flower Extract Preparation After collection, shade-dried flowers of C. ternatea were powdered to obtain 240.5 g and were macerated with 3.0 L of 80% methanol for 45 days with occasional shaking. Afterward, with the help of a cotton plug followed by Whatman filter paper, the mixture was filtered and subjected to a rotary evaporator (Heidolph, Saffron Walden, UK), maintaining low-pressure conditions at 40 °C to acquire a concentrated crude methanolic extract, followed by rotary evaporation; to ensure complete removal of the solvent, the concentrated crude extract was left on a fume hood in the presence of a desiccant (SiO[2]) overnight (extraction efficiency 39.9%, 400 mg/gm dry weight of flower extract). 2.3. Phytochemical Screening The phytochemical analysis of C. ternatea was carried out using previously described protocols [[74]28]. The Molish test was performed to identify carbohydrates. Alkaloid identification was conducted by using Mayer’s reagent, Hager’s reagent, Wagner’s reagent, and Dragendroff’s reagent. Flavonoid identification utilized concentrated HCl, and 5% FeCl[3] was added to the crude extract in the presence of distilled water. The presence of blue-black coloration or precipitation confirmed the presence of tannins. Steroid identification was performed by adding chloroform followed by concentrated H[2]SO[4]. The presence of red color in the chloroform layer ensures the presence of steroids. 2.4. Experimental Animals Swiss albino male mice (body weight: 20–25 gm, average weight 23.2 ± 0.6 gm) aged 6 weeks were purchased from an animal resource facility, the International Center for Diarrheal Disease Research, Bangladesh (ICDDR, B). Housing was carried out at the animal house of the Department of Pharmacy, East West University. Standard polycarbonate cages were used to house the mice. ICDDR, B formulated food was fed to the mice. The temperature and humidity of the room were maintained at 25 ± 1 °C and 60 ± 5%, respectively, and the research protocol was approved with a 12 h dark/light cycle. Approval for the research protocol was obtained the Animal Ethics Committee of the State University of Bangladesh, Dhaka (2023-01-04/SUB/A-ERC/004), before initiating the study. Upon commencement of the study, the animals were subjected to euthanasia using intra-peritoneal administration of a 150 mg/kg dose of pentobarbital. Strict maintenance of the protocols of the Care and Use of Laboratory Animals of the National Institute of Health and the ARRIVE (Animal Research Reporting in vivo Experiments) guidelines were followed throughout the study. Human (Homo sapiens) subjects were involved in the in vitro evaluation of membrane-stabilizing activity, for which healthy individuals with no known medical history of each sex were chosen. Blood was collected at the medical center of East West University under the direct supervision of a professional nurse. Informed consent was obtained from all subjects involved in the study. Approval for the research protocol was obtained from the Animal Ethics Committee of the State University of Bangladesh, Dhaka (2023-01-04/SUB/A-ERC/004), before initiating the study. 2.5. Membrane-Stabilizing Activity This study involves the investigation of the membrane-stabilizing properties of the sample solution exposed to heat by involving techniques leveraging the Shimadzu UV spectrophotometer (Shimadzu, Kyoto, Japan). The absorbance of the supernatants was measured at 540 nm [[75]19]. This method acts as an in vitro means to identify whether the test sample possesses anti-inflammatory activity or not [[76]29]. Hemolysis Induced by Heat In two sets of centrifuge tubes, about 2 mg/mL of extract, 30 μL of erythrocyte suspension, and 5 mL of isotonic buffer were taken. A similar preparation was made for two additional sets but excluding the extract. After gently swirling the tubes, one set was cooled in an ice bath at 0–5 °C while the other set was heated at 54 °C in a water bath for 20 min. Afterward, the formed supernatant was diluted, and its absorbance was evaluated at 540 nm. The percentage of inhibition or the rate of hemolysis acceleration was determined by employing the following equation: Percent (%) inhibition of hemolysis = [{1 − (OD2-OD1)}/(OD3 − OD1)] × 100% (1) where OD1 = absorbance of unheated test sample, OD2 = absorbance of heated test sample, and OD3 = absorbance of heated control sample. 2.6. Evaluation of Anti-Inflammatory Activity The carrageenan-induced paw edema method was employed to assess in vivo anti-inflammatory potential of the crude methanolic extract of C. ternatea flower [[77]30]. Here, normal saline was used as control and the crude methanolic extract was given at 200 mg/kg and 400 mg/kg b.w doses. Aceclofenac was given as standard by dissolving it in normal saline using tween 80 as a solubilizing agent (dose 25 mg/kg b.w) [[78]31]. All four groups contained five mice each. Control, test, and standard mice were fed orally, and percentage inhibition of paw edema was measured at the end of the 1st, 2nd, 3rd, and 4th hours, followed by subcutaneous injection of carrageenan in the right hind paw of the test mice. 2.7. Evaluation of Peripheral Analgesic Activity The acetic acid-induced-writhing method was employed to evaluate the peripheral analgesic activity of the test samples [[79]32]. Crude methanolic extract of C. ternatea flower (200 mg/kg and 400 mg/kg b.w doses) and standard Aceclofenac (25 mg/kg b.w) were given orally to the test animals [[80]33]. Normal saline was used as control to compare and nullify the effects of any residual solvent. Standard, control, and two test groups consisted of five test animals each. Acetic acid was injected intra-peritoneally to induce pain in the form of writhing. Percentage inhibition of writhing for test and standard was evaluated and compared. 2.8. Network Pharmacology-Based Analysis 2.8.1. Collection of Constituents and Targets for C. ternatea The Indian Medicinal Plants, Phytochemistry and Therapeutics (IMPPAT 2.0) database (retrieved from [81]https://cb.imsc.res.in/imppat/ (accessed on 2 February 2024)) was used for the initial listing of the phytoconstituents, and eleven phytoconstituents were found to be present in the C. ternatea flower petals [[82]34]. Additionally, an extensive literature search was carried out using scholarly databases such as PubMed and web search engines such as Google Scholar, using the keywords “Phytoconstituents of Clitoria ternatea flower”, “compound isolated from Butterfly pea flower”, and “bioactive molecules separated from C. ternatea flower”. Extensive literature searches contributed to the enrichment of the phytoconstituent list. Overall, fifty-nine compounds were reported to be present in the C. ternatea flower. Further screening of the listed constituents was performed based on the bioavailability score and drug-likeness properties using the swissADME online tool (retrieved from [83]http://www.swissadme.ch/ (accessed on 2 February 2024) [[84]35]). Compounds with a high bioavailability score (≥0.5) and not violating Lipinski’s rule of five were selected for further analysis. Subsequently, the SwissTargetPrediction (retrieved from [85]http://www.swisstargetprediction.ch/predict.php/ (accessed on 22 February 2024)) web database was searched to identify potential targets of the compounds [[86]36]. The compound target genes were selected based on probability score. Target genes having probability values equal to or greater than 0.2 were selected. The targeted genes of the studied phytochemicals were then subjected to removal of redundancy and preparation of an exclusive gene list. 2.8.2. Collection of Targets for Endometriosis, Inflammation and Endometrial Pain Gene targets for endometriosis, inflammatory pain, and inflammation were collected using two databases—GeneCards (retrieved from [87]https://www.genecards.org/ (accessed on 22 February 2024)) and DisGeNET (retrieved from [88]https://www.disgenet.org/ (accessed on 22 February 2024)) [[89]37,[90]38]. Keywords like endometriosis, inflammatory pain, and inflammation were used to search putative genes related to the selected maladies. For the genes obtained from DisGeNET, a Gene–Disease Association score (sGDA) of 0.2 was chosen as the cutoff score. The gene list obtained from GeneCards was refined by selecting genes with a relevance score higher than or equal to 50%. Gene redundancies were removed from these two lists, and a final list of exclusive genes for endometrial pain was prepared. The compound target genes ([91]Section 2.4) of C. ternatea were matched with the disease target genes of concern through Venny 2.1 (retrieved from [92]https://csbg.cnb.csic.es/BioinfoGP/venny.html (accessed on 22 February 2024)) for intersection analysis, and the genes common for both disease and compounds were then considered as prospective target genes for further analysis [[93]39]. 2.8.3. Analysis of Common Targets for Endometriosis The common gene targets were imported to the STRING platform (retrieved from [94]https://string-db.org/ (accessed on 22 February 2024)) where protein–protein interaction was studied [[95]40]. Homo sapiens was chosen as the species, and a high confidence score (>0.9) was set as the minimum confidence threshold. The resultant protein–protein interaction (PPI) was then visualized using Cytoscape 3.10.1 [[96]41]. Cytohubba plugin was used to identify the core targets based on node connection degree [[97]42]. 2.8.4. GO and KEGG Pathway Enrichment Analysis by Target Genes Shared gene targets of the isolated compounds and the studied diseases were analyzed using the Database for Annotation Visualization and Integrated Discovery ((DAVID) (retrieved from [98]https://david.ncifcrf.gov (accessed on 25 February 2024)) for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses [[99]43]. The official gene symbols of the shared target genes were imported into the DAVID web server, and Homo sapiens was selected as the species of interest. For the GO enrichment analysis, the “Functional Annotation Tool” was utilized, and the GO functions were annotated using the terms biological process (BP), cellular components (CCs), and molecular functions (MFs), and the respective charts were downloaded and processed. The KEGG pathway chart was downloaded from the “Annotation Result Summary”. The obtained GO and KEGG enrichment analysis charts were rearranged by filtering the results with a p-value less than 0.05 and sorted from the largest to smallest enrichment scores in terms of −log10 (p-value) and fold enrichment, respectively. The top ten results of the GO enrichment analysis and top 20 KEGG pathway enrichment analyses were graphed and visualized with bar diagrams and bubble diagrams, respectively, with the help of the SR Plot web server (retrieved from [100]https://www.bioinformatics.com.cn/srplot (accessed on 25 February 2024)). 2.9. Molecular Docking Three-dimensional structures of the selected ligands were searched and collected using the PubChem database ([101]https://pubchem.ncbi.nlm.nih.gov/ (accessed on 22 February 2024)) [[102]44]. To compare the binding capacity of the selected phytochemicals, elagolix (PubChem Id-11250647), which is an FDA-approved drug for relieving endometriosis, was chosen. The structures were saved in SDF format, and energy minimization was performed using PyRx [[103]45]. RCSB Protein Data Bank (retrieved from [104]https://www.rcsb.org/ (accessed on 22 February 2024)) was used for structure retrieval of the top three hub targets i.e., SRC (PDB ID: 2H8H), ESR1 (PDB ID: 3ERT), and PIK3R1 (PDB ID: 5XGJ), using pdb format. Protein preparation was performed by removing co-crystallized water and ligands, followed by adding hydrogen. Energy minimization of the target proteins was performed using SwissPDBviewer (version 4.1) [[105]46]. The active site of the proteins was identified using a literature survey and utilizing exiting co-crystallized ligand coordinates. The Autodock Vina (version 0.8) plugin from PyRx was used to study molecular docking interactions between the target protein’s active site and selected ligands [[106]47]. Visualization of the ligand–target interactions was achieved using Biovia Discovery Studio version 2021 [[107]48]. 2.10. In Silico ADMET Analysis The absorption, distribution, metabolism, excretion and toxicity (ADMET) profile of the selected phytoconstituents based on the molecular docking score was studied using the online server pkCSM ([108]https://biosig.lab.uq.edu.au/pkcsm/ (accessed on 22 February 2024)) [[109]49]. Preliminary pharmacokinetic and toxicity data generated by the server help to identify potential lead compounds in terms of safety and druggability. 2.11. Molecular Dynamics Simulation Molecular dynamics (MD) simulation was performed for the top protein (SRC, PDB ID: 2H8H) and ligand complex showing the highest docking interaction score. The Schrodinger Desmond package (Desmond Molecular Dynamics System version 1.9.1, D. E. Shaw Research, New York, NY, USA, 2021) was utilized to analyze the movements of the proteins docked with ligands and the outcomes were compared to the apo form, generated by the MD simulation. As a force field, OPLS2005 was applied while a simple point charge model was used for the task. All the analyzed target–ligand complexes were neutralized by adding counter ions (47 Na^+ and 44 Cl^−), and to simulate the human physiological system, a 0.15M Na^+ level was maintained. The prepared complexes underwent relaxation through the Desmond default protocol [[110]50]. The NpT ensemble was set at 300 K temperature and 1 bar of pressure for the MD simulation run for 100 ns. Following the completion of the simulation, the results of the ligand–target complexes were acquired as RMSD, RMSF, and ligand RMSF. [111]Figure 2 demonstrates the methodology adopted in the present study. Figure 2. [112]Figure 2 [113]Open in a new tab Workflow diagram for the identification of drug-like phytoconstituents of Clitoria ternatea flower against endometrial pain using network pharmacology analysis followed by in vitro, in vivo, and in silico validation. 2.12. Statistical Analysis The data obtained from biological evaluation were subjected to statistical analysis using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. All the observed values were expressed as mean ± SEM (n = 5). Data for which p values were found as *** p < 0.001 and ** p < 0.01 were considered statistically significant as compared to the control group. The analysis was carried out using Microsoft Excel (version 2010) and GraphPad version 10.2.2. 3. Results 3.1. Phytochemical Screening Phytochemical analysis of CT exposed the presence of alkaloids, flavonoids, glycosides, steroids, and tannins ([114]Table 1). Table 1. Phytochemical screening of the crude methanolic extract of C. ternatea flower. Phytochemicals Observation Alkaloids + Carbohydrates − Flavonoids + Glycosides + Saponins − Steroids + Tannins + [115]Open in a new tab (+) = present, (−) = absent. 3.2. Evaluation of Membrane-Stabilizing Property As the lysosomal membrane and the membrane of red blood cells resemble one another, the effects of medications that stabilize the erythrocyte membrane might have a similar impact on cells responsible for releasing chemicals that can cause inflammation. Therefore, they may be capable of showing anti-inflammatory effects. The capacity of the crude methanolic extract of C. ternatea flower to stabilize membranes in vitro was studied under heat-induced conditions. The absorbance for the determination of the anti-inflammatory activity of the crude methanolic extract of C. ternatea flower in heat-induced conditions is summarized in [116]Table 2. Each test was repeated three times for better precision. Percentage inhibition of hemolysis by the standard and test sample is shown in [117]Figure 3. Table 2. Absorbance of heat-induced erythrocyte suspension upon treatment with control, standard and test sample at 540 nm. Sample Concentration (mg/mL) OD1 OD2 OD3 Control - - - 1.09 ± 0.01 Aspirin 0.1 0.095 ± 0.000 0.171 ± 0.001 - Methanolic extract of CT flower 2.0 0.082 ± 0.001 0.160 ± 0.000 - [118]Open in a new tab OD = observed data; OD1 = absorbance of unheated test sample, OD2 = absorbance of heated test sample, and OD3 = absorbance of heated control sample. All data for OD1, OD2 and OD3 are expressed as mean ± S.E.M. Figure 3. [119]Figure 3 [120]Open in a new tab Effect of crude methanolic extract of C. ternetea flower on heat-induced hemolysis of normal RBC. Here, *** p < 0.001 and ** p < 0.01. This study revealed that methanolic flower extract of C. ternatea demonstrated significant inhibition of hemolysis of RBCs in heat-induced conditions, having 91.47% inhibition of hemolysis in comparison to 92.90% inhibition by the standard aspirin (0.10 mg/mL). Based on these results, it can clearly be stated that the sample under experimentation might have the ability to stabilize the RBC membrane and, by extension, the membranes of the inflammation-causing lysosomal cells, thereby providing a potential anti-inflammatory effect. 3.3. Evaluation of In Vivo Anti-Inflammatory Activity The carrageenan-induced paw edema test induces acute inflammation in test animals. The mean paw volume in each test group for four consecutive hours upon administration of carrageenan is shown in [121]Figure 4. The control group (normal saline) showed a gradual increase in paw volume with time, whereas both standard and test samples showed a gradual decrease in paw volume over the four-hour test period. All the test data were found to be statistically significant except for a 200 mg/kg dose of the test sample right after the first hour of treatment. Moreover, no adverse effects were observed among test animals, thus ensuring safety of the prepared extract. Figure 4. [122]Figure 4 [123]Open in a new tab Effects of C. ternatea flower methanolic extract and Aceclofenac on paw diameter after carrageenan injection. Each value represents the mean ± SEM (n = 5), *** p < 0.001 and ** p < 0.01, compared with control (one-way ANOVA followed by Dunnet’s multiple comparison test). # indicates control, against which comparison was made. The standard Aceclofenac showed 52.79%, 63.22%, 70.56%, and 80.38% inhibition of paw edema. In contrast, the crude extract at the 200 mg/ kg dose showed 21.03%, 31.81%, 48.80%, and 65.28%, and at the 400 mg/kg, dose it showed 56.22%, 64.885, 74.09%, and 84.89% inhibition of paw edema of at the end of the first, second, third, and fourth hours, respectively ([124]Table 3). Table 3. Evaluation of anti-inflammatory activity of C. ternatea flower extract by measuring percentage inhibition of paw edema at different time intervals. Group % Paw Edema Inhibition 1st Hour 2nd Hour 3rd Hour 4th Hour Control No edema inhibition No edema inhibition No edema inhibition No edema inhibition Standard 52.79 63.22 70.56 80.38 Test sample (200 mg/kg) 21.03 31.81 48.80 65.28 Test sample (400 mg/kg) 56.22 64.88 74.19 81.89 [125]Open in a new tab 3.4. Evaluation of Peripheral Analgesic Activity The peripheral analgesic activity of the crude methanolic extract of C. ternatea flower was evaluated using the acetic acid-induced-writhing method, where writhing times were the evaluation index. Aceclofenac at a dose of 25 mg/kg b.w. showed 77.49% inhibition, whereas methanolic extract showed 75.6% and 76.68% inhibition of writhing at a dose of 200 mg/kg and 400 mg/kg, respectively, when compared to the control group ([126]Table 4). No signs of adverse effects were evident among test animals, which ensured the safety of the prepared extract. Table 4. Evaluation of the analgesic activity of crude methanolic extract of C. ternatea flower using acetic acid-induced-writhing test. Sample Code Dose (mg/kg) Number of Writhing Actions (Mean ± SEM) % of Inhibition of Writhing Control 0 84.4 ± 3.78 - Standard 25 16.8 ± 2.52 *** 77.49 Test sample 200 20.6 ± 3.70 *** 75.60 400 19.6 ± 3.262 *** 76.78 [127]Open in a new tab Each value represents the mean ± SEM (n = 5), *** p < 0.001, compared with control (one-way ANOVA followed by Dunnet’s test). 3.5. Collection of Plant Active Constituents and Target Genes In total, 59 different bioactive constituents were identified ([128]Supplementary File) following an extensive literature search, from which 18 were selected as they showed no more than one violation of Lipinski’s rule of five [[129]11,[130]15,[131]16,[132]34,[133]51]. The selected constituents were also predicted to have a high bioavailability score ([134]Table 5). Table 5. List of phytochemicals reported to be present in the flower petal of C. ternatea. Phytochemicals PubChem ID Predicted Oral Bioavailability Score Method of Identification References of Identification Method