Protective effect of Tecomastans (L.) Juss.exKunth in CFA-induced arthritic rat model

Protective effect of Tecomastans (L.) Juss.exKunth in CFA-induced arthritic rat model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protective effect of Tecomastans (L.) Juss.exKunth in CFA-induced arthritic rat model Chandan Das, Pritam Kar, Priyanka Dash, Deepak Pradhan, Vineet Kumar Rai, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4224044/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Tecoma stans (L.) Juss.exKunth (Bignoniaceae) is mainly found in tropical and subtropical regions of Africa and Asia. The leaves, flowers, roots, and bark are used to treat various aliments includes, skin infections, kidney problems, intestinal disorders, jaundice, toothaches, joint pain and repair cracked bones, antidotes for snake, scorpion, and rat bites. The aim of the study is to assess the anti-arthritic properties of T. stans leaf using Complete Freund's adjuvant (CFA)-induced rat model. The ethanol extract of T. stans leaf (ETSL) was taken for Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography-Mass Spectrometry (LC-MS) analysis for the identification of potential bioactive. The in vitro antioxidant and anti-arthritic activity was studied at concentrations of 25, 50, 100, 200, 400, and 500 μg/ml. In vivo anti-arthritic activity was carried out by administering CFA (0.1 ml) into the sub-plantar surface of the right hind paw. The experimental animals were treated with indomethacin (10 mg/kg) and ETSL (250, 500 mg/kg) once a daily for fourteen days. The arthritic parameters such as paw thickness, arthritic index, arthritic score, body weight, organ weight, and hematological and biochemical parameters were evaluated. Pro-inflammatory cytokines; tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, anti-inflammatory cytokines; IL-4 and IL-10 and inflammatory mediator cyclooxygenase-2 (COX-2) were examined in blood serum. In vivo antioxidants parameters; superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and lipid peroxidation (LPO) was carried out in liver and joint. Radiological and histopathological analysis of joint was performed.A computational molecular docking investigation of the phytoconstituents was conducted against COX-2, IL-1β, IL-6, and TNF-α receptors by utilizing AutoDock 4.2 and BIOVIA-Discovery Studio Visualizer software. The in vitro result showed concentration dependent antioxidant activity with highest percentage of inhibition at 500 µg/ml. The in vivo result demonstrated significant restoration of arthritic parameters, hematological and biochemical indices and oxidative stress in CFA-induced rat which was further supported by radiological histological examination at ETSL 500 mg/kg. In addition, there was significant (p<0.05) reduction in pro-inflammatory cytokines, inflammatory mediators and up-regulation of anti-inflammatory cytokines was observed in the treated group. Verbascoside was found to exhibit better biding affinities -10.4, -7.4, -7 and -6.2 kcal/mol against COX-2, IL-1β, TNF-α, and IL-6 respectively, confirmed through in silico study. The observed outcome suggests that ETSL at a dosage of 500 mg/kg demonstrated notable anti-arthritic effects by suppressing pro-inflammatory cytokines and oxidative stress biomarkers. This effect could potentially be attributed to the presence of bioactive verbascoside identified in the LC-MS analysis. Tecomastans Anti-oxidant Complete Freund’s Adjuvant Cytokines GC-MS LC-MS in-silico. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Introduction Rheumatoid arthritis (RA), an autoimmune disease, causes persistent inflammation of the synovium inside joints. This inflammation affects the lining of the joints and may affect the ligaments and bones within the joints, leading to stiffness, discomfort, and degeneration of the articular cartilage(Jain et al. 2023). The risk of developing RA between 30 and 50 years of age is 2 to 3 times higher for women than men (Shamlan et al. 2021). Compared to the global prevalence of 0.46%, India's prevalence of RA is estimated at 0.7% (Bagepally et al. 2023). As a result of arthritis, cartilage, which generally protects joints, breaks down, causing synovial fluid to accumulate in the joints and causing inflammation. Pro-inflammatory modulator stimulates the production of inflammatory cells in the synovial membrane, resulting in bone damage(Shamlan et al. 2021). The inflammatory response associated with arthritis is accompanied by hyperplasia of synovial cells. Cartilage damage and joint alkalosishave been involved in the pathology of disease progression (Jain et al. 2023). The cause of joint damage has been found to be increased levels of pro-inflammatory cytokinessuch as tumor necrosis factor-alpha(TNF-α), interleukin-1-beta (IL-1β), interleukin-6 (IL-6), metabolic enzymes, and decreased levels of anti-inflammatory cytokines; interleukin-4(IL-4) and interleukin-10(IL-10) (Jain et al. 2023). According to recent research, pro-inflammatory mediators cyclooxygenase-2 (COX-2), an inducible enzyme secreted from fibroblasts, macrophages, leukocytes, and cytokines, were found in higher concentrations in the synovial fluid of RA patients and play a crucial in theetiology of disease(Hassan et al. 2019).Specific pathological diseases such as inflammation and RA cause an increase in the expression of this enzyme due to a variety of stimuli, including growth factors and cytokines. The degradation of joint cartilage associated with RA is brought on by increased expression of COX-2 in subchondralbone(Tu et al. 2019).Studies demonstrate that pro-inflammatory cytokines are probably responsible for the overexpression of COX-2(Sokolove and Lepus 2013). Thus, a novel strategy for treating RA may involve by decreasing these pro-inflammatory cytokinesandup regulating of anti-inflammatory cytokines(Hong et al. 2021). Though several numbers of molecules, such as prostaglandins, TNF-α, IL-1β, and IL-6 are implicated in the pathophysiology of RA, reactive oxygen species (ROS) trigger the inflammatory processes by damaged articular cartilage, membrane lipids, proteins, and DNA (Jain et al. 2023). It is observed that inflammatory cells such as neutrophils, lymphocytes, macrophages, and endothelial cells found in the inflammatory joint tissue are the main source of ROS(Sindhu et al. 2012). The primary cause of RA can be identified as oxidative stress. By generating pro-inflammatory cytokines and COX-2, ROS induces oxidative stress, which is a crucial modulator of inflammatory reactions at the inflamed joint during the development of arthritis (Goldring 2003). The defense mechanism against the damaging effects of free radicals is comprised of a variety of detoxifying enzymes and metabolites. The two main antioxidant enzymes involved in detoxification are catalase (CAT) and superoxide dismutase (SOD) (Phull et al. 2017). As a result, many research studies have been investigated to explore the anti-inflammatory properties of antioxidants. Most antioxidants are derived from dietary sources and are crucial for preventing the harmful effects of ROS on living systems. Antioxidants possess the capacity to protect an organism from the harmful effects of free radical damage, thereby reducing or delaying the emergence of multiple pathologies like cardiovascular, cancer, atherosclerosis, RA, and neurodegenerative illnesses(Silva et al. 2023). Currently available therapies such as non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and disease-modifying anti-rheumatic drugs (DMARDs) to treat RA are associated with several side effects, including damage to the liver, cardiovascular problems, and nausea (Su et al. 2022). The current state of medicines has limited their wide application in the treatment of RA due to the high cost and unpredictable response. Thus, there is an urgent desire to identify drugs with fewer adverse effects and established therapeutic efficacy(Lv et al. 2022). According to published research, natural products are a promising alternative source for treating RA because they are less toxic and have fewer adverse effects(Zhang et al. 2008). The unique chemical compositions and biological activities of herbal products allow them to treat various ailments. Manyphytocomponents, such as flavonoids, terpenoids, glycosides, and alkaloids, have been reported with anti-arthritic properties(Jain 2014). Therefore, there is an increasing focus on using natural substances and their products in managing arthritis. Tecomastans (L.) Juss. ex Kunth (Bignoniaceae) is amedium-sized ornamental blooming perennial deciduous shrub known as yellow-elder, yellow trumpet bush, and yellow-bells and found in tropical and subtropical regions of Africa and Asia (Anand and Basavaraju 2021). It is also found as an invasive weed in South Africa(Madire 2013). It is extensively dispersed throughout India, from the plains extending to the southernmost point of the country to the Shiwalik levels in the Himalayas. It has been observed in several regions of the nation, including Shimla in Himachal Pradesh(Thakur 2012); Nalgonda, Baptala, Tirumala hills, Put-taparthi, Simhachalam, and Warangal in Andhra Pradesh; and in Orissa (Dash 2011). The genus Tecoma is rich in phytoconstituent; prior research has identified and isolated numerous constituents belonging to various chemical classes, including phenolics, indolic compounds, iridoids, flavonoids, alkaloids, and triterpenoids. This genus also has a wide range of pharmacological properties, including antibacterial, diuretic, spasmolytic, hypoglycemic, anti-inflammatory, and protozoal illnesses. Other biological activities also have been described, such as anti-diarrhea, antioxidant, anti-proliferative, insecticidal, and astringent properties(Abdel-Mageed et al. 2012). With expressivepantropical distribution, the Bignoniaceae family of plants has around 830 species, where T. stans is recognized as an exotic in Brazil and extensively utilized as ornamental plants. This species produces a variety of compounds, including unsaturated fatty acids, carbohydrates, proteins, resins, phenolic acids, flavonoids, carotenoids, terpenoids, saponins, glycosides, and phytosterols(Raju 2011). Several alkaloids were reported from the leaf of T. stans , such as tecomine, tecostanine, boschniakine, 4-hydroxytecomanine, N-normethylskytanthine, 5-hydroskytanthine, 7-hydroskytanthine, γ-skytanthine, tecomanine, and 4-noractinidine (Hammouda and Amer 1966). Leaf also reported with phenolic acids, namely, chlorogenic, cinnamic acid, ferulic acid, gallic acid, caffeic, vanillic, o-coumaric, and sinapic acids, sitosterols, triterpenoids, and flavonoids, namely, flavonone, apigenin, chrysoeriol, kaempferol, luteolin, quercetin, rutin, 7,8-dihy-droxy-4,6-dimethoxy flavone, and verbascoside. The different parts of the plants, including leaves, flowers, roots, and bark, are used to treat a variety of disorders and ailments, including skin infections, kidney problems, intestinal disorders, jaundice, heart pain, joint pains, and antidotes for snake, scorpion, and rat bites(Anand and Basavaraju 2021). The ancient methods of preparing herbal remedies from T. stans that were most frequently cited were the decoction of aerial parts, infusion of leaves, flowers, and young leaves. The leaf is utilized in the rural Kyaing Tong Township, Myanmar, to treat arthritic joint pain and repair cracked bones. A paste was prepared by combining the leaf with other medicinal plants and rice water and applied as a poultice to the affected area. A week is needed for the fractured bones to heal for children and 15 days for adults over 25(Anand and Basavaraju 2021). Besides that, a wide range of biological activities such as hypoglycemic(Lozoya-Meckes and Mellado-Campos 1985);cardioprotective(Villar 1997); anticancer (Thirumal M 2012); hepatoprotective(Kameshwaran et al. 2012); antimicrobial (Binutu and Lajubutu 1994); antinociceptive and anti-inflammatory ; wound healing (Das 2010); antispasmodic (Gharib-Naseri 2007); antidiarrheal (Kameshwaran et al. 2012); antiulcer (Shanmukha 2013), gastroprotective, anti-obesity and insecticidal (Silva et al. 2023)have also been reported. A literature survey revealed that T. stans was reported with many phenolic and flavonoid compounds. These compounds effectively prevent and manage RA by interacting with different target sites (Ali et al. 2023). The anti-arthritic potential of T. stans leaf has not been documented to date. Hence, this current study aimed to explore the anti-arthritic potential in rats induced with Complete Freund's adjuvant (CFA) by assessing parameters such as paw diameter, arthritic index and score, inflammatory cytokines, and oxidative biomarkers. Moreover, GC-MS and LC-MS analyses were conducted to characterize the bioactive compounds accountable for the anti-arthritic activity. Additionally, computational methods were employed to assess the anti-arthritic potential of bioactive compounds identified from LC-MS analysis against COX-2, IL-1β, IL-6, and TNF-α protein. Materials and Methods Chemicals and reagents Complete Freund's adjuvant (CFA), TNF-α, IL-6, IL-1β, IL-4, IL-10 and COX-2, were obtained from Sigma Aldrich, USA. Indomethacin was acquired from Micro Lab Pvt. Ltd, India. Sodium carboxyl methyl cellulose, 2,2-diphenyl-1-picrylhydrazyl, Tris-HCl, Ethylenediaminetetraacetic acid, Perchloric acid, Ascorbic acid, n-hexane, and Ethanol were purchased from Merck Pvt. Ltd, Germany. Plant collection The leafof T. stans were collected from Bhubaneswar during August 2023with demographic details N 20° 17.1548' and E 85° 46.3195'and authenticated by Dr. Pratap Chandra Panda, Taxonomist, Siksha ‘O’ AnusandhanDeemed to be University, Bhubaneswar, Odisha, India with voucher specimen CBT/2457. Preparation of extract The collected leaf material was shade dried and then subjected to coarse powder. The powder material was successively extracted with n-hexane and ethanol for 48 hours using soxhlet apparatus andconcentrated using a rotary evaporator. The percentage yield of n-hexane and ethanol extract were found to be 7.45 and 18.29 w/w, respectively, and refrigerated until further use. Analysis of ETSL by GC-MS Gas chromatography and mass spectroscopy(GC-MS) analysis of ethanol extract of T. stans leaf (ETSL) was performed using modified procedure(Das et al. 2022a). A mass detector (MS TSQ 8000) was attached to the Thermo Trace 1300GC and Thermo TSQ 800 Triple Quadrupole MS which connected to the BP-5MS (5% Phenyl Polysilphenylene-Siloxane) capillary column (30m X 0.25mm, 0.25µm film thickness).1 µl of the ETSL was added to the column at a split flow rate of 30 ml/minute. The mass spectra from m/z 35 to 650 were obtained at intervals of 0.5 seconds. The temperatures of the injector, MS transmission line, and ion source were changed to 250℃, 280℃, and 230℃, respectively. A steady carrier flow of 1 ml/min was maintained. The oven was heated to 280℃ at a rate of 10℃ per minute after keeping at 60 ℃ for six minutes. The metabolites were identified by comparing the GC-MS investigation results with Wiley and the NIST 2.0 library database (National Institute of Standards and Technology). Analysis of ETSL by LC-MS The LC-MS analysis of ETSL was carried outusing Water Alliance e2695/HPLC-TQD mass spectrometer. The compounds were separated on an RP High Strength Silica (HSS) T3 C18 column (250 mm × 4.6 mm, comprising particles with a diameter of 5 μm, Waters). The temperatures of the auto-sampler and column were adjusted at 5 °C and 35 °C, respectively.A gradient solvent system comprising solvents (A) acetonitrile and (B) ammonium acetate were utilized for the chromatographic separation. ETSL was passed through a 0.2 μm nylon membrane filter. The injection volume was kept at 5.0 μl.Total run time was maintained for 40 minutes. The run duration of 1-10 minutes comprises of 5% for (A) and 95% for (B); 30 % for (A) and 70% for (B); 16-24 minutes: 60% for (A) and 40% for (B); 80% for (A) and 20% for (B); 24-32 minutes: 80% for (A) and 20% for (B); 32-35 minutes: 80% (A) and 20% for (B); 5% (A) and 95% for (B); 35-40 minutes: (A) 5%, (B) 95%. The pressure was kept at 1,200 barand the flow rate was maintained at 1.5 ml/min. The capillary voltage, source cone voltage, and extraction cone voltage were kept at 3.5 kV, 30 V, and 3 V, and;at 3.5 kV, 60 V, and 3 Vin positive and negative mode,respectively.Adesolvation gas, nitrogen was added at a flow rate of 650 l/hour. The temperatures of the desolvation and source were kept at 350°C and 120°C, respectively. Both positive and negative ionization modes of electrospray were used to get the mass spectrum data. Mass spectra were obtained by covering the m/z range of 150-750Da. Assessment of in vitro antioxidant potential of ETSL In vitro antioxidant property of ETSLat25, 50, 100, 200, 400 and 500μg/ml was performed using DPPH (2, 2-diphenyl-1-picrylhydrazyl), ferric reducing, nitric oxideradicalscavengingand reducing power assay, and ascorbic acid used as a standard(Dash and Ghosh 2017). IC 50 value was calculated by plotting the graph, scavenging activity versus the concentration. Assessment of in vitro anti-arthritic potential of ETSL Protein denaturation efficacy of ETSL The protein denaturation efficacy of ETSL was assessed as per the standard method(Alamgeer et al. 2017). The test mixture comprises of Fresh hen's egg albumin (0.2 ml), phosphate-buffered saline (2.8 ml), various concentration (25, 50, 100, 200, 400 and 500 µg/ml) of ETSL and indomethacin (2 ml). Double-distilled water was used as control.Thereaction mixture was incubatedfor 15 minutes at 37 ± 2 °C, followed by heating for 5 minutes at 70 °C. The absorbance of these solutions was recordedat 660 nm. The percentage of inhibition was calculated using following formula. Membrane stabilization efficacy of ETSL Human red blood cell (HRBC) was used to evaluate membrane stabilization potential of ETSL. HRBC(10 ml) wastaken in heparin centrifuge and added with equal volume of Alsever’s solution. The resultant mixture was centrifuged with 0.85% isosaline. Equal quantities of ETSL (25-500 µg/ml) were added to 1ml of HRBC suspension and incubatedfor 30 minutes at 37 °C. Then it was subjected to centrifugation. Mixture without extract was considered as control. The amount of haemoglobin in the supernatant solution was measured at 560 nm(Ajithkumar et al. 2020).The following formula was used to calculate the percentage of hemolysis. Proteinase inhibitory efficacy of ETSL The method outlined by (Sivapalan et al. 2024)was used to evaluate the proteinase inhibition efficacy of ETSL. Thereaction mixture (1 ml of 20mMtris HCL buffer(pH 7.4), 1 ml of ETSL(25-500 µg/ml), and 0.06 ml of trypsin) was incubated for five minutes at 37 °C. Casein (1 ml, 0.8%) was added to the above mixture and kept for twenty minutes. Then 2 ml of perchloric acid (70%) was added and centrifuged (2500 rpm) for five minutes.The absorbance was measured at 280 nm. The proteinase inhibitory activity was estimated using the following formula: Animal Healthy young adult Wistar rats (6-8 weeks) weighing 150-180 g of both sexes were used. Before animal experiment, all animals were acclimatized in animal house for seven days. The animal house was maintainedlight-dark cycle (12 hours), temperature (22 ± 3 °C), and a relative humidity (30-70%). Feeding was done using a standard lab diet.The experimental protocol was approved by Institutional Animal Ethic Committee (IAEC/SPS/SOA/128). Assessment of in vivo anti-arthritic potential of ETSL Experimental protocol Animals were divided into five groups (n=6). Group I: Normal control (NC) received carboxy methyl cellulose-sodium (CMC-Na, 1% w/v); Group II: Disease control received CFA 0.1 ml; Group III: positive control received indomethacin (IND) 10 mg/kg;Group IV and V: test control received ETSL 250 and 500 mg/kg, respectively. 0.1 ml of CFA (10 mg) of heat-killed mycobacterium tuberculosis per ml of paraffin oil was injected into the sub-plantar surface of the right hind paws for the induction of RA (Das et al. 2021). The doseof ETSL was selected as previously reported elsewhere(S. Kameshwaran 2013). The inflammatory edema of the injected paw was observed after 6 to 8 days of CFA immunization, and the maximum severity was noticed on the 14 th day.Thetreatment was commenced from day 15 to 28. Evaluation of arthritis was performed by measuring paw diameter on days 0, 7, 14, 21, and 28 by using Verniercalipers; arthritic score (0-4) with maximum possible score for each animal was 16; arthritic index; body weightandorgan (liver, kidney, and spleen) weightexamination(Das et al. 2021). Examination of hematological and biochemical parameters Experimental animals were sacrificed by cervical dislocation method on 29 th day. Blood sample was collected through cardiac puncture and examined for hematological parameters like red blood cells (RBC), white blood cells (WBC), haemoglobin (Hb), platelet count, erythrocyte sedimentation rate (ESR) and biochemical indicators; rheumatoid factor (RF), aspartate aminotransferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), creatinine, urea, and C-reactive protein (CRP)(Jain et al. 2023). Analysis of pro-inflammatory and anti-inflammatory cytokines The pro-inflammatory cytokinesTNF-α, IL-6, IL-1β,anti-inflammatory cytokines; IL-4 and IL-10, and inflammatory mediator COX-2were measured in the serum of experimental animals using Enzyme-linked immunosorbent assay (ELISA) (Uttra et al. 2018). Determination of oxidative stress biomarker The tissue homogenate of liver and joint were analyzed to measure the oxidative stress biomarkerssuch as SOD, CAT, reduced glutathione (GSH), and lipid peroxidation (LPO).The tissues were washed with cold saline and homogenized in Tris-HCl buffer (0.05 M, pH 7.4). Then, 10% w/v tissue homogenatewascentrifuged at 12,000 rpm for 30 minutes and the supernatant was used to measure the stress biomarkers(Sundaram et al. 2019). Radiological and histopathologicalanalysis of joint Radiographic analysis ofexperimental joint rats was carried out andimages were takenfor the evaluation of joint deformation.Further, histopathology of ankle joints wasperformed after fixing with 10% v/v formalin. Boneswere decalcified by immersing in 10% ethylenediaminetetraacetic acid (EDTA) for 30 days. The microtome sections were taken at 5μm thickness by embedding joints in paraffin wax followed by stainedwith eosin and hemotoxyline.The images were captured at 400x magnification by photomicroscope. Thehistopathological changes in the ankle joints, such as bone degradation, swelling, and pannus formation were examined(Uttra et al. 2019). Histological score Histological score of arthritic rats were measured to assess the extremity of RA in terms of proliferation of synovial vascularity, hyperplasia, inflammation, and expansion of the synovial lining. The score was assessed using a semi-quantitative 4-point grading method; absent (0), mild (1), moderate (2), severe (3) with highest score of 21(El-Tanbouly and Abdelrahman 2022). Molecular docking study The phytocompounds tentatively identified through LC-MS analysis from ETSL were taken for in silico analysis to establish a correlation between the observed anti-arthritic activity and their active constituents. Accordingly, 19 compounds were selected as ligands for molecular docking studies. Based on literature findings, four potential targets COX-2, IL-1β, IL-6, and TNF-α were selected to evaluate the effectiveness of each ligand individually. The structures of all ligands, including indomethacin, were obtained from the PubChem website in SDF format. OPEN BABEL software was employed to convert all the ligands into PDB format. Furthermore, the software Avogadro was utilized to optimize the 3D structures of all compounds, which were then saved as dot pdb (PDB) files. These optimized structures were subsequently used to derive reliable docking scores (kcal/mol).The 3D crystal structures of the target proteins with PDB IDs 2AZ5 (TNF-α), 4G6J (IL-1β), 1ALU (IL-6), and 1PXX (COX-2) were retrieved from the Protein Data Bank (PDB) at http://www.rcsb.org/pdb. The fragmented protein structures were remodelled using SWISS MODEL and saved in dot PDB format for subsequent molecular docking investigations. The binding affinity (Docking Score) of the compounds was computationally determined using AutodockVina 4.2 software. The visualizations of protein-ligand binding characteristics were conducted using BIOVIA-DISCOVERY VISUALIZER 2024 software(Das et al. 2024). Statistical analysis Data were collected in triplicate for the in vitro investigation and expressed as mean ± standard deviation(SD). Using unpaired student t-tests, the statistical difference between the groups was analyzed. All data for the in vivo experiment were presented as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison tests were used to determine the statistical significance of the differences between each of the groups. Using two-way ANOVA and Bonferroni's post hoc test, the findings of paw edoema, arthritic index, arthritic score, and body weight were analyzed.Statistical analysis was performed by GraphPad Prism 5.0, where P˂0.05 was considered as the level of significant difference. Results GC-MS analysis of ETSL GC-MS analysis of ETSL revealed 18 compounds (Fig. 1). The retention time, molecular formula and weight, and chemical nature of the compounds were presented in Table 1. The compound with highest percentage of area was identified as 13-Docosenamide, (Z)-(18.55 %). LC-MS analysis of ETSL The LC/MS chromatogram of ETSL in positive and negative mode was presented in Fig. 2&3, respectively. The phytocomponent of ETSL were tentatively identified based on their retention time, MS/MS fragments, experimental m / z , and metabolite classes and then confirmed using literature data. Total 20 secondary metabolites were identified with 14 in positive mode and 6 in negative mode of ionization (Table 2). For the purpose of determining the m/z ratio of sample, it demonstrated that the positive and negative modes of ionization in mass spectrometry involve the production of positive ions [M+H] + (m/z) and negative ions [M+H] - (m/z), respectively. All the phytochemicals were first time reported through LC-MS analysis. In vitro antioxidant potential of ETSL In vitro antioxidant potential of ETSL (25-500 µg/ml) was presented in Fig4. The result revealed that, ETSL exhibited highest percentage of inhibition at concentration 500µg/ml against reducing power assay (75.23±1.15), DPPH (74.23±1.05), nitric oxide (69.56±2.08) and ferric reducing assay (72.66±2.60) with corresponding IC 50 values of 292.06 ±3.12, 263.36±2.36, 324.34±4.89 and 267.64±3.65µg/ml, respectively. Standard ascorbic acid at concentration 500µg/ml also exhibited greater percentage of inhibition 92.33±0.88, 86.33±1.33, 81.33±1.20 and 83.89±2.08 against reducing power assay, DPPH, nitric oxide and ferric reducing assay, with corresponding IC 50 values of 148.80±1.45, 172.79±2.58, 235.24±2.74 and 260.80±3.14 µg/ml, respectively. Assessment of in vitro anti-arthritic potential of ETSL In vitro, the anti-arthritic property of ETSL was assessed by protein denaturation, membrane stabilization, and proteinase inhibition assay at concentrations 25, 50, 100, 200, and 500µg/ml. ETSL exhibited protein denaturation (78.32±1.36), membrane stabilization (71.10±1.08), and proteinase inhibition (79.33±0.82) with corresponding IC 50 values of 305.57±2.84, 320.35±3.12 and 280.12±3.65µg/ml at a concentration of 500µg/ml, respectively. The standard indomethacin also exhibited remarkable in vitro anti-arthritic potential with 92.88±1.31, 91.33±1.45 and 95.33±2.88 percentage of inhibition against these parameters with corresponding IC 50 values of 197.45 ±1.24, 212.54±2.58 and 209.20±1.86 µg/ml, respectively (Fig. 5). Assessment of in vivo anti-arthritic potential of ETSL Effect of ETSL on paw thickness Sub-plantar injection of CFA into the right hind paw causes a remarkable (13.41±1.38, P<0.001) rise in paw thickness of CFA rats on the 14 th day in contrast to normal control rats. The increase in paw thickness of disease-induced rats was also noticed on day 28. Administration of IND (10 mg/kg) and ETSL (250 & 500 mg/kg) declined the paw diameter compared to CFA-induced rats. A remarkable decrease in paw thickness by IND (4.35±1.29, p<0.001) and ETSL (500 mg/kg) (6.38±1.20, p<0.01) was observed on the 28 th day. However, ETSL (250 mg/kg) on day 28 also significantly (9.43±1.35, p<0.05) decreased the paw thickness. The study indicates that ETSL (500 mg/kg) shows a better effect than 250 mg/kg (Fig. 6A). Effect of ETSL on arthritic index The arthritic index of experimental animals is presented in Fig 6B. Treatment with IND (10 mg/kg) (32.25±1.75, p˂0.001) and ETSL (500 mg/kg) (57.05±2.18, p˂0.01) showed a noticeable reduction in the arthritic index on day 28 in contrast to CFA-induced animal (277.51±1.16). Considerable decline was also marked by ETSL (250 mg/kg) (166.60±1.64, p˂ 0.05) on day 28, indicating dose-dependent response of ETSL. Effect of ETSL on arthritic score Based on the arbitrary score method, the arthritic score of the disease control animals was found to be maximum (10.83± 1.6) on the 28 th day. The group administered with IND (10 mg/kg) noticeably (p˂0.001) reduced the arthritic score (5.5 ± 1.34). However, administration of ETSL (250 and 500 mg/kg) from the 15 th to the 28 th day showed a dose-dependent effect in depletion (p˂0.05, p˂0.01) of the arthritic score (8.5 ± 1.15, 7.5 ± 1.56) as compared to CFA induced rats, respectively(Fig.6C). Effect of ETSL on body weight CFA-induced rats were found to have a remarkable reduction (p˂0.001) in body weight (147.83 ± 1.71) as compared to normal control rats (186.23 ± 2.25) on the 28 th day. Contrarily, treatment with the IND (10 mg/kg) and ETSL (500 mg/kg) noticeably raised the body weight by 170.83 ± 2.86 (p<0.001) and 167.76 ± 1.82 p<0.01), respectively, in contrast to disease control rats on the 28th day. However, ETSL (250 mg/kg) also considerably (p<0.05) raised the weight of the body (161.16 ± 2.83) on the 28 th day as compared to the CFA group. This suggests that a higher dose of ETSL was found to have more effect than a lower dose (Fig.6D). Effect of ETSL on organ weight The disease control group was observed with a remarkable (p<0.001) increase in weight of the kidney (9.14±0.76) (Fig.7B) and spleen (7.59±0.46) (Fig.7C) and decline in the liver (5.85±0.41) (Fig.7A) as compared to normal control rats (4.52±0.46, 3.69±0.55, 9.13±0.21), respectively. Treatment with IND (10 mg/kg, p˂0.001), ETSL (250 mg/kg, p˂0.05), and ETSL (500 mg/kg, p˂0.01) noticeably restored the weight as compared to the disease control group. Effect of ETSL on hematological and biochemical parameters Significant elevation in the levels of WBC, Platelets, ESR, CRP, RF, AST, ALT, ALP, urea, and creatinine, and a decline in RBC and Hb were observed in the disease control group in contrast to normal control rats. Supplementation of IND (10 mg/kg, p˂0.001), ETSL (500 mg/kg, p˂0.01), and ETSL (250 mg/kg, p˂0.05) significantly normalized these altered values in contrast to disease control animal. The data of RBC, WBC, platelets, AST, ALT, ALP, urea, and creatinine were given in Table 3, whereas hemoglobin, ESR, CRP, and RF were presented in Fig. 8. Effect of ETSL on TNF-α, IL-1β, IL-6 and COX-2 The result indicated significant increases in TNF-α (7.58 ± 0.85, p<0.001) in the CFA-induced group as compared to the normal group (1.91±0.16). However, animals treated with IND (10 mg/kg) (2.40±0.59, P˂0.001), ETSL (250 mg/kg) (5.33±0.96, P˂0.05),and ETSL 500 mg/kg (4.33±1.16, P˂0.01) noticed with remarkable decline in TNF-α expression (Fig.9A). Similarly, CFA-induced group was marked with significant (P˂ 0.001) increased in IL-1β expression (191.28 ± 6.19) in contrast to healthy animals (45.85±3.94). Treatment with IND (10 mg/kg) (79.89±3.92, P˂ 0.001), ETSL (250 mg/kg) (141.48±6.75, p˂0.05), and ETSL (500 mg/kg) (100.24±6.80, p˂0.01) demonstrated notable declined in the IL-1β expression (Fig.9B). Remarkable elevated level of IL-6 was noticed with disease control rat (6.81±0.69, p˂0.001) as compared to normal rats (1.59±0.12). Administration of IND (10 mg/kg) (2.78±0.60, p˂ 0.001) and ETSL (500 mg/kg) (3.49±0.48, p˂0.01) notably reduced the IL-6 level in treatment rats as compared to disease control rats (Fig.9C). ETSL at 250 mg/kg (5.51±0.49, P˂ 0.05) also reduced the level of IL-6 in comparison to CFA group. The CFA-induced disease control group observed a remarkable (p<0.001) rise in COX-2 (15.01±2.44) in contrast to normal control rats (4.16±0.60). Treatment with IND (10 mg/kg) (8.08±2.17, p<0.001), ETSL (250 mg/kg) (10.57±1.96, p<0.05), and ETSL (500 mg/kg) (8.76±2.01, p<0.01) significantly declined the level of COX-2 as compared to disease control rats(Fig.9D). The observed result indicated that ETSL at dose of 500 mg/kg was found more effective in reducing the TNF-α, IL-6, IL-1β and COX-2 level than 250 mg/kg. Effect of ETSL on IL-4 and IL-10 The CFA-induced disease control group was noticed with a remarkable (56.14±5.86, p<0.001) decline in IL-4 expression in contrast to normal control animals (86.62±3.16). Group administered with IND (10 mg/kg) (79.60±3.71, p<0.001), ETSL (250 mg/kg) (72.41±2.92, p<0.5), and ETSL (500 mg/kg) (76.03 ± 3.04, p<0.01) were observed with significant up-regulation in IL-4 expression in contrast to CFA-induced rats (Fig.9E). Similarly, the expression of IL-10 in disease control group (2.39±0.47, p<0.001) was notably declined in contrast to normal rats (6.75±0.44). The expression of IL-10 was remarkably up-regulated after administering with IND (10 mg/kg) (6.13±0.37, p< 0.001), ETSL (250 mg/kg) (3.87±0.26, p<0.05), and ETSL (500 mg/kg) (4.97±0.31, p<0.01) as compared to CFA-induced group revealed dose-dependent effect in up-regulation of IL-4 and IL-10 level(Fig.9F). Effect of ETSL on oxidative stress biomarker The antioxidant effect of ETSL in the joint and liver of the disease control group was presented in Fig. 10&11.Significant (p<0.001) decreases in SOD (3.17±0.17, 0.96±0.12), CAT (16.18±2.19, 62.32±2.12), GSH (2.78±0.41, 2.94±0.58) and increased in LPO (34.12±3.35, 35.65±2.74) in joint and liver in CFA disease induced rats was noticed as compared to the normal control rats, respectively. Supplementation with IND (10 mg/kg) caused a remarkable (p<0.001) increase in SOD (6.12±0.36, 1.89±0.13), CAT (37.73±1.96, 107.16±2.56), GSH (7.35±0.83, 5.94±0.40) and decreased in LPO (12.67±2.42, 20.67±1.34) in joint and liver, respectively in contrast to CFA control group. Similarly, supplementation of ETSL (500 mg/kg) also remarkably (p<0.01) augmented the level of SOD (5.01±0.33, 1.73±0.12), CAT (33.65±2.44, 89.60±2.96), GSH (6.44±0.56, 5.54±0.42) and decreased in LPO content (19.24±1.64, 24.48±2.34) in joint and liver, respectively as compared to CFA-induced rats. However, ETSL (250 mg/kg)also causes noticeable (p<0.05) increased in SOD, CAT, GSH and decreased in LPO in contrast to disease control rats. The observed result showed that a higher dose of ETSL was found to be more effective than a lower 250 mg/kg in controlling the oxidative marker. Effect of ETSL on histopathology of joint The histology result of the joint of disease-control rats showed cartilage and bone erosion,pannus formation, and development of synovial hyperplasia. Supplementation with IND (10 mg/kg) and ETSL (250, 500 mg/kg) from day 15-28 considerably reduced the histological changes, as shown in Fig.12. However, the ankle joint of the normal control group did not observe any kind of histological changes. Effect of ETSL on radiology of joint An X-ray analysis of the ankle joints of experimental rats was presented in Fig. 12. Compared to normal control rats, the diseased control rats showed signs of soft tissue swelling, joint space constriction, and bone loss. A noticeable effect was observed in the group treated with IND (10 mg/kg) and ETSL (250, 500 mg/kg). Effect of ETLS on histological score Several parameters, such as the degree of enlargement of the synovial lining cell layer, synovial hyperplasia, synovial inflammation, synovial vascularity proliferation, pannus development, and bone and cartilage erosion, were assessed to determine the histological score in CFA-treated rats. The increase in histological score in the disease control rat (19.33±0.49) was remarkably declined by administration of IND (10 mg/kg) (12.00±0.73, p<0.001), ETSL (250 mg/kg) (16.50±0.42, p<0.05) and ETSL (500 mg/kg) (14.16±0.47, p<0.01) (Fig.13). Molecular docking study Molecular docking methodology facilitates the representation of molecular interactions between a ligand and protein molecule at the atomic scale. In the present investigation, molecular docking was conducted to elucidate the binding interactions of compounds identified through LC/MS analysis of ETSL with potential key targets, including TNF-α, IL-1β, IL-6, and COX-2. The results of the docking analysis conducted in this study are summarized in Table 4. The docking scores indicated that all ligands displayed activity ranging from -6.2 to -7 against TNF-α, -6.5 to -7.7 against IL-1β, -6 to -6.6 against IL-6, and -6.3 to -10.4 against COX-2. Analysis of the docking results revealed that phytoconstituents exhibited relatively stronger binding affinity towards COX-2, followed by IL-1β, TNF-α, and IL-6. Verbascoside, rutin, and naringin demonstrated the highest docking scores (-10.4, -10.3, and -10.2 kcal/mol, respectively) compared to indomethacin (-8.7 kcal/mol) against the COX-2 receptor. Sitosterols, naringin, and verbascoside recorded similar docking scores (-7 kcal/mol) against the TNF-α protein but relatively lower than indomethacin (-8.1 kcal/mol). Quercitrin displayed the highest docking score against IL-6 at -6.6 kcal/mol, surpassing the standard indomethacin (-6.4 kcal/mol) (Fig.14). Discussion The GC-MS analysis of ETSL revealed several bioactive compounds that exhibited anti-inflammatory and antioxidant activities. These compounds include pentadecane(Okechukwu 2020); heptadecane, 2,6,10,15-tetramethyl- (Jeha F. Dela Pena 2019); 1-Hexadecanol (Murugan Prasathkumar 2022); cetene(Murugan Prasathkumar 2022); 3-Eicosene, (E)- (Okechukwu 2020); methyl 9-cis,11-trans-octadecadienoate (Alabi A. Adenike 2019); cis-13-Octadecenoic acid, methyl ester (Diab AT 2021); 13-Docosenamide, (Z)- (Tareq et al. 2020); 9-Octadecenamide, (Z)- (Awakan et al. 2018); 7,8-Epoxylanostan-11-ol, 3-acetoxy- (Never Zekeya 2014); hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester (Markkas 2015); ethyl iso-allocholate(Johnson et al. 2020) and oleic acid, eicosyl ester (N. Habeela Jainab 2017). LC-MS analysis of ETSL revealed 20 compounds: amino acids (2), alkaloid (1), phenolic (11), and flavonoid compounds (6). The amino acids tryptamine(Vitalini et al. 2020) and tryptophan (Wang et al. 2019) showed molecular ions [M+H] + with a mass of 160.2 m/z and 203.3 m/z at retention time (RT) 25.89 and 16.79 minutes, respectively. The peak shown at RT 7.13 minutes indicates a molecular ion [M+H] - of alkaloid,Boschniakine, with a mass of 161.1 m/z (Wang et al. 2021). The peaks at RT of 5.56,6.65, 7.69,29.0, 10.95, 13.52, 16.13, 26.49, and 29.0 minutes revealed phenolic compounds such as p-coumaric acid (Cui et al. 2010), luteolin(Shi et al. 2018), gallic acid (Yu et al. 2018), naringin(Fang et al. 2006), ferulic acid (Jain and Surana 2016), chlorogenic acid (Clifford et al. 2003), (-)-catechin(Kaigongi et al. 2020) and caffeic acid (Wang et al. 2015) with molecular ions [M+H] + and mass of 163.0 m/z , 285.2 m/z , 169.3 m/z , 580.5 m/z , 195.3 m/z , 353.3 m/z , 291.4 m/z and 181.3 m/z , respectively. Compounds hesperetin(Tong et al. 2012), rosmarinic acid (Ma et al. 2013)and sitosterols(Kim et al. 2016) showed molecular ions [M+H] - with a mass of 301.2 m/z , 353.3 m/z and 415.3 m/z at RT of 6.65,8.38 and 12.66 minutes, respectively. Flavonoid compounds such as apigenin(Shaker et al. 2022), kaempferol(Chen et al. 2015), quercetin(Panchal and Shah 2017), quercitrin(da Costa et al. 2019), rutin(Wang et al. 2003) and verbascoside(Luca et al. 2019)at RT of 6.31, 7.14,8.10, 9.76,8.93 and 11.29 minutes showed molecular ions [M+H] + with mass of 288.2 m/z , 287.2 m/z , 302.2 m/z , 447.4 m/z , 617.5 m/z and 624.5 m/z , respectively. ROS is one of the main causes of RA. The overabundance of pro-inflammatory cytokines prompts neutrophils and activated macrophages to release ROS into the synovial fluid and serve as a mediator of tissue damage. ROS attacks biological components like lipids, proteins, enzymes, DNA, and RNA in adjuvant-induced arthritis, causing damage to cells or tissues (Kumar et al. 2016). According to (Arulselvan et al. 2016), an imbalance between antioxidants and free radicals leads to oxidative stress, which damages cellular components and induces inflammation. In the current study, ETSL exhibited in vitro antioxidant properties in a concentration-dependent manner against DPPH, ferric-reducing assay, reducing power, and nitric oxide radicals. Several phenolic and flavonoid compounds are known to possess antioxidant properties. Protocatechuic acid (3,4-Dihydroxybenzoic acid) was reported to scavenge free radicals of H 2 O 2 and DPPH (Semaming et al. 2015). Tryptamine possesses an antioxidant activity by metal chelating effect(M. E. Neganova 2011). Ferulic acid and apigenin were known to scavenge free radicals such as hydroxyl radicals, superoxide, hydrogen peroxide, and nitrogen radicals(Kashyap et al. 2022; Zhu et al. 2020). Hesperidin (Adefegha et al. 2020)and quercetin(Haleagrahara et al. 2017)were also found to decrease the reactive oxygen species levels. In the current study, these phenolic and flavonoid compounds were identified by LC-MS analysis of ETSL and could be responsible for significant in vitro antioxidant properties. The free radical scavenging property of T. stans leaf was also reported earlier by (Anand and Basavaraju 2021). Denaturation of protein is considered a good indication of RA. The process in which proteins lose their secondary and tertiary structures in response to external pressures, such as changes in temperature and pH and exposure to organic or inorganic substances, is known as denaturation. The hydrophobic, electrostatic hydrogen, and disulfide bonds are altered throughout the degradation process of proteins (Das et al. 2022b). It is noted that RA is associated with the development of auto-antigens as a result of protein denaturation. Denaturation of intercellular material or components of cell proteins may result in tissue damage (Osman et al. 2016). Therefore, the ability of the substance to stop protein denaturation suggests its anti-inflammatory properties. In the current investigation, ETSL inhibited protein denaturation in a concentration-dependent manner, which might be due to its defense against protein denaturation and auto-antigen formation. Phytochemicals, including alkaloids, flavonoids, tannins, and phenolic acids, were reported to inhibit the protein denaturation reported by (Pavithra 2015). Therefore, the presence of these phytoconstituents in ETSL may potentiate its protein denaturation efficacy. Protein denaturation inhibitory activity of ethanol leaf extract of T. stans by bovine serum albumin and egg albumin was also studied earlier (Priyanka Sivasubramanian 2021). Proteases are a class of proteolytic enzymes that break down polypeptide links and degrade the proteins. Proteases are crucial regulators of physiological processes because proteins are important for preserving homeostasis (Agbowuro et al. 2018). Proteinases have the ability to control the inflammatory cytokines by regulating the expression and function of inflammatory mediators such as chemokines, immune cells, and cytokines. These cytokines and macrophages increase in the inflammatory phase of RA produce excessive amount of proteinase, and causes tissue deterioration. Thus, it is essential to control proteinase secretion. Therefore, a considerable degree of protection was provided by proteinase inhibitors (Jyothilakshmi et al. 2017). In the current investigation, ETSL remarkably prevented the secretion of proteinase in a concentration-dependent manner. The phytochemicals such as alkaloids, flavonoids, tannins, and phenolic acids were identified in ETSL could be effective against proteinase secretion(Dharmeshkumar D. Prajapati 2015). The findings of the present investigation were also in agreement with the previously reported work of(Govindappa M 2011). The process of protecting biological membranes, such as lysosomal and erythrocyte membranes, from heat and osmotic-induced lysis is known as membrane stabilization (Anosike et al. 2019). The lysosomal membrane lyses during the inflammatory process and releases active neutrophils, proteases, and bactericidal enzymes that harm the surrounding organelles and tissues. Thus, controlling tissue damage during inflammation requires blocking the release of lysosomal contents by stabilizing the lysosomal membrane (Ghosh et al. 2018). Human red blood cell (RBC) membranes are analogous to lysosomal membranes. Thus, stabilizing an RBC membrane implies stabilizing a lysosomal membrane. Hemolysis of RBC in a hyposaline solution results in the release of internal hemoglobin into the surrounding solution. Hence, the protection or stabilization of the RBC membrane by anti-inflammatory drugs during hyposaline-induced hemolysis is necessary (Daram et al. 2021). In the current investigation, ETSL exhibited the membrane stabilization potential in a concentration-dependent way. The binding of the ETSL to the erythrocyte membranes and the resulting modification of the cell surface charges may be the cause of the membrane stabilization potentials. This could hinder direct contact with aggregating agents or encourage dispersion through the mutual repulsion of similar charges, which are necessary for red blood cell hemolysis. Studies have demonstrated that flavonoids and saponins have a significant stabilizing impact on the lysosomal membrane. Also, tannins and saponins have the capacity to bind cations and stabilize erythrocyte membranes (Anosike et al. 2019). Therefore, the presence of these phytochemicals in T. stans leaf might be responsible for protecting the erythrocytes membranes effectively (Dharmeshkumar D. Prajapati 2015). Paw edema is linked with increased fluid discharge, vascular penetrability, and cellular penetration in the inflammatory region. This indicator is simple to quantify and offers a quick way to assess the anti-arthritic potency of the drug. The decline in paw thickness indicates a reduction in the release of inflammatory mediators and indicates the anti-inflammatory efficacy of the drugs in adjuvant arthritis (Manan et al. 2020). The disease control group experienced a 28-day period of swelling due to increased mast cells, neutrophils, and macrophages (Zheng et al. 2014). Rats in the ETSL (500 mg/kg) treated groups from days 15 to 28 displayed a notable reduction (p< 0.01) in paw diameter in contrast to disease-control rats. Several phenolic and flavonoid compounds such as caffeic acid (Fikry et al. 2019); ferulic acid (Zhu et al. 2020); hesperidin (Adefegha et al. 2020); rosmarinic acid (Jain 2021);β-sitosterol(Zhang et al. 2020), quercetin(El-Said et al. 2022); naringin(Ahmad et al. 2014);verbascoside(Gutiérrez-Rebolledo et al. 2018) and catechin(Tang et al. 2007)were found to decrease the pain severity and inflammatory paw puffiness in adjuvant arthritis. These compounds were identified by LC-MS analysis of ETSL. Hence, the reduction in paw diameter in the group treated with ETSL might be due to the presence of these phytoconstituents. In CFA-induced arthritis, the arthritis score serves as a standard for joint puffiness. The severity of arthritis was monitored visually by the scoring method. The immunosuppressive and anti-inflammatory effects of T. stans can be distinguished by a reduction in the arthritic index (Gautam et al. 2018). In the present investigation, ETSL-treated rats noticed a significant (p<0.05) decrease in the arthritic index and arthritic score as compared to the disease control group and protected against morphological changes by lowering the swelling and redness of the paw. Hesperidin(Adefegha et al. 2020); β-sitosterol(Zhang et al. 2020); catechin(Tang et al. 2007)and rosmarinic(Gautam et al. 2019)acid were reported to decrease arthritis score and arthritic index in CFA arthritis.Identification of these compounds by LC-MS analysis in ETSL might be responsible for the reduction in arthritic index and arthritic score in CFA-induced rats. Rheumatoid cachexia is recognized as a loss of weight and lean body mass in RA (Roubenoff et al. 1994). Previous studies suggested that a decline in the weight of the body is linked to inflammation because it inhibits the gut's ability to absorb nutrients. The overexpression of inflammatory cytokines, such as TNF-α, promotes energy use by improving lipid and protein metabolism and lowering calorie intake through their anorectic action, which causes cachexia. Also, it has been shown that anti-inflammatory drugs can enhance the compromised absorption ability of the intestine (Alamgeer et al. 2017). The results of the current investigation revealed that ETSL 500 mg/kg noticeably (p<0.01) increased the body weight in the treated group due to enhanced absorption of nutrients and inhibiting the level of inflammatory cytokines compared to the disease control group. The decrease in body weight in CFA rats was significantly up-regulated by phytocompoundsferulic acid(Zhu et al. 2020), β-sitosterol(Zhang et al. 2020), verbascoside(Gutiérrez-Rebolledo et al. 2018),rosmarinic(Gautam et al. 2019)and quercetin(El-Said et al. 2022)as reported earlier which were confirmed in our LC-MS analysis might be responsible for increase in body weight. In the current study, the CFA-induced group noticed a decrease in liver weight and an increase in kidney and spleen weight. These findings may be the result of changes in the cell populations in the organs, which are linked to immune function (Phull et al. 2017). The supplementation of ETSL significantly (p <0.01) reversed the change in organ weight in relation to the disease control group. Polyphenols are substances that have the ability to regulate various immune system functions and minimize the severity of autoimmune diseases (Shakoor et al. 2021). Therefore, the identification of different phenolic compounds by LC-MS in ETSL might be responsible for correcting this altered organ weight. A decrease in erythropoietin, bone marrow failure, and damage of erythrocytes causes a decline in Hb and RBC levels in CFA-induced rats, which leads to anemia(Alamgeer et al. 2017). Pathogenic microorganisms may trigger an immunological response in arthritic animals, leading to an increase in platelets, WBCs, and ESR, essential hematological indicators for the diagnosis of inflammatory conditions (Phull et al. 2017). The increased level of inflammatory proteins in the blood due to increased IL-6, TNF-α, and IL-1β concentration also causes an increase in ESR count in the CFA group (Manan et al. 2020). Previous studies have shown that cytokines such as IL-6, TNF-α, and IL-1β control the production of CRP from the liver (Littman et al. 1995). In the present study, administration with ETSL considerably (p<0.05) restored the hematological abnormalities in RA in contrast to the disease control group. The compounds ferulic acid(Zhu et al. 2020), hesperidin (Adefegha et al. 2020)and rosmarinic acid (Gautam et al. 2019)have been reported to increase the Hb and RBC and reduced WBC, platelets, ESR and CRP in CFA-induced arthritic rat. The identification of these compounds using LC-MS study in ETSL might be responsible for restoring hematological abnormalities. Serum biochemical enzyme levels (ALP, ALT, and AST) were measured to determine the role of kidney and liver damage in inflammatory arthritic diseases. The increased ALP, ALT, and AST in the blood of CFA-induced animals suggest abnormal liver function. Furthermore, the liver is the primary organ involved in inflammatory diseases like RA, where changes in the synthesis, release, and catabolism of enzymes are important mediators and indicators of cellular damage caused by oxidative stress (Kumari and Anbarasu 2014). In the present study, treatment with ETSL (500 mg/kg) considerably (p<0.05) reduced the levels of biochemical enzymes compared to CFA-induced rats. The decreased kidney function in disease control rats led to increased blood levels of urea and creatinine(Chanda 2015). Groups under treatment with ETSL (500 mg/kg) marked a reduction in serum urea and creatinine levels. According to (Alamgeer et al. 2017) RF is an auto-antibody that targets the Fc (fragment crystallizable form) of IgG (immunoglobulin G) and creates immunological complexes that help in the development of RA. Administration of rats with ETSL (500 mg/kg) showed a significant (p<0.05) decrease in RF as compared to disease control rats. The changes in the biochemical parameters were reversed to normal state by phenolic compound ferulic acid in CFA-induced rats, as reported previously by (Zhu et al. 2020)which confirmed by LC-MS analysis of ETSL. Previous study cited that administration of CFA in experimental animals activates T-cells, which in turn initiate monocytes and macrophages, resulting in generations of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) in the subchondral bone layer, cartilage, synovial fluid, and cartilage membrane. These cytokines are responsible for bone degradation, joint degeneration, production of auto-antibodies, and cell death (Xiao et al. 2024). Inflammation and autoimmune diseases are largely caused by COX-2, which is involved in the production of pro-inflammatory substances, vasodilation, and cartilage degradation (Shabbir et al. 2016; Xiao et al. 2024). However, IL-10 and IL-4 are the immunoregulatory cytokines. In RA, it minimizes the effects of antigen-presenting cells and protects the tissue integrity of the joints (Lin et al. 2013). In the current investigation, it was found that the disease control group had noticed higher expressions of TNF-α, IL-1β, IL-6, and COX-2 and lower expressions of IL-4 and IL-10. According to (Cheng et al. 2015), inhibiting the synthesis and function of pro-inflammatory mediators and up-regulating the expression of anti-inflammatory mediators is considered an efficient way to treat RA. In the present study, treatment with ETSL (500 mg/kg) remarkably (p<0.05) boosted anti-inflammatory and lowered pro-inflammatory expression as compared to disease control, thereby reducing inflammation, bone loss, and cartilage damage in RA. Different phenolic and flavonoid compounds were reported to inhibit these cytokines. Protocatechuic acid (3,4-Dihydroxybenzoic acid)(Semaming et al. 2015), tryptamine(Agista et al. 2022),p-coumaric acid(Zhu et al. 2018), apigenin(Lee et al. 2007), luteolin(Shi et al. 2015), β-sitosterol(Zhang et al. 2020), hesperetin(Lin et al. 2020), naringin(Ahmad et al. 2014),catechin(Tang et al. 2007), gallic acid (Bai et al. 2021)and ferulic acid(Zhai et al. 2023)inhibited TNF-α, IL-1β, IL-6 and suppress COX-2 expression in carrageenan-induced inflammation in animal model.Caffeic acid(Kim et al. 2023), quercetin(Guan et al. 2021); and kaempferol(Tian et al. 2021)exhibited anti-inflammatory effects through significant suppression of IL-6, IL-1β, and TNF-α in lipopolysaccharide (LPS) stimulated RAW 264.7 cells. In the present investigation, confirmation of these phenolic and flavonoid compounds by LC-MS analysis in ETSL proves its anti-inflammatory activity in CFA rats by inhibiting pro-inflammatory cytokines. In the progression of arthritis, endogenous antioxidant enzymes play a vital role in combating oxidative damage. SOD is thought to be the first line of defense against the production of free radicals and is necessary for the conversion of superoxide radicals into oxygen and hydrogen peroxide, which results in the inactivation of CAT and glutathione peroxidase. As a result of ROS-mediated degradation, SOD activity may have decreased during detoxification of these radicals(El-barbary et al. 2011). According to earlier research, a decrease in CAT activity in RA patients may be due to inactivation of CAT by hydrogen peroxide (Kalpakcioglu and Senel 2008). The increased production of ROS tends to proliferate abundantly during chronic inflammation and consequently causes excessive damage to tissues and is responsible for lipid peroxidation. Reduced membrane fluidity and inactivation of membrane-bound proteins cause lipid peroxidation, which breaks down into harmful malondialdehydes (MDA) (Sahu et al. 2017). Therefore, detoxification of these free radicals can stop cell damage and finally stop lipid peroxidation (Sadiq Umar 2012). GSH, a non-protein sulfhydryl molecule, is regarded as an essential non-enzymatic antioxidant defense system against the production of hydrogen peroxide and organic peroxides. A reduction in GSH level may worsen the clearance of H 2 O 2 and encourage the creation of OH, which raises the free radical load and disturbs homeostasis. Furthermore, the lower content of GSH has been noted in the liver of CFA-induced rats(Comar et al. 2013). In the current investigation, supplementation of experimental rats with ETSL (500 mg/kg) significantly reduced the level of LPO and increased SOD, CAT, and GSH content in the liver and joints of CFA-induced rats as compared to disease-control rats. The reduction in lipid peroxide and increase in level of SOD, CAT and GSH in CFA-induced rat have been reported by several phenolic and flavonoid compounds such as Protocatechuic acid (3,4-Dihydroxybenzoic acid)(Semaming et al. 2015), caffeic acid(Kim et al. 2023), β-sitosterol(Zhang et al. 2020), naringin(Ahmad et al. 2014), p-Coumaric acid(Shen et al. 2019), hesperidin (Adefegha et al. 2020)and rosmarinic acid(Jain 2021). Rutin significantly restored the GSH level and SOD activity in collagen-immunized rats (Sadiq Umar 2012). The identification of these compounds in ETSL by LC-MS study supports the potent antioxidant activity of T. stans leaf . Histopathological analyses of arthritic joints were carried out in the current investigation to support the anti-arthritic effect of ETSL. The specific etiopathogenesis of RA is still unknown, but the research has shown that increased pro-inflammatory cytokine expression and oxidative stress are the main causes of synovial hyperplasia, pannus formation and, the cartilage and bone destruction (Xiao et al. 2024). In the present study, the administration of ETSL (500 mg/kg) noticeably prevented cartilage degradation and bone erosion, as evidenced by a decrease in histological score. This might be due to the prevention of pro-inflammatory cytokines and scavenging of free radicals by ETSL. The compounds such as caffeic acid(Fikry et al. 2019); luteolin(Shi et al. 2015); naringin(Ahmad et al. 2014)and rutin(Sadiq Umar 2012)were known to alleviate cartilage and bone destruction, synovial hyperplasia, decreased joint inflammation in CFA-induced rats. Hence, the identification of these phytoconstituents by LC-MS analysis of ETSL might be responsible for protective effects against cartilage and bone destruction. The advancement of computational methodologies offers a rapid and reliable process for screening the enormous number of these natural compounds. To identify phytochemicals potentially responsible for the therapeutic effects of ETSL on RA, various compounds were subjected to a docking study to interact with TNF-α, IL-1β, IL-6, and COX-2 using computer-assisted modeling techniques.During the initiation of arthritis, numerous inflammatory mediators such as TNF-α, IL-6, and IL-1β play pivotal roles(Aldossari et al. 2023). COX-2 plays a significant role in the synthesis of prostaglandins, which serve as pain mediators and contribute to the inflammatory process(Simon 1999). Hence, the inhibition of TNF-α, IL-1β, IL-6, and COX-2 proteins with PDB IDs 2AZ5, 4G6J, 1ALU, and 1PXX, respectively, is anticipated to constitute a potential mechanism for anti-arthritis activity. In the present study, indomethacin was selected as the standard drug due to its capacity to inhibit the production of IL-6, IL-1β, and COX-2(Bour et al. 2000; Tonby et al. 2016).After evaluating individual docking scores, it was noticed that verbascoside emerged as the most promising candidate against all four targets, followed by naringin and rutin. In the drug development process, hydrogen bonding plays a crucial role in determining drug specificity, as it offers essential insights into protein-ligand interactions(Aldossari et al. 2023). The highest docking score achieved by verbascoside against COX-2 can be elucidated by its hydrogen bond interactions with amino acids HIS133, ASN39, GLU465, GLN461, CYS47, ALA156, and CYS36, as well as its hydrophobic interactions with MET48 and LEU152. In IL-1β, verbascoside exhibited a better docking score due to its hydrogen bond interactions with amino acids LYS103, VAL155, GLU153, GLN166, and ALA85 and electrostatic interactions with GLU165 and hydrophobic interactions with ALA83, ALA84, PRO40, ALA83, ALA85, and LYS103.In TNF-α, verbascoside demonstrated the highest docking score primarily due to its hydrogen bond interaction with TYR151 and hydrophobic interactions with TYR59 and LEU157.Likewise, verbascoside achieved a superior docking score against IL-6, which is attributed to its hydrogen bond interaction with GLU23 and hydrophobic interaction with LYS27.This aligns with punicalagin, a polyphenolic compound that has shown substantial binding affinities to identical protein targets via hydrogen bonding and hydrophobic interactions(Jghef et al. 2023).Furthermore, verbascoside has been reported to exhibit potent anti-inflammatory activities by suppressing the expression of COX-2 (Pongkitwitoon et al. 2024). A decrease in TNF-α, IL-1β, and IL-6 levels also has been observed in rats administered with verbascoside(Rossi et al. 2023). Therefore, based on molecular docking analysis, it is conceivable to consider that the compound verbascoside could be a novel potential candidate for the management of RA. Conclusion The anti-arthritic potential of ethanol extract of T. stans leaf was evaluated by in vitro, in vivo and in silico approaches. ETSL exhibited significant in vitro antioxidant properties as well as in vitro anti-arthritic potential in a dose-dependent manner. Further, the inhibition of pro-inflammatory cytokines and increase in expression of anti-inflammatory cytokines support its in vivo anti-arthritic efficacy. The molecular docking analysis identified verbascoside as having the highest docking score against COX-2 and IL-1β among the various phenolic, flavonoid, and anti-inflammatory compounds detected through LC-MS and GC-MS analyses. This finding provides robust evidence for the potential clinical application of Tecoma stans leaf in managing RA, and affirming its traditional medicinal efficacy. Abbreviations ALP: Alkaline phosphatase; ALT: Alanine amino transferase; AST: Aspartate aminotransferase; CAT: Catalase; CFA: Complete Freund's Adjuvant; CMC-Na: Carboxy methyl cellulose-sodium; COX-2: Cyclooxygenase-2;CRP: C-reactive protein; DMARDs: Disease-modifying anti-rheumatic drugs; DPPH: 2, 2-diphenyl-1-picrylhydrazyl; EDTA: Ethylenediaminetetraacetic acid; ELISA: Enzyme-linked immunosorbent assay; ESR: Erythrocyte sedimentation rate; ETSL: Ethanol extract of T. stans leaf; Fc: Fragment crystallizable form; GC-MS: Gas Chromatography-Mass Spectroscopy; GSH: Reduced glutathione; Hb: Haemoglobin; HRBC: Human red blood cells; IgG: Immunoglobulin G; IL-10: Interleukin-10;IL-1β: Interleukin-1-beta; IL-4: Interleukin-4;IL-6: Interleukin-6; LC-MS: Liquid Chromatography-Mass Spectrometry; LPO: Lipid peroxidation; LPS: Lipopolysaccharide; MDA: Malondialdehydes; NIST: National Institute of Standards and Technology; NSAIDs: Non-steroidal anti-inflammatory drugs; RA: Rheumatoid arthritis ; RBC: Red blood cell; RBC: Red blood cells; RF: Rheumatoid factor; ROS: Reactive oxygen species; RT: Retention time; SD: Standard deviation; SOD: Superoxide dismutase; TNF-α: Tumor necrosis factor-alpha;WBC: White blood cells Declarations Funding: The research was funded by the DBT Builder project with order no (BT/INF/22/SP45078/2022). 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Inflammopharmacology 28:463-473 doi:10.1007/s10787-019-00642-0 Tables Table 1 Identification of compounds from ethanol extract of T. stans leaf by GC-MS analysis Sl.no RT Compound name M.F M.W (g/mol) Nature of compound Area (%) 1 11.15 Pentadecane C 15 H 32 212.41 Alkane 1.84 2 16.62 Heptadecane, 2,6,10,15-tetramethyl- C 21 H 44 296.6 Alkane 2.52 3 19.14 1-Hexadecanol C 16 H 34 O 242.44 Alcohol 2.23 4 24.04 Cetene C 16 H 32 224.42 Alkene 2.07 5 27.33 Dimethyl palmitamine C 18 H 39 N 269.5 Amine 3.62 6 29.59 3-Eicosene, (E)- C 20 H 40 280.5 Alkene 2.18 7 31.80 Methyl 9-cis,11-trans-octadecadienoate C 19 H 34 O 2 294.5 Ester 2.20 8 31.97 cis-13-Octadecenoic acid, methyl ester C 19 H 36 O 2 296.5 Ester 2.49 9 37.23 13-Docosenamide, (Z)- C 22 H 43 NO 337.6 Carboxamide 18.55 10 43.91 9-Octadecenamide, (Z)- C 18 H 35 NO 281.5 Carboxamide 7.03 11 49.72 7,8-Epoxylanostan-11-ol, 3-acetoxy- C 32 H 54 O 4 502.8 Epoxide 2.03 12 49.80 Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester C 35 H 68 O 5 568.9 Ester 2.39 13 49.90 Eicosanoic acid, 9-octadecenyl ester, (Z)- C 38 H 74 O 2 563.00 Ester 1.82 14 50.04 Ethyl iso-allocholate C 26 H 44 O 5 436.6 Ester 1.60 15 50.25 9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester C 21 H 40 O 4 356.5 Ester 1.59 16 50.31 9-Octadecenoic acid, 1,2,3-propanetriyl ester, (E,E,E)- C 57 H 104 O 6 885.4 Ester 0.96 17 50.35 Oleic acid, eicosyl ester C 38 H 74 O 2 563 Ester 0.78 18 50.46 9-Octadecenoic acid (Z)-, 2-hydroxy-3-[(1-oxohexadecyl)oxy]propyl ester C 37 H 70 O 5 594.9 Ester 1.91 RT: Retention time; M.F: Molecular formula; M.W: Molecular weight Table 2 Identification of compounds from ethanol extract of T. stans leaf by LC-MS analysis Sl.no Peak no. RT [M+H] + ( m/z ) [M+H] - ( m/z ) Exact mass M.F Proposed metabolite Chemical nature 1 59 25.89 160.2 - 160.216 C 10 H 12 N 2 Tryptamine Amino acid 2 18 7.13 - 161.1 161.20 C 10 H 11 NO Boschniakine Alkaloid 3 13 5.56 - 163.0 164.16 C 9 H 8 O 3 p-Coumaric acid Phenolic acid 4 22 7.69 169.3 - 170.12 C 7 H 6 O 5 Gallic acid Phenolic acid 5 66 29.0 181.3 180.16 C 9 H 8 O 4 Caffeic acid Phenolic acid 6 44 13.52 195.3 - 194.18 C 10 H 10 O 4 Ferulic acid Phenolic acid 7 49 16.79 203.3 - 204.229 C 11 H 12 N 2 O 2 Tryptophan Amino acid 8 16 6.31 288.2 - 270.24 C 15 H 10 O 5 Apigenin Flavonoid 9 19 7.14 287.2 - 286.24 C 15 H 10 O 6 Kaempferol Flavonoid 10 17 6.65 285.2 - 286.24 C 15 H 10 O 6 Luteolin Phenolic acid 11 61 26.49 291.4 - 290.27 C 15 H 14 O 6 (-)-Catechin Phenolic acid 12 23 8.10 302.2 - 302.23 C 15 H 10 O 7 Quercetin Flavonoid 13 17 6.65 - 301.2 302.28 C 16 H 14 O 6 Hesperetin Phenolic acid 14 24 8.38 - 353.3 360.3 C 18 H 16 O 8 Rosmarinic acid Phenolic acid 15 47 16.13 353.3 - 354.31 C 16 H 18 O 9 Chlorogenic acid Phenolic acid 16 41 12.66 - 415.3 414.7 C 29 H 50 O Sitosterols Phenolic acid 17 28 9.76 447.4 - 448.4 C 21 H 20 O 11 Quercitrin Flavonoid 18 32 10.95 580.5 - 580.54 C 27 H 32 O 14 Naringin Phenolic acid 19 26 8.93 - 617.5 610.5 C 27 H 30 O 16 Rutin Flavonoid 20 34 11.29 624.5 - 624.6 C 29 H 36 O 15 Verbascoside Flavonoid RT: Retention time; M.F: Molecular formula Table 3 Effect of ethanol extract of T. stans leaf on haematological and biochemical parameters in CFA-induced arthritis in rat Parameters Treatment groups NC CFA (0.1 ml) IND (10 mg/kg) ETSL (250 mg/kg) ETSL (500 mg/kg) RBC (X10 6 /mm 3 ) 6.31±0.88 2.78±1.48## 5.24±1.56** 3.78±1.52 ns 4.67±0.94* WBC (X10 3 /mm 3 ) 5.02±1.38 12.13±0.76## 6.96±1.42*** 10.47±1.02* 8.06±1.65* Platelets (X 10 3 /µl) 948.65±7.16 1323.92±1.99## 1014.86±5.50** 1191.77±4.14* 1062.07±5.21** AST (IU/L) 23.28±2.30 52.30±3.16### 26.97±2.36*** 39.30±2.39** 35.17±2.59*** ALT (IU/L) 109.14±5.47 152.82±7.05## 119.16±3.87*** 130.27±3.95** 124.92±3.17** ALP (IU/L) 154.40±2.62 299.07±5.60### 181.47±6.22*** 234.88±9.33** 203.06±6.74*** UREA (mg/dL) 0.51±0.55 1.03±0.47## 0.61±0.35*** 0.77±0.88** 0.69±0.46** CREAT (mg/dL) 19.25±1.52 48.32±4.66### 26.39±2.56*** 36.57±2.91** 31.07±2.92*** RBC; Red blood cells, WBC; White blood cell, AST; Aspartate aminotransferase, ALT; Alanine transaminase, ALP; Alkaline phosphatase; CFREAT-Creatinine. Values are expressed as mean ± SD (n=6). All data were expressed as mean ± standard deviation (SD), (n= 6). The statistical significance of difference between various groups was tested by one-way ANOVA followed by Tukey’s multiple comparison tests. Comparisons were made between NC vs CFA, and CFA vs IND (10 mg/kg), ETSL (250 mg/kg) and ETSL (500 mg/kg). The level of significant difference was checked between NC vs CFA at ## P˂ 0.01 and ### P˂ 0.001 and between CFA and IND, ETSL (250 mg/kg) and ETSL (500 mg/kg) at *P˂0.05, **P˂0.01 and ***P˂0.001. Table 4 In silico docking studies of 2AZ5 (TNF-α), 4G6J (IL-1β), 1ALU (IL-6) and 1PXX (COX-2) by various ligands. Protein Ligand/inhibitor Binding affinity (kcal/mol) Interacting residues Hydrogen-bond interacting active binding site residues Hydrophobic Interaction 2AZ5 (TNF-α) Apigenin -6.5 VAL91, LEU93, GLN125, ARG82 ARG82 VAL91, LEU93, GLN125 Kaempferol -6.3 GLY121, LEU120, SER95, ALA96, LYS98 GLY121 LEU120, SER95, ALA96, LYS98 (-)-Catechin -6.4 GLY121, LEU120, SER95, ALA96, TYR119, PRO117 SER95, GLY121 LEU120, ALA96, TYR119, PRO117 Hesperetin -6.7 LEU55, ALA96, GLY121, LEU120, TYR119, PRO117 GLY121, PRO117 LEU55, ALA96, LEU120, TYR119 Rosmarinic acid -6.2 VAL91, ARG82, LEU93, GLN125 ARG82, LEU93 VAL91, GLN125 Chlorogenic acid -6.4 LEU57, TYR59, HIS15, TYR151 TYR151, TYR59, HIS15 LEU57 Sitosterols -7 TYR151, TYR59, LEU57, ILE155, LEU157, VAL123 TYR151, TYR59, LEU57, ILE155, LEU157, VAL123 Quercitrin -6.4 LYS98, PRO117, TYR119, LEU120, GLY121, LEU55 PRO117, TYR119, LEU55, GLY121 LYS98, LEU120 Naringin -7 LYS11, GLN61, TYR59, TYR151 GLN61, TYR151 LYS11, TYR59 Rutin -6.7 TYR119, PRO117, ILE118, LEU120, GLY121, LEU55, GLN125, GLY54, GLU53, ILE97, LYS98 GLU53, PRO117, TYR119, LYS98, ILE97, GLN125, GLY121 TYR119, ILE118, LEU120, LEU55, GLY54 Verbascoside -7 TYR151, TYR59, LEU157 TYR151 TYR59, LEU157 Indomethacin -8.1 VAL123, LEU157, TYR59 - VAL123, LEU157, TYR59 4G6J (IL-1β) Kaempferol -7.6 TRP47, GLU46, PHE98, THR97, PHE96, ALA61, ASP62, VAL3 TRP47, PHE98 GLU46, THR97, PHE96, ALA61, ASP62, VAL3 (-)-Catechin -7.1 ALA61, ASP62, TRP47, GLU46, PHE96, THR97, PHE98, PRO100 PHE96, PHE98, ASP62 ALA61, TRP47, GLU46, THR97, PRO100 Quercetin -7.5 ASP62, ALA61, TRP47, GLU46, THR97, PHE98, PRO100, LEU4 PHE98, LEU4, TRP47, ASP62 ALA61, GLU46, THR97, PRO100 Hesperetin -7.5 TRY95, VAL93 TRY95, VAL93 Rosmarinic acid -6.5 TYR87, GLN39, LYS43, ALA85, LYS103, ALA84, ASP41, PRO40, GLU165, ALA83, GLN166 ASP41, GLN39, TYR87, ALA85 LYS43, LYS103, ALA84, PRO40, GLU165, ALA83, GLN166 Chlorogenic acid -6.9 GLN166, GLU165, LYS103, ALA85, ALA84, ALA83, PRO40, ASP41, GLN38 GLN166 GLU165, LYS103, ALA85, ALA84, ALA83, PRO40, ASP41, GLN38 Sitosterols -7.3 TYR87, GLN39, PRO41, VAL93, LEU113 TYR87, GLN39 PRO41, VAL93, LEU113 Quercitrin -7.4 LYS39, GLN42, ASP41, GLN110, PRO41, VAL93, LEU113, GLU153 LYS39, GLN42, GLN110, GLU153 ASP41, PRO41, VAL93, LEU113 Naringin -7.7 VAL155, GLU153, ALA173, ALA42, PRO172, GLN110, PRO40, ALA85 GLN110, ALA85, ALA42, VAL155, GLU153 ALA173, PRO172, PRO40 Rutin -7.7 PRO172, VAL155, GLU153, GLU165, PRO41, LEU113, ASP41, VAL93, GLN110, GLN42, LYS39 GLU165, GLN42, LYS39, GLU153, GLN110 PRO172, VAL155, PRO41, LEU113, ASP41, VAL93 Verbascoside -7.4 ALA85, LYS103, ALA84, ALA83, GLN166, GLU165, PRO40, ASP41, ALA173, VAL155, GLU153 LYS103, VAL155, GLU153, GLN166, ALA85 ALA84, ALA83, GLU165, PRO40, ASP41, ALA173 Indomethacin -7.1 LYS39, GLN42, PRO172, ALA42, GLN39 LYS39, GLN42, GLN39 PRO172, ALA42 1ALU (IL-6) Hesperetin -6 LEU19, LYS27 - LEU19, LYS27 Quercitrin -6.6 LEU19, GLU23, ASP26, LYS27, ARG30 - LEU19, GLU23, ASP26, LYS27, ARG30 Naringin -6.4 LEU19, LYS27, GLU23 - LEU19, LYS27, GLU23 Rutin -6.2 ARG30, LYS27, LEU19, GLU23, ASP26 - ARG30, LYS27, LEU19, GLU23, ASP26 Verbascoside -6.2 GLU23, LEU19, LYS27, ARG30 GLU23 LEU19, LYS27, ARG30 Indomethacin -6.4 ALA114, TYR31 ALA114, TYR31 1PXX (COX-2) Tryptamine -6.3 LEU390, ALA199, ALA202, GLN203 GLN203, ALA199 LEU390, ALA202 p-Coumaric acid -6.7 ASN382, HIS388, TRP387, ALA202 ASN382 HIS388, TRP387, ALA202 Gallic acid -7.1 ARG469, ILE124, ALA151, ASP125, THR149, THR129 ALA151, THR149 ARG469, ILE124, ASP125, THR129 Caffeic acid -6.9 TRP387, HIS386, TYR148, TYR385, ASN382, THR206, HIS207 TYR148, ASN382, THR206, TRP387 HIS386, TYR385, HIS207 Ferulic acid -6.6 HIS388, TRP387, ASN382, ALA202, ALA199 ASN382, ALA199 HIS388, TRP387, ALA202 Tryptophan -7 ARG120, TRP1, VAL523, GLY526 GLY526 ARG120, VAL523 Apigenin -9.1 ARG44, CYS47, ARG469, LYS468, GLU465, PRO153, LEU152 CYS47, GLU465 ARG44, ARG469, LYS468, PRO153, LEU152 Kaempferol -8.9 LYS468, ARG469, ARG44, GLN42, CYS47, LEU152, PRO153 GLN42, LYS468 ARG469, ARG44, CYS47, LEU152, PRO153 (-)-Catechin -8.9 ASN39, CYS37, ASN34, ALA156, PRO154, PRO153 CYS37, ASN39, ASN34 ALA156, PRO154, PRO153 Hesperetin -9.1 ARG469, ARG44, CYS47, CYS36, PRO153 CYS47 ARG469, ARG44, CYS36, PRO153 Rosmarinic acid -9.1 ARG44, GLY45, CYS47, CYS36, ASN39, PRO153, LEU152, CYS37, ALA156, VAL155 ARG44, GLY45, CYS47, ASN39 CYS36, PRO153, LEU152, CYS37, ALA156, VAL155 Chlorogenic acid -9.2 ARG44, GLY45, CYS41, ASN34, ALA156, GLU465, PRO153 ASN34, GLY45, ARG44, GLU465 CYS41, ALA156, PRO153 Sitosterols -8.1 ARG469, ARG44, TYR122, SER471, LYS79, LEU80, LYS83, LEU82 ARG469 ARG44, TYR122, SER471, LYS79, LEU80, LYS83, LEU82 Quercitrin -9 HIS386, PHE210, LYS211, THR212, ARG222, GLN289, GLU290, VAL291, ILE274 GLN289, ARG222, THR212, LYS211 HIS386, PHE210, GLU290, VAL291, ILE274 Naringin -10.2 SER1518, GLU46, CYS47, CYS36, ASN39, GLN461, ALA156, PRO154, GLY135, GLN1327, TYR130 GLU46, PRO154, CYS47, ASN39, GLN461, CYS36 SER1518, ALA156, GLY135, GLN1327, TYR130 Rutin -10.3 TYR130, GLY45, CYS41, CYS47, MET48, CYS36, SER49, ALA156, TYR136, HIS133, GLN1327 HIS133, SER49, CYS47, CYS36 TYR130, GLY45, CYS41, MET48, ALA156, TYR136, GLN1327 Verbascoside -10.4 MET48, CYS47, CYS36, ASN39, GLU465, GLN461, LEU152, ALA156, HIS133, GLN1327 HIS133, ASN39, GLU465, GLN461, CYS47, ALA156 MET48, CYS36, LEU152, GLN1327 Indomethacin -8.7 CYS1036, CYS1047, PRO1153, TYR1130 - CYS1036, CYS1047, PRO1153, TYR1130 Supplementary Files GRAPHICALABSTRACT.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4224044","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289367601,"identity":"5b411f9e-657c-44b0-a627-9be70a3ad637","order_by":0,"name":"Chandan Das","email":"","orcid":"","institution":"Siksha O Anusandhan University School of Pharmaceutical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chandan","middleName":"","lastName":"Das","suffix":""},{"id":289367602,"identity":"80b83a6e-f2b0-4eea-afb1-2718caa2c737","order_by":1,"name":"Pritam Kar","email":"","orcid":"","institution":"Siksha O Anusandhan University School of Pharmaceutical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Pritam","middleName":"","lastName":"Kar","suffix":""},{"id":289367603,"identity":"f563efdd-23a6-41eb-bd8b-2217c417bad9","order_by":2,"name":"Priyanka Dash","email":"","orcid":"","institution":"Siksha O Anusandhan University School of Pharmaceutical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Priyanka","middleName":"","lastName":"Dash","suffix":""},{"id":289367604,"identity":"4e9acdd8-41ef-4ad8-b6bb-b3c7b5f33b0f","order_by":3,"name":"Deepak Pradhan","email":"","orcid":"","institution":"Siksha O Anusandhan University School of Pharmaceutical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Deepak","middleName":"","lastName":"Pradhan","suffix":""},{"id":289367605,"identity":"28e93ee2-e51d-442f-a98f-777e789a7608","order_by":4,"name":"Vineet Kumar Rai","email":"","orcid":"","institution":"Siksha O Anusandhan University School of Pharmaceutical 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16:19:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4224044/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4224044/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54557032,"identity":"976755f9-1b79-4bef-aae7-156ba4574075","added_by":"auto","created_at":"2024-04-12 08:50:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":45355,"visible":true,"origin":"","legend":"\u003cp\u003eGC-MS chromatogram of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/9149bd30fd8eaea3bf441304.jpg"},{"id":54557044,"identity":"5561f6e3-0cb1-4518-a22e-6847765a3566","added_by":"auto","created_at":"2024-04-12 08:50:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":58438,"visible":true,"origin":"","legend":"\u003cp\u003eLC-MS chromatogram of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf at positive mode of ionization\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/3da0accb8bbeaac027339c83.jpg"},{"id":54557663,"identity":"8102254b-414e-4b18-a239-d19b533a280c","added_by":"auto","created_at":"2024-04-12 08:58:56","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42425,"visible":true,"origin":"","legend":"\u003cp\u003eLC-MS chromatogram of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf at negative mode of ionization\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/15d63c129175842bb15846e9.jpg"},{"id":54557038,"identity":"bfde9cbe-9ad0-41fa-9086-7966e7fb43fd","added_by":"auto","created_at":"2024-04-12 08:50:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74714,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003e leaf(ETSL) and ascorbic acid (ASC) on reducing power assay (A), DDPH (B), nitric oxide (C) and ferric reducing assay (D).\u0026nbsp; IC\u003csub\u003e50\u003c/sub\u003e value is depicted in (E). All data are taken in triplicate. The data were calculated as mean ± standard deviation (SD).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/97a051baca6af2a2b76a8461.jpg"},{"id":54557662,"identity":"78eda7c5-7b9c-4319-a0c3-1bac9c1f6775","added_by":"auto","created_at":"2024-04-12 08:58:56","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74714,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003e leaf (ETSL) and indomethacin (IND) on protein denaturation (A), membrane stabilization (B) and proteinase inhibition (C). IC\u003csub\u003e50\u003c/sub\u003e value is depicted in (D). All data are taken in triplicate. The data were calculated as mean ± standard deviation (SD).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/dc3d9587374d084a79804a5a.jpg"},{"id":54557037,"identity":"a33beffc-d5dc-4b4f-998d-657e99dffe78","added_by":"auto","created_at":"2024-04-12 08:50:56","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":244642,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf (ETSL 250 and 500 mg/kg) and indomethacin (IND 10 mg/kg) on paw diameter (A), arthritic index (B), arthritic score (C) and body weight (D) in CFA-induced rats.All data were expressed as mean ± standard deviation (SD), (n= 6). The statistical significance of difference between the groups was tested by two-way ANOVA followed by Bonferroni’s post hoc test. Comparisons were made between the groups: NC vs CFA (0.1 ml); CFA (0.1 ml) vsIND (10mg/kg), ETSL 250 and 500 mg/kg; IND (10mg/kg) vs ETSL 250 and 500 mg/kg; ETSL 250 mg/kg vs ETSL 500 mg/kg. The level of significant was checked at *P˂ 0.05, **P˂ 0.01 and ***P˂ 0.001.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/75f644c4624f115518bd9821.jpg"},{"id":54557036,"identity":"bba06480-cda4-43bd-aca0-2ddafb4b5efe","added_by":"auto","created_at":"2024-04-12 08:50:56","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":70241,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf (ETSL 250 and 500 mg/kg) and indomethacin (IND 10 mg/kg) on liver weight (A), kidney weight (B) and spleen weight (C) in CFA-induced rats. All data were expressed as mean ± standard deviation (SD), (n= 6). The statistical significance of difference between various groups was tested by one-way ANOVA followed by Tukey’s multiple comparison tests. Comparisons were made between the groups: NC vs CFA (0.1 ml); CFA (0.1 ml) vs IND (10mg/kg), ETSL 250 and 500 mg/kg; IND (10mg/kg) vs ETSL 250 and 500 mg/kg; ETSL 250 mg/kg vs ETSL 500 mg/kg. The level of significant was checked at *P˂ 0.05, **P˂ 0.01 and ***P˂ 0.001.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/9ae0abd94fc12a9da37f0c78.jpg"},{"id":54557047,"identity":"a13686d2-7960-4873-872e-4c7ee1c3b101","added_by":"auto","created_at":"2024-04-12 08:51:00","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":81824,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf (ETSL 250 and 500 mg/kg) and indomethacin (IND 10 mg/kg) on Hb (A), ESR (B), CRP (C) and RF (D) in CFA-induced rats. All data were expressed as mean ± standard deviation (SD), (n= 6). The statistical significance of difference between various groups was tested by one-way ANOVA followed by Tukey’s multiple comparison tests. Comparisons were made between the groups: \u003cbr\u003e\nNC vs CFA (0.1 ml); CFA (0.1 ml) vs IND (10mg/kg), ETSL 250 and 500 mg/kg; IND (10mg/kg) vs ETSL 250 and 500 mg/kg; ETSL 250 mg/kg vs ETSL 500 mg/kg. The level of significant was checked at *P˂ 0.05, **P˂ 0.01 and ***P˂ 0.001.\u003c/p\u003e\n\u003cp\u003eHb; Hemoglobin, ESR; Erythrocyte sedimentation rate, CRP; C-reactive protein, RF; Rheumatoid factor.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/e94c6b532d7fb5c771fff234.jpg"},{"id":54557665,"identity":"eb0e2c10-781d-48b5-a508-9fa27c8f64c4","added_by":"auto","created_at":"2024-04-12 08:58:58","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":101254,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ethanol extract of \u003cem\u003eT. stans \u003c/em\u003eleaf (ETSL 250 and 500 mg/kg) and indomethacin (IND 10 mg/kg) on pro-inflammatory cytokines TNF-α (A); IL-β (B), IL-6 (C), inflammatory mediator COX-2 (D), and anti-inflammatory cytokines IL-4 (E), and IL-10 (F) in CFA-induced rats. All data were expressed as mean ± standard deviation (SD), (n=6). The statistical significance of difference between various groups was tested by one-way ANOVA followed by Tukey’s multiple comparison tests. Comparisons were made between the groups: NC vs CFA (0.1 ml); CFA (0.1 ml) vs IND (10mg/kg), ETSL 250 and 500 mg/kg; IND (10mg/kg) vs ETSL 250 and 500 mg/kg; ETSL 250 mg/kg vs ETSL 500 mg/kg. The level of significant was checked at *P˂ 0.05, **P˂ 0.01 and ***P˂ 0.001.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/6ec1399ba0f7be115301082b.jpg"},{"id":54557042,"identity":"e34565ac-f1de-4439-a7cc-50dae9f19984","added_by":"auto","created_at":"2024-04-12 08:50:57","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":91764,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf (ETSL 250 and 500 mg/kg) and indomethacin (IND 10 mg/kg) on in vivo antioxidant enzymes SOD (A), Catalase (B), LPO (C) and GSH (D) in joint of CFA-induced rats. All data were expressed as mean ± standard deviation (SD), (n= 6). The statistical significance of difference between various groups was tested by one-way ANOVA followed by Tukey’s multiple comparison tests. Comparisons were made between the groups: NC vs CFA (0.1 ml); CFA (0.1 ml) vs IND (10mg/kg), ETSL 250 and 500 mg/kg; IND (10mg/kg) vs ETSL 250 and 500 mg/kg; ETSL 250 mg/kgvs ETSL 500 mg/kg. The level of significant was checked at *P˂ 0.05, **P˂ 0.01 and ***P˂ 0.001.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/5fb13970ff56b9ba3b35b528.jpg"},{"id":54557664,"identity":"c92ae3f1-def9-4930-9a4e-852cbff9cb9e","added_by":"auto","created_at":"2024-04-12 08:58:57","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":76514,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf (ETSL 250 and 500 mg/kg) and indomethacin (IND 10 mg/kg) on in vivo antioxidant enzymes SOD (A), Catalase (B), LPO (C) and GSH (D) in liver of CFA-induced rats. All data were expressed as mean ± standard deviation (SD), (n= 6). The statistical significance of difference between various groups was tested by one-way ANOVA followed by Tukey’s multiple comparison tests. Comparisons were made between the groups: NC vs CFA(0.1 ml); CFA (0.1 ml) vs IND (10mg/kg), ETSL 250 and 500 mg/kg; IND(10mg/kg) vs ETSL 250 and 500 mg/kg; ETSL 250 mg/kgvs ETSL 500 mg/kg. The level of significant was checked at *P˂ 0.05, **P˂ 0.01 and ***P˂ 0.001.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/0636e373c844d8380eb2b125.jpg"},{"id":54557046,"identity":"6ce24870-42c7-4a7d-86cc-0d5532799fa4","added_by":"auto","created_at":"2024-04-12 08:50:59","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":97991,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopical examination of (A) NC group showed normal paw without any sign of inflammation; (B) CFA (0.1 ml/kg) induced marked with greater inflammation; (C) IND (10 mg/kg) treated group mark with reduction in paw diameter and redness; (D) ETSL 250 mg/kg treated group mark with moderate decrease in paw diameter; (E) ETSL 500 mg/kg noticed with greater reduction in paw diameter. Histological sections (400X) of tarsotibial joints of right hind paw of (A) NC group showed normal joint space, articular cartilage, bone structure and absence of pannus formation and synovial hyperplasia; (B) CFA-induced arthritic group marked with reduce in joint space, bone and cartilage erosion, pannus formation and synovial hyperplasia; (C) IND (10 mg/kg) revealed increase in joint space, decrease in bone and cartilage erosion, synovial hyperplasia; (D) ETSL (250 mg/kg) showed moderate decrease in bone and cartilage erosion, pannus formation, joint space than ETSL 500 mg/kg (E).X-ray radiographic image of proximal interphalangeal joints of right hind pawof (A)NC rat do not show any inflammation; (B) CFA (0.1 ml) marked with swelling of soft tissue and degenerative changes; (C) IND (10 mg/kg) showed decrease in swelling and degenerative changes; (D) ETSL 250 mg/kg showed comparatively mild swelling in soft tissue and degenerative changes than ETSL 500 mg/kg (E).Soft tissue swelling (red arrow) and degenerative changes (yellow arrow).\u003c/p\u003e\n\u003cp\u003eAbbreviations:\u003c/p\u003e\n\u003cp\u003eB: Bone; C: Cartilage; BE: Bone erosion; CE: Cartilage erosion; JS: Joint space; SH: Synovial hyperplasia; SM: Synovial membrane.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/4e32a12ab5503580fd68c9a2.jpg"},{"id":54557034,"identity":"b2ac117d-5e7a-4c53-9dba-c2346c8f1a26","added_by":"auto","created_at":"2024-04-12 08:50:56","extension":"jpg","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":38196,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf (ETSL 250 and 500 mg/kg) and indomethacin (IND 10 mg/kg) on histological score in CFA-induced rats. All data were expressed as mean ± standard deviation (SD), (n= 6). The statistical significance of difference between various groups was tested by one-way ANOVA followed by Tukey’s multiple comparison tests. Comparisons were made between the groups: CFA (0.1 ml) vs IND (10mg/kg), ETSL 250 and 500 mg/kg; IND (10mg/kg) vs ETSL 250 and 500 mg/kg; ETSL 250 mg/kg vs ETSL 500 mg/kg. The level of significant was checked at *P˂ 0.05, **P˂ 0.01 and ***P˂ 0.001.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/b9e2cc452ec2c82e966ad2c4.jpg"},{"id":54557043,"identity":"0911b23f-1830-40c9-9427-4ad87207ed43","added_by":"auto","created_at":"2024-04-12 08:50:58","extension":"jpg","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":51260,"visible":true,"origin":"","legend":"\u003cp\u003eVisualization of 3D models of docked complexes depicting interactions of verbascoside of ethanol extract of \u003cem\u003eT. stans \u003c/em\u003eleaf with (a) TNF-α protein (PDB ID: 2AZ5), (b) IL-1β (PDB ID: 4G6J), (c) IL-6 (PDB ID: 1ALU), and (d)COX-2 (PDB ID: 1PXX). The primary interactive residue of the corresponding compound was illustrated at the TNF-α, IL-1β, IL-6, and COX-2 binding pockets. Hydrogen bonds were represented by green dashed lines, while hydrophobic interactions were depicted by pink dashed lines.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/a3bc84ebcd0715fd2c72b5bc.jpg"},{"id":59334347,"identity":"7dd26ba3-db07-4f7e-9d86-0e11df4c9fda","added_by":"auto","created_at":"2024-06-29 20:26:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2513072,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/206f866a-35bf-4aab-8332-f1656df5ab65.pdf"},{"id":54557040,"identity":"5c293d13-81d4-484c-bd24-fe13a6f5693d","added_by":"auto","created_at":"2024-04-12 08:50:57","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":329808,"visible":true,"origin":"","legend":"","description":"","filename":"GRAPHICALABSTRACT.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4224044/v1/4932bf69bdca048f208185f7.jpg"}],"financialInterests":"","formattedTitle":"Protective effect of Tecomastans (L.) Juss.exKunth in CFA-induced arthritic rat model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRheumatoid arthritis (RA), an autoimmune disease, causes persistent inflammation of the synovium inside joints. This inflammation affects the lining of the joints and may affect the ligaments and bones within the joints, leading to stiffness, discomfort, and degeneration of the articular cartilage(Jain et al. 2023). The risk of developing RA between 30 and 50 years of age is 2 to 3 times higher for women than men (Shamlan et al. 2021). Compared to the global prevalence of 0.46%, India\u0026apos;s prevalence of RA is estimated at 0.7% (Bagepally et al. 2023). As a result of arthritis, cartilage, which generally protects joints, breaks down, causing synovial fluid to accumulate in the joints and causing inflammation. Pro-inflammatory modulator stimulates the production of inflammatory cells in the synovial membrane, resulting in bone damage(Shamlan et al. 2021). The inflammatory response associated with arthritis is accompanied by hyperplasia of synovial cells. Cartilage damage and joint alkalosishave been involved in the pathology of disease progression (Jain et al. 2023).\u003c/p\u003e\n\u003cp\u003eThe cause of joint damage has been found to be increased levels of pro-inflammatory cytokinessuch as tumor necrosis factor-alpha(TNF-\u0026alpha;), interleukin-1-beta (IL-1\u0026beta;), interleukin-6 (IL-6), metabolic enzymes, and decreased levels of anti-inflammatory cytokines; interleukin-4(IL-4) and interleukin-10(IL-10) (Jain et al. 2023). According to recent research, pro-inflammatory mediators cyclooxygenase-2 (COX-2), an inducible enzyme secreted from fibroblasts, macrophages, leukocytes, and cytokines, were found in higher concentrations in the synovial fluid of RA patients and play a crucial in theetiology of disease(Hassan et al. 2019).Specific pathological diseases such as inflammation and RA cause an increase in the expression of this enzyme due to a variety of stimuli, including growth factors and cytokines. The degradation of joint cartilage associated with RA is brought on by increased expression of COX-2 in subchondralbone(Tu et al. 2019).Studies demonstrate that pro-inflammatory cytokines are probably responsible for the overexpression of COX-2(Sokolove and Lepus 2013). Thus, a novel strategy for treating RA may involve by decreasing these pro-inflammatory cytokinesandup regulating of anti-inflammatory cytokines(Hong et al. 2021).\u003c/p\u003e\n\u003cp\u003eThough several numbers of molecules, such as prostaglandins, TNF-\u0026alpha;, IL-1\u0026beta;, and IL-6 are implicated in the pathophysiology of RA, reactive oxygen species (ROS) trigger the inflammatory processes by damaged articular cartilage, membrane lipids, proteins, and DNA (Jain et al. 2023). It is observed that inflammatory cells such as neutrophils, lymphocytes, macrophages, and endothelial cells found in the inflammatory joint tissue are the main source of ROS(Sindhu et al. 2012). The primary cause of RA can be identified as oxidative stress. By generating pro-inflammatory cytokines and COX-2, ROS induces oxidative stress, which is a crucial modulator of inflammatory reactions at the inflamed joint during the development of arthritis (Goldring 2003). The defense mechanism against the damaging effects of free radicals is comprised of a variety of detoxifying enzymes and metabolites. The two main antioxidant enzymes involved in detoxification are catalase (CAT) and superoxide dismutase (SOD) (Phull et al. 2017). As a result, many research studies have been investigated to explore the anti-inflammatory properties of antioxidants. Most antioxidants are derived from dietary sources and are crucial for preventing the harmful effects of ROS on living systems. Antioxidants possess the capacity to protect an organism from the harmful effects of free radical damage, thereby reducing or delaying the emergence of multiple pathologies like cardiovascular, cancer, atherosclerosis, RA, and neurodegenerative illnesses(Silva et al. 2023).\u003c/p\u003e\n\u003cp\u003eCurrently available therapies such as non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and disease-modifying anti-rheumatic drugs (DMARDs) to treat RA are associated with several side effects, including damage to the liver, cardiovascular problems, and nausea (Su et al. 2022). The current state of medicines has limited their wide application in the treatment of RA due to the high cost and unpredictable response. Thus, there is an urgent desire to identify drugs with fewer adverse effects and established therapeutic efficacy(Lv et al. 2022). According to published research, natural products are a promising alternative source for treating RA because they are less toxic and have fewer adverse effects(Zhang et al. 2008). The unique chemical compositions and biological activities of herbal products allow them to treat various ailments. Manyphytocomponents, such as flavonoids, terpenoids, glycosides, and alkaloids, have been reported with anti-arthritic properties(Jain 2014). Therefore, there is an increasing focus on using natural substances and their products in managing arthritis. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTecomastans\u003c/em\u003e (L.) Juss. ex Kunth (Bignoniaceae) is amedium-sized ornamental blooming perennial deciduous shrub known as yellow-elder, yellow trumpet bush, and yellow-bells and found in tropical and subtropical regions of Africa and Asia (Anand and Basavaraju 2021). It is also found as an invasive weed in South Africa(Madire 2013). It is extensively dispersed throughout India, from the plains extending to the southernmost point of the country to the Shiwalik levels in the Himalayas. It has been observed in several regions of the nation, including Shimla in Himachal Pradesh(Thakur 2012); Nalgonda, Baptala, Tirumala hills, Put-taparthi, Simhachalam, and Warangal in Andhra Pradesh; and in Orissa (Dash 2011). The genus Tecoma is rich in phytoconstituent; prior research has identified and isolated numerous constituents belonging to various chemical classes, including phenolics, indolic compounds, iridoids, flavonoids, alkaloids, and triterpenoids. This genus also has a wide range of pharmacological properties, including antibacterial, diuretic, spasmolytic, hypoglycemic, anti-inflammatory, and protozoal illnesses. Other biological activities also have been described, such as anti-diarrhea, antioxidant, anti-proliferative, insecticidal, and astringent properties(Abdel-Mageed et al. 2012). With expressivepantropical distribution, the Bignoniaceae family of plants has around 830 species, where\u003cem\u003eT. stans\u003c/em\u003e is recognized as an exotic in Brazil and extensively utilized as ornamental plants. This species produces a variety of compounds, including unsaturated fatty acids, carbohydrates, proteins, resins, phenolic acids, flavonoids, carotenoids, terpenoids, saponins, glycosides, and phytosterols(Raju 2011). Several alkaloids were reported from the leaf of \u003cem\u003eT. stans\u003c/em\u003e, such as tecomine, tecostanine, boschniakine, 4-hydroxytecomanine, N-normethylskytanthine, 5-hydroskytanthine, 7-hydroskytanthine, \u0026gamma;-skytanthine, tecomanine, and 4-noractinidine (Hammouda and Amer 1966). Leaf also reported with phenolic acids, namely, chlorogenic, cinnamic acid, ferulic acid, gallic acid, caffeic, vanillic, o-coumaric, and sinapic acids, sitosterols, triterpenoids, and flavonoids, namely, flavonone, apigenin, chrysoeriol, kaempferol, luteolin, quercetin, rutin, 7,8-dihy-droxy-4,6-dimethoxy flavone, and verbascoside. The different parts of the plants, including leaves, flowers, roots, and bark, are used to treat a variety of disorders and ailments, including skin infections, kidney problems, intestinal disorders, jaundice, heart pain, joint pains, and antidotes for snake, scorpion, and rat bites(Anand and Basavaraju 2021). The ancient methods of preparing herbal remedies from \u003cem\u003eT. stans\u003c/em\u003ethat were most frequently cited were the decoction of aerial parts, infusion of leaves, flowers, and young leaves. The leaf is utilized in the rural Kyaing Tong Township, Myanmar, to treat arthritic joint pain and repair cracked bones. A paste was prepared by combining the leaf with other medicinal plants and rice water and applied as a poultice to the affected area. A week is needed for the fractured bones to heal for children and 15 days for adults over 25(Anand and Basavaraju 2021). Besides that, a wide range of biological activities such as hypoglycemic(Lozoya-Meckes and Mellado-Campos 1985);cardioprotective(Villar 1997); anticancer (Thirumal M 2012); hepatoprotective(Kameshwaran et al. 2012); antimicrobial (Binutu and Lajubutu 1994); antinociceptive and anti-inflammatory ; wound healing (Das 2010); antispasmodic (Gharib-Naseri 2007); antidiarrheal (Kameshwaran et al. 2012); antiulcer (Shanmukha 2013), gastroprotective, anti-obesity and insecticidal (Silva et al. 2023)have also been reported.\u003c/p\u003e\n\u003cp\u003eA literature survey revealed that\u003cem\u003eT. stans\u003c/em\u003ewas reported with many phenolic and flavonoid compounds. These compounds effectively prevent and manage RA by interacting with different target sites (Ali et al. 2023). The anti-arthritic potential of \u003cem\u003eT. stans\u003c/em\u003eleaf has not been documented to date. Hence, this current study aimed to explore the anti-arthritic potential in rats induced with Complete Freund\u0026apos;s adjuvant (CFA) by assessing parameters such as paw diameter, arthritic index and score, inflammatory cytokines, and oxidative biomarkers. Moreover, GC-MS and LC-MS analyses were conducted to characterize the bioactive compounds accountable for the anti-arthritic activity. Additionally, computational methods were employed to assess the anti-arthritic potential of bioactive compounds identified from LC-MS analysis against COX-2, IL-1\u0026beta;, IL-6, and TNF-\u0026alpha; protein.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eChemicals and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComplete Freund\u0026apos;s adjuvant (CFA), TNF-\u0026alpha;, IL-6, IL-1\u0026beta;, IL-4, IL-10 and COX-2, were obtained from Sigma Aldrich, USA. Indomethacin was acquired from Micro Lab Pvt. Ltd, India. Sodium carboxyl methyl cellulose, 2,2-diphenyl-1-picrylhydrazyl, Tris-HCl, Ethylenediaminetetraacetic acid, Perchloric acid, Ascorbic acid, n-hexane, and Ethanol were purchased from Merck Pvt. Ltd, Germany.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe leafof \u003cem\u003eT. stans\u003c/em\u003ewere collected from Bhubaneswar during August 2023with demographic details N 20\u0026deg; 17.1548\u0026apos; and E 85\u0026deg; 46.3195\u0026apos;and authenticated by Dr. Pratap Chandra Panda, Taxonomist, Siksha \u0026lsquo;O\u0026rsquo; AnusandhanDeemed to be University, Bhubaneswar, Odisha, India with voucher specimen CBT/2457.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of extract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collected leaf material was shade dried and then subjected to coarse powder. The powder material was successively extracted with n-hexane and ethanol for 48 hours using soxhlet apparatus andconcentrated using a rotary evaporator. The percentage yield of n-hexane and ethanol extract were found to be 7.45 and 18.29 w/w, respectively, and refrigerated until further use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of ETSL by GC-MS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGas chromatography and mass spectroscopy(GC-MS) analysis of\u0026nbsp;ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf (ETSL) was performed using modified procedure(Das et al. 2022a). A mass detector (MS TSQ 8000) was attached to the Thermo Trace 1300GC and Thermo TSQ 800 Triple Quadrupole MS which connected to the BP-5MS (5% Phenyl Polysilphenylene-Siloxane) capillary column (30m X 0.25mm, 0.25\u0026micro;m film thickness).1 \u0026micro;l of the ETSL was added to the column at a split flow rate of 30 ml/minute. The mass spectra from m/z 35 to 650 were obtained at intervals of 0.5 seconds. The temperatures of the injector, MS transmission line, and ion source were changed to 250℃, 280℃, and 230℃, respectively. A steady carrier flow of 1 ml/min was maintained. The oven was heated to 280℃ at a rate of 10℃ per minute after keeping at 60 ℃ for six minutes. The metabolites were identified by comparing the GC-MS investigation results with Wiley and the NIST 2.0 library database (National Institute of Standards and Technology).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of ETSL by LC-MS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LC-MS analysis of ETSL was carried outusing Water Alliance e2695/HPLC-TQD mass spectrometer. The compounds were separated on an RP High Strength Silica (HSS) T3 C18 column (250 mm \u0026times; 4.6 mm, comprising particles with a diameter of 5 \u0026mu;m, Waters). The temperatures of the auto-sampler and column were adjusted at 5 \u0026deg;C and 35 \u0026deg;C, respectively.A gradient solvent system comprising solvents (A) acetonitrile and (B) ammonium acetate were utilized for the chromatographic separation. ETSL was passed through a 0.2 \u0026mu;m nylon membrane filter. The injection volume was kept at 5.0 \u0026mu;l.Total run time was maintained for 40 minutes. The run duration of 1-10 minutes comprises of 5% for (A) and 95% for (B); 30 % for (A) and 70% for (B); 16-24 minutes: 60% for (A) and 40% for (B); 80% for (A) and 20% for (B); 24-32 minutes: 80% for (A) and 20% for (B); 32-35 minutes: 80% (A) and 20% for (B); 5% (A) and 95% for (B); 35-40 minutes: (A) 5%, (B) 95%. The pressure was kept at 1,200 barand the flow rate was maintained at 1.5 ml/min. The capillary voltage, source cone voltage, and extraction cone voltage were kept at 3.5 kV, 30 V, and 3 V, and;at 3.5 kV, 60 V, and 3 Vin positive and negative mode,respectively.Adesolvation gas, nitrogen was added at a flow rate of 650 l/hour. The temperatures of the desolvation and source were kept at 350\u0026deg;C and 120\u0026deg;C, respectively. Both positive and negative ionization modes of electrospray were used to get the mass spectrum data. Mass spectra were obtained by covering the m/z range of 150-750Da.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of in vitro antioxidant potential of ETSL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn vitro antioxidant property of ETSLat25, 50, 100, 200, 400 and 500\u0026mu;g/ml was performed using DPPH (2, 2-diphenyl-1-picrylhydrazyl), ferric reducing, nitric oxideradicalscavengingand reducing power assay, and ascorbic acid used as a standard(Dash and Ghosh 2017). IC\u003csub\u003e50\u003c/sub\u003e value was calculated by plotting the graph, scavenging activity versus the concentration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of in vitro anti-arthritic potential of ETSL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein denaturation efficacy of ETSL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein denaturation efficacy of ETSL was assessed as per the standard method(Alamgeer et al. 2017). The test mixture comprises of Fresh hen\u0026apos;s egg albumin (0.2 ml), phosphate-buffered saline (2.8 ml), various concentration (25, 50, 100, 200, 400 and 500 \u0026micro;g/ml) of ETSL and indomethacin (2 ml). Double-distilled water was used as control.Thereaction mixture was incubatedfor 15 minutes at 37 \u0026plusmn; 2 \u0026deg;C, followed by heating for 5 minutes at 70 \u0026deg;C. The absorbance of these solutions was recordedat 660 nm. The percentage of inhibition was calculated using following formula.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" style=\"width: 651px; height: 46.2996px;\" width=\"651\" height=\"46.2996\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMembrane stabilization efficacy of ETSL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman red blood cell (HRBC) was used to evaluate membrane stabilization potential of ETSL. HRBC(10 ml) wastaken in heparin centrifuge and added with equal volume of Alsever\u0026rsquo;s solution. The resultant mixture was centrifuged with 0.85% isosaline. Equal quantities of ETSL (25-500 \u0026micro;g/ml) were added to 1ml of HRBC suspension and incubatedfor 30 minutes at 37 \u0026deg;C. Then it was subjected to centrifugation. Mixture without extract was considered as control. The amount of haemoglobin in the supernatant solution was measured at 560 nm(Ajithkumar et al. 2020).The following formula was used to calculate the percentage of hemolysis.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" style=\"width: 475px; height: 64.5717px;\" width=\"475\" height=\"64.5717\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteinase inhibitory efficacy of ETSL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method outlined by (Sivapalan et al. 2024)was used to evaluate the proteinase inhibition efficacy of ETSL. Thereaction mixture (1 ml of 20mMtris HCL buffer(pH 7.4), 1 ml of ETSL(25-500 \u0026micro;g/ml), and 0.06 ml of trypsin) was incubated for five minutes at 37 \u0026deg;C. Casein (1 ml, 0.8%) was added to the above mixture and kept for twenty minutes. Then 2 ml of perchloric acid (70%) was added and centrifuged (2500 rpm) for five minutes.The absorbance was measured at 280 nm. The proteinase inhibitory activity was estimated using the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" style=\"width: 500px; height: 50.578px;\" width=\"500\" height=\"50.578\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHealthy young adult Wistar rats (6-8 weeks) weighing 150-180 g of both sexes were used. Before animal experiment, all animals were acclimatized in animal house for seven days. The animal house was maintainedlight-dark cycle (12 hours), temperature (22 \u0026plusmn; 3 \u0026deg;C), and a relative humidity (30-70%). Feeding was done using a standard lab diet.The experimental protocol was approved by Institutional Animal Ethic Committee (IAEC/SPS/SOA/128).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of in vivo anti-arthritic potential of ETSL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals were divided into five groups (n=6). Group I: Normal control (NC) received carboxy methyl cellulose-sodium (CMC-Na, 1% w/v); Group II: Disease control received CFA 0.1 ml; Group III: positive control received indomethacin (IND) 10 mg/kg;Group IV and V: test control received ETSL 250 and 500 mg/kg, respectively. 0.1 ml of CFA (10 mg) of heat-killed mycobacterium tuberculosis per ml of paraffin oil was injected into the sub-plantar surface of the right hind paws for the induction of RA (Das et al. 2021). The doseof ETSL was selected as previously reported elsewhere(S. Kameshwaran 2013). The inflammatory edema of the injected paw was observed after 6 to 8 days of CFA immunization, and the maximum severity was noticed on the 14\u003csup\u003eth\u003c/sup\u003eday.Thetreatment was commenced from day 15 to 28.\u003c/p\u003e\n\u003cp\u003eEvaluation of arthritis was performed by measuring paw diameter on days 0, 7, 14, 21, and 28 by using Verniercalipers; arthritic score (0-4) with maximum possible score for each animal was 16; arthritic index; body weightandorgan (liver, kidney, and spleen) weightexamination(Das et al. 2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExamination of hematological and biochemical parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental animals were sacrificed by cervical dislocation method on 29\u003csup\u003eth\u003c/sup\u003e day. Blood sample was collected through cardiac puncture and examined for hematological parameters like red blood cells (RBC), white blood cells (WBC), haemoglobin (Hb), platelet count, erythrocyte sedimentation rate (ESR) and biochemical indicators; rheumatoid factor (RF), aspartate aminotransferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), creatinine, urea, and C-reactive protein (CRP)(Jain et al. 2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of pro-inflammatory and anti-inflammatory cytokines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pro-inflammatory cytokinesTNF-\u0026alpha;, IL-6, IL-1\u0026beta;,anti-inflammatory cytokines; IL-4 and IL-10, and inflammatory mediator COX-2were measured in the serum of experimental animals using Enzyme-linked immunosorbent assay (ELISA) (Uttra et al. 2018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of oxidative stress biomarker\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tissue homogenate of liver and joint were analyzed to measure the oxidative stress biomarkerssuch as SOD, CAT, reduced glutathione (GSH), and lipid peroxidation (LPO).The tissues were washed with cold saline and homogenized in Tris-HCl buffer (0.05 M, pH 7.4). Then, 10% w/v tissue homogenatewascentrifuged at 12,000 rpm for 30 minutes and the supernatant was used to measure the stress biomarkers(Sundaram et al. 2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRadiological and histopathologicalanalysis of joint\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRadiographic analysis ofexperimental joint rats was carried out andimages were takenfor the evaluation of joint deformation.Further, histopathology of ankle joints wasperformed after fixing with 10% v/v formalin. Boneswere decalcified by immersing in 10% ethylenediaminetetraacetic acid (EDTA) for 30 days. The microtome sections were taken at 5\u0026mu;m thickness by embedding joints in paraffin wax followed by stainedwith eosin and hemotoxyline.The images were captured at 400x magnification by photomicroscope. Thehistopathological changes in the ankle joints, such as bone degradation, swelling, and pannus formation were examined(Uttra et al. 2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological score\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistological score of arthritic rats were measured to assess the extremity of RA in terms of proliferation of synovial vascularity, hyperplasia, inflammation, and expansion of the synovial lining. The score was assessed using a semi-quantitative 4-point grading method; absent (0), mild (1), moderate (2), severe (3) with highest score of 21(El-Tanbouly and Abdelrahman 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phytocompounds tentatively identified through LC-MS analysis from ETSL were taken for \u003cem\u003ein silico\u003c/em\u003e analysis to establish a correlation between the observed anti-arthritic activity and their active constituents. Accordingly, 19 compounds were selected as ligands for molecular docking studies. Based on literature findings, four potential targets COX-2, IL-1\u0026beta;, IL-6, and TNF-\u0026alpha; were selected to evaluate the effectiveness of each ligand individually. The structures of all ligands, including indomethacin, were obtained from the PubChem website in SDF format. OPEN BABEL software was employed to convert all the ligands into PDB format. Furthermore, the software Avogadro was utilized to optimize the 3D structures of all compounds, which were then saved as dot pdb (PDB) files. These optimized structures were subsequently used to derive reliable docking scores (kcal/mol).The 3D crystal structures of the target proteins with PDB IDs 2AZ5 (TNF-\u0026alpha;), 4G6J (IL-1\u0026beta;), 1ALU (IL-6), and 1PXX (COX-2) were retrieved from the Protein Data Bank (PDB) at http://www.rcsb.org/pdb. The fragmented protein structures were remodelled using SWISS MODEL and saved in dot PDB format for subsequent molecular docking investigations. The binding affinity (Docking Score) of the compounds was computationally determined using AutodockVina 4.2 software. The visualizations of protein-ligand binding characteristics were conducted using BIOVIA-DISCOVERY VISUALIZER 2024 software(Das et al. 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were collected in triplicate for the in vitro investigation and expressed as mean \u0026plusmn; standard deviation(SD). Using unpaired student t-tests, the statistical difference between the groups was analyzed. All data for the in vivo experiment were presented as mean \u0026plusmn; SD. One-way ANOVA followed by Tukey\u0026apos;s multiple comparison tests were used to determine the statistical significance of the differences between each of the groups. Using two-way ANOVA and Bonferroni\u0026apos;s post hoc test, the findings of paw edoema, arthritic index, arthritic score, and body weight were analyzed.Statistical analysis was performed by GraphPad Prism 5.0, where P˂0.05 was considered as the level of significant difference.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGC-MS analysis of ETSL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGC-MS analysis of ETSL revealed 18 compounds (Fig. 1). The retention time, molecular formula and weight, and chemical nature of the compounds were presented in Table 1. The compound with highest percentage of area was identified as 13-Docosenamide, (Z)-(18.55 %).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC-MS analysis of ETSL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LC/MS chromatogram of ETSL in positive and negative mode was presented in Fig. 2\u0026amp;3, respectively. The phytocomponent of ETSL were tentatively identified based on their retention time, MS/MS fragments, experimental \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e, and metabolite classes and then confirmed using literature data. Total 20 secondary metabolites were identified with 14 in positive mode and 6 in negative mode of ionization (Table 2). For the purpose of determining the m/z ratio of sample, it demonstrated that the positive and negative modes of ionization in mass spectrometry involve the production of positive ions [M+H]\u003csup\u003e+\u003c/sup\u003e (m/z) and negative ions [M+H]\u003csup\u003e-\u003c/sup\u003e (m/z), respectively. All the phytochemicals were first time reported through LC-MS analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro antioxidant potential of ETSL\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn vitro antioxidant potential of ETSL (25-500 µg/ml) was presented in Fig4. The result revealed that, ETSL exhibited highest percentage of inhibition at concentration 500µg/ml against reducing power assay (75.23±1.15), DPPH (74.23±1.05), nitric oxide (69.56±2.08) and ferric reducing assay (72.66±2.60) with corresponding IC\u003csub\u003e50\u003c/sub\u003e values of 292.06 ±3.12, 263.36±2.36, 324.34±4.89 and 267.64±3.65µg/ml, respectively. Standard ascorbic acid at concentration 500µg/ml also exhibited greater percentage of inhibition 92.33±0.88, 86.33±1.33, 81.33±1.20 and 83.89±2.08 against reducing power assay, DPPH, nitric oxide and ferric reducing assay, with corresponding IC\u003csub\u003e50\u003c/sub\u003e values of 148.80±1.45, 172.79±2.58, 235.24±2.74 and 260.80±3.14 µg/ml, respectively. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of in vitro anti-arthritic potential of ETSL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn vitro, the anti-arthritic property of ETSL was assessed by protein denaturation, membrane stabilization, and proteinase inhibition assay at concentrations 25, 50, 100, 200, and 500µg/ml. ETSL exhibited protein denaturation (78.32±1.36), membrane stabilization (71.10±1.08), and proteinase inhibition (79.33±0.82) with corresponding IC\u003csub\u003e50\u003c/sub\u003evalues of 305.57±2.84, 320.35±3.12 and 280.12±3.65µg/ml at a concentration of 500µg/ml, respectively. The standard indomethacin also exhibited remarkable in vitro anti-arthritic potential with 92.88±1.31, 91.33±1.45 and 95.33±2.88 percentage of inhibition against these parameters with corresponding IC\u003csub\u003e50\u003c/sub\u003e values of 197.45 ±1.24, 212.54±2.58 and 209.20±1.86 µg/ml, respectively (Fig. 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of in vivo anti-arthritic potential of ETSL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ETSL on paw thickness\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSub-plantar injection of CFA into the right hind paw causes a remarkable (13.41±1.38,\u0026nbsp;P\u0026lt;0.001) rise in paw thickness of CFA rats on the 14\u003csup\u003eth\u003c/sup\u003eday in contrast to normal control rats. The increase in paw thickness of disease-induced rats was also noticed on day 28. Administration of IND (10 mg/kg) and ETSL (250 \u0026amp; 500 mg/kg) declined the paw diameter compared to CFA-induced rats. A remarkable decrease in paw thickness by IND (4.35±1.29, p\u0026lt;0.001) and ETSL (500 mg/kg) (6.38±1.20, p\u0026lt;0.01) was observed on the 28\u003csup\u003eth\u003c/sup\u003e day. However, ETSL (250 mg/kg) on day 28 also significantly (9.43±1.35, p\u0026lt;0.05) decreased the paw thickness. The study indicates that ETSL (500 mg/kg) shows a better effect than 250 mg/kg (Fig. 6A). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ETSL on arthritic index\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe arthritic index of experimental animals is presented in Fig 6B. Treatment with IND (10 mg/kg) (32.25±1.75, p˂0.001) and ETSL (500 mg/kg) (57.05±2.18, p˂0.01) showed a noticeable reduction in the arthritic index on day 28 in contrast to CFA-induced animal (277.51±1.16). Considerable decline was also marked by ETSL (250 mg/kg) (166.60±1.64, p˂ 0.05) on day 28, indicating dose-dependent response of ETSL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ETSL on arthritic score\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the arbitrary score method, the arthritic score of the disease control animals was found to be maximum (10.83± 1.6) on the 28\u003csup\u003eth\u003c/sup\u003eday. The group administered with IND (10 mg/kg) noticeably (p˂0.001) reduced the arthritic score (5.5 ± 1.34). However, administration of ETSL (250 and 500 mg/kg) from the 15\u003csup\u003eth\u003c/sup\u003e to the 28\u003csup\u003eth\u003c/sup\u003e day showed a dose-dependent effect in depletion (p˂0.05, p˂0.01) of the arthritic score (8.5 ± 1.15, 7.5 ± 1.56) as compared to CFA induced rats, respectively(Fig.6C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ETSL on body weight\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCFA-induced rats were found to have a remarkable reduction (p˂0.001) in body weight (147.83 ± 1.71) as compared to normal control rats (186.23 ± 2.25) on the 28\u003csup\u003eth\u003c/sup\u003e day. Contrarily, treatment with the IND (10 mg/kg) and ETSL (500 mg/kg) noticeably raised the body weight by 170.83 ± 2.86 (p\u0026lt;0.001) and 167.76 ± 1.82 p\u0026lt;0.01), respectively, in contrast to disease control rats on the 28th day. However, ETSL (250 mg/kg) also considerably (p\u0026lt;0.05) raised the weight of the body (161.16 ± 2.83) on the 28\u003csup\u003eth\u003c/sup\u003e day as compared to the CFA group. This suggests that a higher dose of ETSL was found to have more effect than a lower dose (Fig.6D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ETSL on organ weight\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe disease control group was observed with a remarkable (p\u0026lt;0.001) increase in weight of the kidney (9.14±0.76) (Fig.7B) and spleen (7.59±0.46) (Fig.7C) and decline in the liver (5.85±0.41) (Fig.7A) as compared to normal control rats (4.52±0.46, 3.69±0.55, 9.13±0.21), respectively. Treatment with IND (10 mg/kg, p˂0.001), ETSL (250 mg/kg, p˂0.05), and ETSL (500 mg/kg, p˂0.01) noticeably restored the weight as compared to the disease control group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ETSL on hematological and biochemical parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificant elevation in the levels of WBC, Platelets, ESR, CRP, RF, AST, ALT, ALP, urea, and creatinine, and a decline in RBC and Hb were observed in the disease control group in contrast to normal control rats. Supplementation of IND (10 mg/kg, p˂0.001), ETSL (500 mg/kg, p˂0.01), and ETSL (250 mg/kg, p˂0.05) significantly normalized these altered values in contrast to disease control animal. The data of RBC, WBC, platelets, AST, ALT, ALP, urea, and creatinine were given in Table 3, whereas hemoglobin, ESR, CRP, and RF were presented in Fig. 8.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ETSL on TNF-α, IL-1β, IL-6 and COX-2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe result indicated significant increases in TNF-α (7.58 ± 0.85, p\u0026lt;0.001) in the CFA-induced group as compared to the normal group (1.91±0.16). However, animals treated with IND (10 mg/kg) (2.40±0.59, P˂0.001), ETSL (250 mg/kg) (5.33±0.96, P˂0.05),and ETSL 500 mg/kg (4.33±1.16, P˂0.01) noticed with remarkable decline in TNF-α expression (Fig.9A). Similarly, CFA-induced group was marked with significant (P˂ 0.001) increased in IL-1β expression (191.28 ± 6.19) in contrast to healthy animals (45.85±3.94). Treatment with IND (10 mg/kg) (79.89±3.92, P˂ 0.001), ETSL (250 mg/kg) (141.48±6.75, p˂0.05), and ETSL (500 mg/kg) (100.24±6.80, p˂0.01) demonstrated notable declined in the IL-1β expression (Fig.9B). Remarkable elevated level of IL-6 was noticed with disease control rat (6.81±0.69, p˂0.001) as compared to normal rats (1.59±0.12). Administration of IND (10 mg/kg) (2.78±0.60, p˂ 0.001) and ETSL (500 mg/kg) (3.49±0.48, p˂0.01) notably reduced the IL-6 level in treatment rats as compared to disease control rats (Fig.9C). ETSL at 250 mg/kg (5.51±0.49, P˂ 0.05) also reduced the level of IL-6 in comparison to CFA group. The CFA-induced disease control group observed a remarkable (p\u0026lt;0.001) rise in COX-2 (15.01±2.44) in contrast to normal control rats (4.16±0.60). Treatment with IND (10 mg/kg) (8.08±2.17, p\u0026lt;0.001), ETSL (250 mg/kg) (10.57±1.96, p\u0026lt;0.05), and ETSL (500 mg/kg) (8.76±2.01, p\u0026lt;0.01) significantly declined the level of COX-2 as compared to disease control rats(Fig.9D). The observed result indicated that ETSL at dose of 500 mg/kg was found more effective in reducing the TNF-α, IL-6, IL-1β and COX-2 level than 250 mg/kg.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ETSL on IL-4 and IL-10\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CFA-induced disease control group was noticed with a remarkable (56.14±5.86, p\u0026lt;0.001) decline in IL-4 expression in contrast to normal control animals (86.62±3.16). Group administered with IND (10 mg/kg) (79.60±3.71, p\u0026lt;0.001), ETSL (250 mg/kg) (72.41±2.92, p\u0026lt;0.5), and ETSL (500 mg/kg) (76.03 ± 3.04, p\u0026lt;0.01) were observed with significant up-regulation in IL-4 expression in contrast to CFA-induced rats (Fig.9E). Similarly, the expression of IL-10 in disease control group (2.39±0.47, p\u0026lt;0.001) was notably declined in contrast to normal rats (6.75±0.44). The expression of IL-10 was remarkably up-regulated after administering with IND (10 mg/kg) (6.13±0.37, p\u0026lt; 0.001), ETSL (250 mg/kg) (3.87±0.26, p\u0026lt;0.05), and ETSL (500 mg/kg) (4.97±0.31, p\u0026lt;0.01) as compared to CFA-induced group revealed dose-dependent effect in up-regulation of IL-4 and IL-10 level(Fig.9F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ETSL on oxidative stress biomarker\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antioxidant effect of ETSL in the joint and liver of the disease control group was presented in Fig. 10\u0026amp;11.Significant (p\u0026lt;0.001) decreases in SOD (3.17±0.17, 0.96±0.12), CAT (16.18±2.19, 62.32±2.12), GSH (2.78±0.41, 2.94±0.58) and increased in LPO (34.12±3.35, 35.65±2.74) in joint and liver in CFA disease induced rats was noticed as compared to the normal control rats, respectively. Supplementation with IND (10 mg/kg) caused a remarkable (p\u0026lt;0.001) increase in SOD (6.12±0.36, 1.89±0.13), CAT (37.73±1.96, 107.16±2.56), GSH (7.35±0.83, 5.94±0.40) and decreased in LPO (12.67±2.42, 20.67±1.34) in joint and liver, respectively in contrast to CFA control group. Similarly, supplementation of ETSL (500 mg/kg) also remarkably (p\u0026lt;0.01) augmented the level of SOD (5.01±0.33, 1.73±0.12), CAT (33.65±2.44, 89.60±2.96), GSH (6.44±0.56, 5.54±0.42) and decreased in LPO content (19.24±1.64, 24.48±2.34) in joint and liver, respectively as compared to CFA-induced rats. However, ETSL (250 mg/kg)also causes noticeable (p\u0026lt;0.05) increased in SOD, CAT, GSH and decreased in LPO in contrast to disease control rats. The observed result showed that a higher dose of ETSL was found to be more effective than a lower 250 mg/kg in controlling the oxidative marker.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ETSL on histopathology of joint\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe histology result of the joint of disease-control rats showed cartilage and bone erosion,pannus formation, and development of synovial hyperplasia. Supplementation with IND (10 mg/kg) and ETSL (250, 500 mg/kg) from day 15-28 considerably reduced the histological changes, as shown in Fig.12. However, the ankle joint of the normal control group did not observe any kind of histological changes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ETSL on radiology of joint\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn X-ray analysis of the ankle joints of experimental rats was presented in Fig. 12. Compared to normal control rats, the diseased control rats showed signs of soft tissue swelling, joint space constriction, and bone loss. A noticeable effect was observed in the group treated with IND (10 mg/kg) and ETSL (250, 500 mg/kg).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of ETLS on histological score\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral parameters, such as the degree of enlargement of the synovial lining cell layer, synovial hyperplasia, synovial inflammation, synovial vascularity proliferation, pannus development, and bone and cartilage erosion, were assessed to determine the histological score in CFA-treated rats. The increase in histological score in the disease control rat (19.33±0.49) was remarkably declined by administration of IND (10 mg/kg) (12.00±0.73, p\u0026lt;0.001), ETSL (250 mg/kg) (16.50±0.42, p\u0026lt;0.05) and ETSL (500 mg/kg) (14.16±0.47, p\u0026lt;0.01) (Fig.13).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular docking methodology facilitates the representation of molecular interactions between a ligand and protein molecule at the atomic scale. In the present investigation, molecular docking was conducted to elucidate the binding interactions of compounds identified through LC/MS analysis of ETSL with potential key targets, including TNF-α, IL-1β, IL-6, and COX-2. The results of the docking analysis conducted in this study are summarized in Table 4. The docking scores indicated that all ligands displayed activity ranging from -6.2 to -7 against TNF-α, -6.5 to -7.7 against IL-1β, -6 to -6.6 against IL-6, and -6.3 to -10.4 against COX-2. Analysis of the docking results revealed that phytoconstituents exhibited relatively stronger binding affinity towards COX-2, followed by IL-1β, TNF-α, and IL-6. Verbascoside, rutin, and naringin demonstrated the highest docking scores (-10.4, -10.3, and -10.2 kcal/mol, respectively) compared to indomethacin (-8.7 kcal/mol) against the COX-2 receptor. Sitosterols, naringin, and verbascoside recorded similar docking scores (-7 kcal/mol) against the TNF-α protein but relatively lower than indomethacin (-8.1 kcal/mol). Quercitrin displayed the highest docking score against IL-6 at -6.6 kcal/mol, surpassing the standard indomethacin (-6.4 kcal/mol) (Fig.14).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe GC-MS analysis of ETSL revealed several bioactive compounds that exhibited anti-inflammatory and antioxidant activities. These compounds include pentadecane(Okechukwu 2020); heptadecane, 2,6,10,15-tetramethyl- (Jeha F. Dela Pena 2019); 1-Hexadecanol (Murugan Prasathkumar 2022); cetene(Murugan Prasathkumar 2022); 3-Eicosene, (E)- (Okechukwu 2020); methyl 9-cis,11-trans-octadecadienoate (Alabi A. Adenike 2019); cis-13-Octadecenoic acid, methyl ester (Diab AT 2021); 13-Docosenamide, (Z)- (Tareq et al. 2020); 9-Octadecenamide, (Z)- (Awakan et al. 2018); 7,8-Epoxylanostan-11-ol, 3-acetoxy- (Never Zekeya 2014); hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester (Markkas 2015); ethyl iso-allocholate(Johnson et al. 2020) and oleic acid, eicosyl ester (N. Habeela Jainab 2017). \u003c/p\u003e\n\u003cp\u003eLC-MS analysis of ETSL revealed 20 compounds: amino acids (2), alkaloid (1), phenolic (11), and flavonoid compounds (6). The amino acids tryptamine(Vitalini et al. 2020) and tryptophan (Wang et al. 2019) showed molecular ions [M+H]\u003csup\u003e+ \u003c/sup\u003ewith a mass of 160.2 \u003cem\u003em/z \u003c/em\u003eand 203.3 \u003cem\u003em/z \u003c/em\u003eat retention time (RT) 25.89 and 16.79 minutes, respectively. The peak shown at RT 7.13 minutes indicates a molecular ion [M+H]\u003csup\u003e-\u003c/sup\u003e of alkaloid,Boschniakine, with a mass of 161.1\u003cem\u003em/z \u003c/em\u003e(Wang et al. 2021). The peaks at RT of 5.56,6.65, 7.69,29.0, 10.95, 13.52, 16.13, 26.49, and 29.0 minutes revealed phenolic compounds such as p-coumaric acid (Cui et al. 2010), luteolin(Shi et al. 2018), gallic acid (Yu et al. 2018), naringin(Fang et al. 2006), ferulic acid (Jain and Surana 2016), chlorogenic acid (Clifford et al. 2003), (-)-catechin(Kaigongi et al. 2020) and caffeic acid (Wang et al. 2015) with molecular ions [M+H]\u003csup\u003e+ \u003c/sup\u003e and mass of 163.0 \u003cem\u003em/z\u003c/em\u003e, 285.2 \u003cem\u003em/z\u003c/em\u003e, 169.3 \u003cem\u003em/z\u003c/em\u003e, 580.5 \u003cem\u003em/z\u003c/em\u003e, 195.3 \u003cem\u003em/z\u003c/em\u003e, 353.3 \u003cem\u003em/z\u003c/em\u003e, 291.4 \u003cem\u003em/z\u003c/em\u003e and 181.3 \u003cem\u003em/z\u003c/em\u003e, respectively. Compounds hesperetin(Tong et al. 2012), rosmarinic acid (Ma et al. 2013)and sitosterols(Kim et al. 2016) showed molecular ions [M+H]\u003csup\u003e- \u003c/sup\u003ewith a mass of 301.2 \u003cem\u003em/z\u003c/em\u003e, 353.3 \u003cem\u003em/z\u003c/em\u003e and 415.3 \u003cem\u003em/z\u003c/em\u003e at RT of 6.65,8.38 and 12.66 minutes, respectively. Flavonoid compounds such as apigenin(Shaker et al. 2022), kaempferol(Chen et al. 2015), quercetin(Panchal and Shah 2017), quercitrin(da Costa et al. 2019), rutin(Wang et al. 2003) and verbascoside(Luca et al. 2019)at RT of 6.31, 7.14,8.10, 9.76,8.93 and 11.29 minutes showed molecular ions [M+H]\u003csup\u003e+ \u003c/sup\u003ewith mass of 288.2 \u003cem\u003em/z\u003c/em\u003e, 287.2 \u003cem\u003em/z\u003c/em\u003e, 302.2 \u003cem\u003em/z\u003c/em\u003e, 447.4 \u003cem\u003em/z\u003c/em\u003e, 617.5 \u003cem\u003em/z\u003c/em\u003e and 624.5 \u003cem\u003em/z\u003c/em\u003e, respectively.\u003c/p\u003e\n\u003cp\u003eROS is one of the main causes of RA. The overabundance of pro-inflammatory cytokines prompts neutrophils and activated macrophages to release ROS into the synovial fluid and serve as a mediator of tissue damage. ROS attacks biological components like lipids, proteins, enzymes, DNA, and RNA in adjuvant-induced arthritis, causing damage to cells or tissues (Kumar et al. 2016). According to (Arulselvan et al. 2016), an imbalance between antioxidants and free radicals leads to oxidative stress, which damages cellular components and induces inflammation. In the current study, ETSL exhibited in vitro antioxidant properties in a concentration-dependent manner against DPPH, ferric-reducing assay, reducing power, and nitric oxide radicals. Several phenolic and flavonoid compounds are known to possess antioxidant properties. Protocatechuic acid (3,4-Dihydroxybenzoic acid) was reported to scavenge free radicals of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and DPPH (Semaming et al. 2015). Tryptamine possesses an antioxidant activity by metal chelating effect(M. E. Neganova 2011). Ferulic acid and apigenin were known to scavenge free radicals such as hydroxyl radicals, superoxide, hydrogen peroxide, and nitrogen radicals(Kashyap et al. 2022; Zhu et al. 2020). Hesperidin (Adefegha et al. 2020)and quercetin(Haleagrahara et al. 2017)were also found to decrease the reactive oxygen species levels. In the current study, these phenolic and flavonoid compounds were identified by LC-MS analysis of ETSL and could be responsible for significant in vitro antioxidant properties. The free radical scavenging property of \u003cem\u003eT. stans\u003c/em\u003eleaf was also reported earlier by (Anand and Basavaraju 2021).\u003c/p\u003e\n\u003cp\u003eDenaturation of protein is considered a good indication of RA. The process in which proteins lose their secondary and tertiary structures in response to external pressures, such as changes in temperature and pH and exposure to organic or inorganic substances, is known as denaturation. The hydrophobic, electrostatic hydrogen, and disulfide bonds are altered throughout the degradation process of proteins (Das et al. 2022b). It is noted that RA is associated with the development of auto-antigens as a result of protein denaturation. Denaturation of intercellular material or components of cell proteins may result in tissue damage (Osman et al. 2016). Therefore, the ability of the substance to stop protein denaturation suggests its anti-inflammatory properties. In the current investigation, ETSL inhibited protein denaturation in a concentration-dependent manner, which might be due to its defense against protein denaturation and auto-antigen formation. Phytochemicals, including alkaloids, flavonoids, tannins, and phenolic acids, were reported to inhibit the protein denaturation reported by (Pavithra 2015). Therefore, the presence of these phytoconstituents in ETSL may potentiate its protein denaturation efficacy. Protein denaturation inhibitory activity of ethanol leaf extract of \u003cem\u003eT. stans\u003c/em\u003e by bovine serum albumin and egg albumin was also studied earlier (Priyanka Sivasubramanian 2021).\u003c/p\u003e\n\u003cp\u003eProteases are a class of proteolytic enzymes that break down polypeptide links and degrade the proteins. Proteases are crucial regulators of physiological processes because proteins are important for preserving homeostasis (Agbowuro et al. 2018). Proteinases have the ability to control the inflammatory cytokines by regulating the expression and function of inflammatory mediators such as chemokines, immune cells, and cytokines. These cytokines and macrophages increase in the inflammatory phase of RA produce excessive amount of proteinase, and causes tissue deterioration. Thus, it is essential to control proteinase secretion. Therefore, a considerable degree of protection was provided by proteinase inhibitors (Jyothilakshmi et al. 2017). In the current investigation, ETSL remarkably prevented the secretion of proteinase in a concentration-dependent manner. The phytochemicals such as alkaloids, flavonoids, tannins, and phenolic acids were identified in ETSL could be effective against proteinase secretion(Dharmeshkumar D. Prajapati 2015). The findings of the present investigation were also in agreement with the previously reported work of(Govindappa M 2011). \u003c/p\u003e\n\u003cp\u003eThe process of protecting biological membranes, such as lysosomal and erythrocyte membranes, from heat and osmotic-induced lysis is known as membrane stabilization (Anosike et al. 2019). The lysosomal membrane lyses during the inflammatory process and releases active neutrophils, proteases, and bactericidal enzymes that harm the surrounding organelles and tissues. Thus, controlling tissue damage during inflammation requires blocking the release of lysosomal contents by stabilizing the lysosomal membrane (Ghosh et al. 2018). Human red blood cell (RBC) membranes are analogous to lysosomal membranes. Thus, stabilizing an RBC membrane implies stabilizing a lysosomal membrane. Hemolysis of RBC in a hyposaline solution results in the release of internal hemoglobin into the surrounding solution. Hence, the protection or stabilization of the RBC membrane by anti-inflammatory drugs during hyposaline-induced hemolysis is necessary (Daram et al. 2021). In the current investigation, ETSL exhibited the membrane stabilization potential in a concentration-dependent way. The binding of the ETSL to the erythrocyte membranes and the resulting modification of the cell surface charges may be the cause of the membrane stabilization potentials. This could hinder direct contact with aggregating agents or encourage dispersion through the mutual repulsion of similar charges, which are necessary for red blood cell hemolysis. Studies have demonstrated that flavonoids and saponins have a significant stabilizing impact on the lysosomal membrane. Also, tannins and saponins have the capacity to bind cations and stabilize erythrocyte membranes (Anosike et al. 2019). Therefore, the presence of these phytochemicals in \u003cem\u003eT. stans\u003c/em\u003e leaf might be responsible for protecting the erythrocytes membranes effectively (Dharmeshkumar D. Prajapati 2015). \u003c/p\u003e\n\u003cp\u003ePaw edema is linked with increased fluid discharge, vascular penetrability, and cellular penetration in the inflammatory region. This indicator is simple to quantify and offers a quick way to assess the anti-arthritic potency of the drug. The decline in paw thickness indicates a reduction in the release of inflammatory mediators and indicates the anti-inflammatory efficacy of the drugs in adjuvant arthritis (Manan et al. 2020). The disease control group experienced a 28-day period of swelling due to increased mast cells, neutrophils, and macrophages (Zheng et al. 2014). Rats in the ETSL (500 mg/kg) treated groups from days 15 to 28 displayed a notable reduction (p\u0026lt; 0.01) in paw diameter in contrast to disease-control rats. Several phenolic and flavonoid compounds such as caffeic acid (Fikry et al. 2019); ferulic acid (Zhu et al. 2020); hesperidin (Adefegha et al. 2020); rosmarinic acid (Jain 2021);\u0026beta;-sitosterol(Zhang et al. 2020), quercetin(El-Said et al. 2022); naringin(Ahmad et al. 2014);verbascoside(Guti\u0026eacute;rrez-Rebolledo et al. 2018) and catechin(Tang et al. 2007)were found to decrease the pain severity and inflammatory paw puffiness in adjuvant arthritis. These compounds were identified by LC-MS analysis of ETSL. Hence, the reduction in paw diameter in the group treated with ETSL might be due to the presence of these phytoconstituents. \u003c/p\u003e\n\u003cp\u003eIn CFA-induced arthritis, the arthritis score serves as a standard for joint puffiness. The severity of arthritis was monitored visually by the scoring method. The immunosuppressive and anti-inflammatory effects of \u003cem\u003eT. stans\u003c/em\u003e can be distinguished by a reduction in the arthritic index (Gautam et al. 2018). In the present investigation, ETSL-treated rats noticed a significant (p\u0026lt;0.05) decrease in the arthritic index and arthritic score as compared to the disease control group and protected against morphological changes by lowering the swelling and redness of the paw. Hesperidin(Adefegha et al. 2020); \u0026beta;-sitosterol(Zhang et al. 2020); catechin(Tang et al. 2007)and rosmarinic(Gautam et al. 2019)acid were reported to decrease arthritis score and arthritic index in CFA arthritis.Identification of these compounds by LC-MS analysis in ETSL might be responsible for the reduction in arthritic index and arthritic score in CFA-induced rats. \u003c/p\u003e\n\u003cp\u003eRheumatoid cachexia is recognized as a loss of weight and lean body mass in RA (Roubenoff et al. 1994). Previous studies suggested that a decline in the weight of the body is linked to inflammation because it inhibits the gut\u0026apos;s ability to absorb nutrients. The overexpression of inflammatory cytokines, such as TNF-\u0026alpha;, promotes energy use by improving lipid and protein metabolism and lowering calorie intake through their anorectic action, which causes cachexia. Also, it has been shown that anti-inflammatory drugs can enhance the compromised absorption ability of the intestine (Alamgeer et al. 2017). The results of the current investigation revealed that ETSL 500 mg/kg noticeably (p\u0026lt;0.01) increased the body weight in the treated group due to enhanced absorption of nutrients and inhibiting the level of inflammatory cytokines compared to the disease control group. The decrease in body weight in CFA rats was significantly up-regulated by phytocompoundsferulic acid(Zhu et al. 2020), \u0026beta;-sitosterol(Zhang et al. 2020), verbascoside(Guti\u0026eacute;rrez-Rebolledo et al. 2018),rosmarinic(Gautam et al. 2019)and quercetin(El-Said et al. 2022)as reported earlier which were confirmed in our LC-MS analysis might be responsible for increase in body weight. \u003c/p\u003e\n\u003cp\u003eIn the current study, the CFA-induced group noticed a decrease in liver weight and an increase in kidney and spleen weight. These findings may be the result of changes in the cell populations in the organs, which are linked to immune function (Phull et al. 2017). The supplementation of ETSL significantly (p \u0026lt;0.01) reversed the change in organ weight in relation to the disease control group. Polyphenols are substances that have the ability to regulate various immune system functions and minimize the severity of autoimmune diseases (Shakoor et al. 2021). Therefore, the identification of different phenolic compounds by LC-MS in ETSL might be responsible for correcting this altered organ weight. \u003c/p\u003e\n\u003cp\u003eA decrease in erythropoietin, bone marrow failure, and damage of erythrocytes causes a decline in Hb and RBC levels in CFA-induced rats, which leads to anemia(Alamgeer et al. 2017). Pathogenic microorganisms may trigger an immunological response in arthritic animals, leading to an increase in platelets, WBCs, and ESR, essential hematological indicators for the diagnosis of inflammatory conditions (Phull et al. 2017). The increased level of inflammatory proteins in the blood due to increased IL-6, TNF-\u0026alpha;, and IL-1\u0026beta; concentration also causes an increase in ESR count in the CFA group (Manan et al. 2020). Previous studies have shown that cytokines such as IL-6, TNF-\u0026alpha;, and IL-1\u0026beta; control the production of CRP from the liver (Littman et al. 1995). In the present study, administration with ETSL considerably (p\u0026lt;0.05) restored the hematological abnormalities in RA in contrast to the disease control group. The compounds ferulic acid(Zhu et al. 2020), hesperidin (Adefegha et al. 2020)and rosmarinic acid (Gautam et al. 2019)have been reported to increase the Hb and RBC and reduced WBC, platelets, ESR and CRP in CFA-induced arthritic rat. The identification of these compounds using LC-MS study in ETSL might be responsible for restoring hematological abnormalities. \u003c/p\u003e\n\u003cp\u003eSerum biochemical enzyme levels (ALP, ALT, and AST) were measured to determine the role of kidney and liver damage in inflammatory arthritic diseases. The increased ALP, ALT, and AST in the blood of CFA-induced animals suggest abnormal liver function. Furthermore, the liver is the primary organ involved in inflammatory diseases like RA, where changes in the synthesis, release, and catabolism of enzymes are important mediators and indicators of cellular damage caused by oxidative stress (Kumari and Anbarasu 2014). In the present study, treatment with ETSL (500 mg/kg) considerably (p\u0026lt;0.05) reduced the levels of biochemical enzymes compared to CFA-induced rats. The decreased kidney function in disease control rats led to increased blood levels of urea and creatinine(Chanda 2015). Groups under treatment with ETSL (500 mg/kg) marked a reduction in serum urea and creatinine levels. According to (Alamgeer et al. 2017) RF is an auto-antibody that targets the Fc (fragment crystallizable form) of IgG (immunoglobulin G) and creates immunological complexes that help in the development of RA. Administration of rats with ETSL (500 mg/kg) showed a significant (p\u0026lt;0.05) decrease in RF as compared to disease control rats. The changes in the biochemical parameters were reversed to normal state by phenolic compound ferulic acid in CFA-induced rats, as reported previously by (Zhu et al. 2020)which confirmed by LC-MS analysis of ETSL.\u003c/p\u003e\n\u003cp\u003ePrevious study cited that administration of CFA in experimental animals activates T-cells, which in turn initiate monocytes and macrophages, resulting in generations of pro-inflammatory cytokines (IL-1\u0026beta;, TNF-\u0026alpha;, and IL-6) in the subchondral bone layer, cartilage, synovial fluid, and cartilage membrane. These cytokines are responsible for bone degradation, joint degeneration, production of auto-antibodies, and cell death (Xiao et al. 2024). Inflammation and autoimmune diseases are largely caused by COX-2, which is involved in the production of pro-inflammatory substances, vasodilation, and cartilage degradation (Shabbir et al. 2016; Xiao et al. 2024). However, IL-10 and IL-4 are the immunoregulatory cytokines. In RA, it minimizes the effects of antigen-presenting cells and protects the tissue integrity of the joints (Lin et al. 2013). In the current investigation, it was found that the disease control group had noticed higher expressions of TNF-\u0026alpha;, IL-1\u0026beta;, IL-6, and COX-2 and lower expressions of IL-4 and IL-10. According to (Cheng et al. 2015), inhibiting the synthesis and function of pro-inflammatory mediators and up-regulating the expression of anti-inflammatory mediators is considered an efficient way to treat RA. In the present study, treatment with ETSL (500 mg/kg) remarkably (p\u0026lt;0.05) boosted anti-inflammatory and lowered pro-inflammatory expression as compared to disease control, thereby reducing inflammation, bone loss, and cartilage damage in RA. Different phenolic and flavonoid compounds were reported to inhibit these cytokines. Protocatechuic acid (3,4-Dihydroxybenzoic acid)(Semaming et al. 2015), tryptamine(Agista et al. 2022),p-coumaric acid(Zhu et al. 2018), apigenin(Lee et al. 2007), luteolin(Shi et al. 2015), \u0026beta;-sitosterol(Zhang et al. 2020), hesperetin(Lin et al. 2020), naringin(Ahmad et al. 2014),catechin(Tang et al. 2007), gallic acid (Bai et al. 2021)and ferulic acid(Zhai et al. 2023)inhibited TNF-\u0026alpha;, IL-1\u0026beta;, IL-6 and suppress COX-2 expression in carrageenan-induced inflammation in animal model.Caffeic acid(Kim et al. 2023), quercetin(Guan et al. 2021); and kaempferol(Tian et al. 2021)exhibited anti-inflammatory effects through significant suppression of IL-6, IL-1\u0026beta;, and TNF-\u0026alpha; in lipopolysaccharide (LPS) stimulated RAW 264.7 cells. In the present investigation, confirmation of these phenolic and flavonoid compounds by LC-MS analysis in ETSL proves its anti-inflammatory activity in CFA rats by inhibiting pro-inflammatory cytokines. \u003c/p\u003e\n\u003cp\u003eIn the progression of arthritis, endogenous antioxidant enzymes play a vital role in combating oxidative damage. SOD is thought to be the first line of defense against the production of free radicals and is necessary for the conversion of superoxide radicals into oxygen and hydrogen peroxide, which results in the inactivation of CAT and glutathione peroxidase. As a result of ROS-mediated degradation, SOD activity may have decreased during detoxification of these radicals(El-barbary et al. 2011). According to earlier research, a decrease in CAT activity in RA patients may be due to inactivation of CAT by hydrogen peroxide (Kalpakcioglu and Senel 2008). The increased production of ROS tends to proliferate abundantly during chronic inflammation and consequently causes excessive damage to tissues and is responsible for lipid peroxidation. Reduced membrane fluidity and inactivation of membrane-bound proteins cause lipid peroxidation, which breaks down into harmful malondialdehydes (MDA) (Sahu et al. 2017). Therefore, detoxification of these free radicals can stop cell damage and finally stop lipid peroxidation (Sadiq Umar 2012). GSH, a non-protein sulfhydryl molecule, is regarded as an essential non-enzymatic antioxidant defense system against the production of hydrogen peroxide and organic peroxides. A reduction in GSH level may worsen the clearance of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and encourage the creation of OH, which raises the free radical load and disturbs homeostasis. Furthermore, the lower content of GSH has been noted in the liver of CFA-induced rats(Comar et al. 2013).\u003c/p\u003e\n\u003cp\u003eIn the current investigation, supplementation of experimental rats with ETSL (500 mg/kg) significantly reduced the level of LPO and increased SOD, CAT, and GSH content in the liver and joints of CFA-induced rats as compared to disease-control rats. The reduction in lipid peroxide and increase in level of SOD, CAT and GSH in CFA-induced rat have been reported by several phenolic and flavonoid compounds such as Protocatechuic acid (3,4-Dihydroxybenzoic acid)(Semaming et al. 2015), caffeic acid(Kim et al. 2023), \u0026beta;-sitosterol(Zhang et al. 2020), naringin(Ahmad et al. 2014), p-Coumaric acid(Shen et al. 2019), hesperidin (Adefegha et al. 2020)and rosmarinic acid(Jain 2021). Rutin significantly restored the GSH level and SOD activity in collagen-immunized rats (Sadiq Umar 2012). The identification of these compounds in ETSL by LC-MS study supports the potent antioxidant activity of \u003cem\u003eT. stans\u003c/em\u003eleaf\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHistopathological analyses of arthritic joints were carried out in the current investigation to support the anti-arthritic effect of ETSL. The specific etiopathogenesis of RA is still unknown, but the research has shown that increased pro-inflammatory cytokine expression and oxidative stress are the main causes of synovial hyperplasia, pannus formation and, the cartilage and bone destruction (Xiao et al. 2024). In the present study, the administration of ETSL (500 mg/kg) noticeably prevented cartilage degradation and bone erosion, as evidenced by a decrease in histological score. This might be due to the prevention of pro-inflammatory cytokines and scavenging of free radicals by ETSL. The compounds such as caffeic acid(Fikry et al. 2019); luteolin(Shi et al. 2015); naringin(Ahmad et al. 2014)and rutin(Sadiq Umar 2012)were known to alleviate cartilage and bone destruction, synovial hyperplasia, decreased joint inflammation in CFA-induced rats. Hence, the identification of these phytoconstituents by LC-MS analysis of ETSL might be responsible for protective effects against cartilage and bone destruction. \u003c/p\u003e\n\u003cp\u003eThe advancement of computational methodologies offers a rapid and reliable process for screening the enormous number of these natural compounds. To identify phytochemicals potentially responsible for the therapeutic effects of ETSL on RA, various compounds were subjected to a docking study to interact with TNF-\u0026alpha;, IL-1\u0026beta;, IL-6, and COX-2 using computer-assisted modeling techniques.During the initiation of arthritis, numerous inflammatory mediators such as TNF-\u0026alpha;, IL-6, and IL-1\u0026beta; play pivotal roles(Aldossari et al. 2023). COX-2 plays a significant role in the synthesis of prostaglandins, which serve as pain mediators and contribute to the inflammatory process(Simon 1999). Hence, the inhibition of TNF-\u0026alpha;, IL-1\u0026beta;, IL-6, and COX-2 proteins with PDB IDs 2AZ5, 4G6J, 1ALU, and 1PXX, respectively, is anticipated to constitute a potential mechanism for anti-arthritis activity. In the present study, indomethacin was selected as the standard drug due to its capacity to inhibit the production of IL-6, IL-1\u0026beta;, and COX-2(Bour et al. 2000; Tonby et al. 2016).After evaluating individual docking scores, it was noticed that verbascoside emerged as the most promising candidate against all four targets, followed by naringin and rutin. In the drug development process, hydrogen bonding plays a crucial role in determining drug specificity, as it offers essential insights into protein-ligand interactions(Aldossari et al. 2023). The highest docking score achieved by verbascoside against COX-2 can be elucidated by its hydrogen bond interactions with amino acids HIS133, ASN39, GLU465, GLN461, CYS47, ALA156, and CYS36, as well as its hydrophobic interactions with MET48 and LEU152. In IL-1\u0026beta;, verbascoside exhibited a better docking score due to its hydrogen bond interactions with amino acids LYS103, VAL155, GLU153, GLN166, and ALA85 and electrostatic interactions with GLU165 and hydrophobic interactions with ALA83, ALA84, PRO40, ALA83, ALA85, and LYS103.In TNF-\u0026alpha;, verbascoside demonstrated the highest docking score primarily due to its hydrogen bond interaction with TYR151 and hydrophobic interactions with TYR59 and LEU157.Likewise, verbascoside achieved a superior docking score against IL-6, which is attributed to its hydrogen bond interaction with GLU23 and hydrophobic interaction with LYS27.This aligns with punicalagin, a polyphenolic compound that has shown substantial binding affinities to identical protein targets via hydrogen bonding and hydrophobic interactions(Jghef et al. 2023).Furthermore, verbascoside has been reported to exhibit potent anti-inflammatory activities by suppressing the expression of COX-2 (Pongkitwitoon et al. 2024). A decrease in TNF-\u0026alpha;, IL-1\u0026beta;, and IL-6 levels also has been observed in rats administered with verbascoside(Rossi et al. 2023). Therefore, based on molecular docking analysis, it is conceivable to consider that the compound verbascoside could be a novel potential candidate for the management of RA.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe anti-arthritic potential of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf was evaluated by in vitro, in vivo and \u003cem\u003ein silico\u0026nbsp;\u003c/em\u003eapproaches. ETSL exhibited significant in vitro antioxidant properties as well as in vitro anti-arthritic potential in a dose-dependent manner. Further, the inhibition of pro-inflammatory cytokines and increase in expression of anti-inflammatory cytokines support its in vivo anti-arthritic efficacy. The molecular docking analysis identified verbascoside as having the highest docking score against COX-2 and IL-1\u0026beta; among the various phenolic, flavonoid, and anti-inflammatory compounds detected through LC-MS and GC-MS analyses. This finding provides robust evidence for the potential clinical application of \u003cem\u003eTecoma stans\u003c/em\u003e leaf in managing RA, and affirming its traditional medicinal efficacy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALP: Alkaline phosphatase; ALT: Alanine amino transferase; AST: Aspartate aminotransferase; CAT: Catalase; CFA: Complete Freund\u0026apos;s Adjuvant; CMC-Na: Carboxy methyl cellulose-sodium;\u0026nbsp;COX-2: Cyclooxygenase-2;CRP: C-reactive protein; DMARDs: Disease-modifying anti-rheumatic drugs; DPPH: 2, 2-diphenyl-1-picrylhydrazyl;\u0026nbsp;EDTA: Ethylenediaminetetraacetic acid; ELISA: Enzyme-linked immunosorbent assay; ESR: Erythrocyte sedimentation rate; ETSL: Ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf; Fc: Fragment crystallizable form; GC-MS: Gas Chromatography-Mass Spectroscopy; GSH: Reduced glutathione; Hb: Haemoglobin; HRBC: Human red blood cells; IgG: Immunoglobulin G; IL-10: Interleukin-10;IL-1\u0026beta;: Interleukin-1-beta; IL-4: Interleukin-4;IL-6: Interleukin-6; LC-MS: Liquid Chromatography-Mass Spectrometry; LPO: Lipid peroxidation; LPS: Lipopolysaccharide; \u0026nbsp;MDA: Malondialdehydes; NIST: National Institute of Standards and Technology; NSAIDs: Non-steroidal anti-inflammatory drugs; RA: Rheumatoid arthritis ; RBC: Red blood cell; RBC: Red blood cells; RF: Rheumatoid factor; ROS: Reactive oxygen species; RT: Retention time; SD: Standard deviation; SOD: Superoxide dismutase; TNF-\u0026alpha;: Tumor necrosis factor-alpha;WBC: White blood cells\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThe research was funded by the DBT Builder project with order no (BT/INF/22/SP45078/2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u0026nbsp;\u003c/strong\u003eThe authors acknowledge the Researchers Supporting Project number (RSPD2024R708), King Saud University, Riyadh, Saudi Arabia for funding this research work. 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Inflammopharmacology 28:463-473 doi:10.1007/s10787-019-00642-0\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eIdentification of compounds from ethanol extract of \u003cem\u003eT. stans\u003c/em\u003e leaf by GC-MS analysis\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003eSl.no\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003eCompound name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eM.F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003eM.W\u0026nbsp;(g/mol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eNature of compound\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003eArea (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e11.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003ePentadecane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e212.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eAlkane\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e1.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e16.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003eHeptadecane, 2,6,10,15-tetramethyl-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e44\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e296.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eAlkane\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e2.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e19.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003e1-Hexadecanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e242.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eAlcohol\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e2.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e24.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003eCetene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e224.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eAlkene\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e2.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e27.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003eDimethyl palmitamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e39\u003c/sub\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e269.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eAmine\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e3.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e29.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003e3-Eicosene, (E)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e40\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e280.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eAlkene\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e2.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e31.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003eMethyl 9-cis,11-trans-octadecadienoate\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e294.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eEster\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e2.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e31.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003ecis-13-Octadecenoic acid, methyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e36\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e296.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eEster\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e2.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e37.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003e13-Docosenamide, (Z)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e43\u003c/sub\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e337.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eCarboxamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e18.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e43.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003e9-Octadecenamide, (Z)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e35\u003c/sub\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e281.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eCarboxamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e7.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e49.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003e7,8-Epoxylanostan-11-ol, 3-acetoxy-\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e32\u003c/sub\u003eH\u003csub\u003e54\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e502.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eEpoxide\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e2.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e49.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003eHexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e35\u003c/sub\u003eH\u003csub\u003e68\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e568.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eEster\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e2.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e49.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003eEicosanoic acid, 9-octadecenyl ester, (Z)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e38\u003c/sub\u003eH\u003csub\u003e74\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e563.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eEster\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e50.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003eEthyl iso-allocholate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e26\u003c/sub\u003eH\u003csub\u003e44\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e436.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eEster\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e50.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003e9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e40\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e356.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eEster\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e1.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e50.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003e9-Octadecenoic acid, 1,2,3-propanetriyl ester,\u003c/p\u003e\n \u003cp\u003e(E,E,E)-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e57\u003c/sub\u003eH\u003csub\u003e104\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e885.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eEster\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e50.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003eOleic acid, eicosyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e38\u003c/sub\u003eH\u003csub\u003e74\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eEster\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.503075030750307%\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.626076260762607%\" valign=\"top\"\u003e\n \u003cp\u003e50.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"45.018450184501845%\" valign=\"top\"\u003e\n \u003cp\u003e9-Octadecenoic acid (Z)-, 2-hydroxy-3-[(1-oxohexadecyl)oxy]propyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.96309963099631%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e37\u003c/sub\u003eH\u003csub\u003e70\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.348093480934809%\" valign=\"top\"\u003e\n \u003cp\u003e594.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.423124231242312%\" valign=\"top\"\u003e\n \u003cp\u003eEster\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.118081180811808%\" valign=\"top\"\u003e\n \u003cp\u003e1.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eRT: Retention time; M.F: Molecular formula; M.W: Molecular weight\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eIdentification of compounds from ethanol extract of \u003cem\u003eT. stans\u003c/em\u003e leaf by LC-MS analysis\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"896\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003eSl.no\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003ePeak no.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003em/z\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e[M+H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003em/z\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003eExact mass\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eM.F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eProposed metabolite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003eChemical nature\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e25.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e160.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e160.216\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eTryptamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003eAmino acid\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e7.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e161.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e161.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eBoschniakine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003eAlkaloid\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e5.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e163.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e164.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003ep-Coumaric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003ePhenolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e7.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e169.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e170.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eGallic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003ePhenolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e29.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e181.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e180.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eCaffeic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003ePhenolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e13.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e195.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e194.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eFerulic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003ePhenolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e16.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e203.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e204.229\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eTryptophan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003eAmino acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e6.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e288.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e270.24\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eApigenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003eFlavonoid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e7.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e287.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e286.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eKaempferol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003eFlavonoid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e6.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e285.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e286.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eLuteolin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003ePhenolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e26.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e291.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e290.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003e(-)-Catechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003ePhenolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e8.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e302.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e302.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eQuercetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003eFlavonoid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e6.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e301.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e302.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eHesperetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003ePhenolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e8.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e353.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e360.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eRosmarinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003ePhenolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e16.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e353.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e354.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eChlorogenic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003ePhenolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e12.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e415.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e414.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e50\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eSitosterols\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003ePhenolic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e9.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e447.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e448.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eQuercitrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003eFlavonoid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e10.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e580.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e580.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eNaringin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003ePhenolic acid\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e8.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e617.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e610.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eRutin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003eFlavonoid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.9284116331096195%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.487695749440716%\" valign=\"top\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.382550335570469%\" valign=\"top\"\u003e\n \u003cp\u003e11.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.507829977628635%\" valign=\"top\"\u003e\n \u003cp\u003e624.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.626398210290828%\" valign=\"top\"\u003e\n \u003cp\u003e624.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.51454138702461%\" valign=\"top\"\u003e\n \u003cp\u003eC\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e36\u003c/sub\u003eO\u003csub\u003e15\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.39821029082774%\" valign=\"top\"\u003e\n \u003cp\u003eVerbascoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.639821029082775%\" valign=\"top\"\u003e\n \u003cp\u003eFlavonoid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eRT: Retention time; M.F: Molecular formula\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eEffect of ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf on haematological and biochemical parameters in CFA-induced arthritis in rat\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.141414141414142%\" rowspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"85.85858585858585%\" colspan=\"5\" valign=\"bottom\"\u003e\n \u003cp\u003eTreatment groups\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.457831325301205%\" valign=\"bottom\"\u003e\n \u003cp\u003eNC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.27710843373494%\" valign=\"bottom\"\u003e\n \u003cp\u003eCFA (0.1 ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.096385542168676%\" valign=\"bottom\"\u003e\n \u003cp\u003eIND (10\u0026thinsp;mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.686746987951807%\" valign=\"bottom\"\u003e\n \u003cp\u003eETSL (250 mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.481927710843372%\" valign=\"bottom\"\u003e\n \u003cp\u003eETSL (500 mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\" valign=\"bottom\"\u003e\n \u003cp\u003eRBC (X10\u003csup\u003e6\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"bottom\"\u003e\n \u003cp\u003e6.31\u0026plusmn;0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\" valign=\"bottom\"\u003e\n \u003cp\u003e2.78\u0026plusmn;1.48##\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\" valign=\"bottom\"\u003e\n \u003cp\u003e5.24\u0026plusmn;1.56**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\" valign=\"bottom\"\u003e\n \u003cp\u003e3.78\u0026plusmn;1.52\u003cstrong\u003e\u003csup\u003ens\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.52577319587629%\" valign=\"bottom\"\u003e\n \u003cp\u003e4.67\u0026plusmn;0.94*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\" valign=\"bottom\"\u003e\n \u003cp\u003eWBC (X10\u003csup\u003e3\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"bottom\"\u003e\n \u003cp\u003e5.02\u0026plusmn;1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\" valign=\"bottom\"\u003e\n \u003cp\u003e12.13\u0026plusmn;0.76##\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\" valign=\"bottom\"\u003e\n \u003cp\u003e6.96\u0026plusmn;1.42***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\" valign=\"bottom\"\u003e\n \u003cp\u003e10.47\u0026plusmn;1.02*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.52577319587629%\" valign=\"bottom\"\u003e\n \u003cp\u003e8.06\u0026plusmn;1.65*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\" valign=\"bottom\"\u003e\n \u003cp\u003ePlatelets\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(X 10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"bottom\"\u003e\n \u003cp\u003e948.65\u0026plusmn;7.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\" valign=\"bottom\"\u003e\n \u003cp\u003e1323.92\u0026plusmn;1.99##\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\" valign=\"bottom\"\u003e\n \u003cp\u003e1014.86\u0026plusmn;5.50**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\" valign=\"bottom\"\u003e\n \u003cp\u003e1191.77\u0026plusmn;4.14*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.52577319587629%\" valign=\"bottom\"\u003e\n \u003cp\u003e1062.07\u0026plusmn;5.21**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\" valign=\"bottom\"\u003e\n \u003cp\u003eAST (IU/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"bottom\"\u003e\n \u003cp\u003e23.28\u0026plusmn;2.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\" valign=\"bottom\"\u003e\n \u003cp\u003e52.30\u0026plusmn;3.16###\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\" valign=\"bottom\"\u003e\n \u003cp\u003e26.97\u0026plusmn;2.36***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\" valign=\"bottom\"\u003e\n \u003cp\u003e39.30\u0026plusmn;2.39**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.52577319587629%\" valign=\"bottom\"\u003e\n \u003cp\u003e35.17\u0026plusmn;2.59***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\" valign=\"bottom\"\u003e\n \u003cp\u003eALT (IU/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"bottom\"\u003e\n \u003cp\u003e109.14\u0026plusmn;5.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\" valign=\"bottom\"\u003e\n \u003cp\u003e152.82\u0026plusmn;7.05##\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\" valign=\"bottom\"\u003e\n \u003cp\u003e119.16\u0026plusmn;3.87***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\" valign=\"bottom\"\u003e\n \u003cp\u003e130.27\u0026plusmn;3.95**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.52577319587629%\" valign=\"bottom\"\u003e\n \u003cp\u003e124.92\u0026plusmn;3.17**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\" valign=\"bottom\"\u003e\n \u003cp\u003eALP (IU/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"bottom\"\u003e\n \u003cp\u003e154.40\u0026plusmn;2.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\" valign=\"bottom\"\u003e\n \u003cp\u003e299.07\u0026plusmn;5.60###\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\" valign=\"bottom\"\u003e\n \u003cp\u003e181.47\u0026plusmn;6.22***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\" valign=\"bottom\"\u003e\n \u003cp\u003e234.88\u0026plusmn;9.33**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.52577319587629%\" valign=\"bottom\"\u003e\n \u003cp\u003e203.06\u0026plusmn;6.74***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\" valign=\"bottom\"\u003e\n \u003cp\u003eUREA (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"bottom\"\u003e\n \u003cp\u003e0.51\u0026plusmn;0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\" valign=\"bottom\"\u003e\n \u003cp\u003e1.03\u0026plusmn;0.47##\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\" valign=\"bottom\"\u003e\n \u003cp\u003e0.61\u0026plusmn;0.35***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\" valign=\"bottom\"\u003e\n \u003cp\u003e0.77\u0026plusmn;0.88**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.52577319587629%\" valign=\"bottom\"\u003e\n \u003cp\u003e0.69\u0026plusmn;0.46**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.43298969072165%\" valign=\"bottom\"\u003e\n \u003cp\u003eCREAT (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"bottom\"\u003e\n \u003cp\u003e19.25\u0026plusmn;1.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.49484536082474%\" valign=\"bottom\"\u003e\n \u003cp\u003e48.32\u0026plusmn;4.66###\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.61855670103093%\" valign=\"bottom\"\u003e\n \u003cp\u003e26.39\u0026plusmn;2.56***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.556701030927837%\" valign=\"bottom\"\u003e\n \u003cp\u003e36.57\u0026plusmn;2.91**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.52577319587629%\" valign=\"bottom\"\u003e\n \u003cp\u003e31.07\u0026plusmn;2.92***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eRBC; Red blood cells, WBC; White blood cell, AST; Aspartate aminotransferase, ALT; Alanine transaminase, ALP; Alkaline phosphatase; CFREAT-Creatinine. Values are expressed as mean \u0026plusmn; SD (n=6). All data were expressed as mean \u0026plusmn; standard deviation (SD), (n= 6). The statistical significance of difference between various groups was tested by one-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison tests. Comparisons were made between NC vs CFA, and CFA vs IND (10 mg/kg), ETSL (250 mg/kg) and ETSL (500 mg/kg). The level of significant difference was checked between NC vs CFA at \u003csup\u003e##\u003c/sup\u003eP˂ 0.01 and \u003csup\u003e###\u003c/sup\u003eP˂ 0.001 and between\u0026nbsp;CFA\u0026nbsp;and IND, ETSL (250 mg/kg) and ETSL (500 mg/kg) at *P˂0.05, **P˂0.01 and ***P˂0.001.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e\u003cem\u003eIn silico\u0026nbsp;\u003c/em\u003edocking studies of 2AZ5 (TNF-\u0026alpha;), 4G6J (IL-1\u0026beta;), 1ALU (IL-6) and 1PXX (COX-2) by various ligands.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.324661810613943%\" valign=\"top\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.735691987513007%\" valign=\"top\"\u003e\n \u003cp\u003eLigand/inhibitor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.781477627471384%\" valign=\"top\"\u003e\n \u003cp\u003eBinding affinity (kcal/mol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.57544224765869%\" valign=\"top\"\u003e\n \u003cp\u003eInteracting residues\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.667013527575442%\" valign=\"top\"\u003e\n \u003cp\u003eHydrogen-bond interacting active binding site residues\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.915712799167533%\" valign=\"top\"\u003e\n \u003cp\u003eHydrophobic Interaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.324661810613943%\" rowspan=\"12\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2AZ5\u003c/p\u003e\n \u003cp\u003e(TNF-\u0026alpha;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.735691987513007%\" valign=\"top\"\u003e\n \u003cp\u003eApigenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.781477627471384%\" valign=\"top\"\u003e\n \u003cp\u003e-6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.57544224765869%\" valign=\"top\"\u003e\n \u003cp\u003eVAL91, LEU93, GLN125, ARG82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.667013527575442%\" valign=\"top\"\u003e\n \u003cp\u003eARG82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.915712799167533%\" valign=\"top\"\u003e\n \u003cp\u003eVAL91, LEU93, GLN125\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eKaempferol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eGLY121, LEU120, SER95, ALA96, LYS98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eGLY121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eLEU120, SER95, ALA96, LYS98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003e(-)-Catechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eGLY121, LEU120, SER95, ALA96, TYR119, PRO117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eSER95, GLY121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eLEU120, ALA96, TYR119, PRO117\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eHesperetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eLEU55, ALA96, GLY121, LEU120, TYR119, PRO117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eGLY121, PRO117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eLEU55, ALA96, LEU120, TYR119\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eRosmarinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eVAL91, ARG82, LEU93, GLN125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eARG82, LEU93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eVAL91, GLN125\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eChlorogenic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eLEU57, TYR59, HIS15, TYR151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eTYR151, TYR59, HIS15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eLEU57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eSitosterols\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eTYR151, TYR59, LEU57, ILE155, LEU157, VAL123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eTYR151, TYR59, LEU57, ILE155, LEU157, VAL123\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eQuercitrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eLYS98, PRO117, TYR119, LEU120, GLY121, LEU55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003ePRO117, TYR119, LEU55, GLY121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eLYS98, LEU120\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eNaringin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eLYS11, GLN61, TYR59, TYR151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eGLN61, TYR151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eLYS11, TYR59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eRutin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eTYR119, PRO117, ILE118, LEU120, GLY121, LEU55, GLN125, GLY54, GLU53, ILE97, LYS98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eGLU53, PRO117, TYR119, LYS98, ILE97, GLN125, GLY121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eTYR119, ILE118, LEU120, LEU55, GLY54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eVerbascoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eTYR151, TYR59, LEU157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eTYR151\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eTYR59, LEU157\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eIndomethacin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-8.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eVAL123, LEU157, TYR59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eVAL123, LEU157, TYR59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.324661810613943%\" rowspan=\"12\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4G6J (IL-1\u0026beta;)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.735691987513007%\" valign=\"top\"\u003e\n \u003cp\u003eKaempferol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.781477627471384%\" valign=\"top\"\u003e\n \u003cp\u003e-7.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.57544224765869%\" valign=\"top\"\u003e\n \u003cp\u003eTRP47, GLU46, PHE98, THR97, PHE96, ALA61, ASP62, VAL3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.667013527575442%\" valign=\"top\"\u003e\n \u003cp\u003eTRP47, PHE98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.915712799167533%\" valign=\"top\"\u003e\n \u003cp\u003eGLU46, THR97, PHE96, ALA61, ASP62, VAL3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003e(-)-Catechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eALA61, ASP62, TRP47, GLU46, PHE96, THR97, PHE98, PRO100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003ePHE96, PHE98, ASP62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eALA61, TRP47, GLU46, THR97, PRO100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eQuercetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eASP62, ALA61, TRP47, GLU46, THR97, PHE98, PRO100, LEU4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003ePHE98, LEU4, TRP47, ASP62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eALA61, GLU46, THR97, PRO100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eHesperetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eTRY95, VAL93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eTRY95, VAL93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eRosmarinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eTYR87, GLN39, LYS43, ALA85, LYS103, ALA84, ASP41, PRO40, GLU165, ALA83, GLN166\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eASP41, GLN39, TYR87, ALA85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eLYS43, LYS103, ALA84, PRO40, GLU165, ALA83, GLN166\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eChlorogenic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eGLN166, GLU165, LYS103, ALA85, ALA84, ALA83, PRO40, ASP41, GLN38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eGLN166\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eGLU165, LYS103, ALA85, ALA84, ALA83, PRO40, ASP41, GLN38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eSitosterols\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eTYR87, GLN39, PRO41, VAL93, LEU113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eTYR87, GLN39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003ePRO41, VAL93, LEU113\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eQuercitrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eLYS39, GLN42, ASP41, GLN110, PRO41, VAL93, LEU113, GLU153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eLYS39, GLN42, GLN110, GLU153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eASP41, PRO41, VAL93, LEU113\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eNaringin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eVAL155, GLU153, ALA173, ALA42, PRO172, GLN110, PRO40, ALA85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eGLN110, ALA85, ALA42, VAL155, GLU153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eALA173, PRO172, PRO40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eRutin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003ePRO172, VAL155, GLU153, GLU165, PRO41, LEU113, ASP41, VAL93, GLN110, GLN42, LYS39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eGLU165, GLN42, LYS39, GLU153, GLN110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003ePRO172, VAL155, PRO41, LEU113, ASP41, VAL93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eVerbascoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eALA85, LYS103, ALA84, ALA83, GLN166, GLU165, PRO40, ASP41, ALA173, VAL155, GLU153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eLYS103, VAL155, GLU153, GLN166, ALA85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eALA84, ALA83, GLU165, PRO40, ASP41, ALA173\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eIndomethacin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eLYS39, GLN42, PRO172, ALA42, GLN39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eLYS39, GLN42, GLN39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003ePRO172, ALA42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.324661810613943%\" rowspan=\"6\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1ALU (IL-6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.735691987513007%\" valign=\"top\"\u003e\n \u003cp\u003eHesperetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.781477627471384%\" valign=\"top\"\u003e\n \u003cp\u003e-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.57544224765869%\" valign=\"top\"\u003e\n \u003cp\u003eLEU19, LYS27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.667013527575442%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.915712799167533%\" valign=\"top\"\u003e\n \u003cp\u003eLEU19, LYS27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eQuercitrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eLEU19, GLU23, ASP26, LYS27, ARG30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eLEU19, GLU23, ASP26, LYS27, ARG30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eNaringin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eLEU19, LYS27, GLU23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eLEU19, LYS27, GLU23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eRutin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eARG30, LYS27, LEU19, GLU23, ASP26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eARG30, LYS27, LEU19, GLU23, ASP26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eVerbascoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eGLU23, LEU19, LYS27, ARG30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eGLU23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eLEU19, LYS27, ARG30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eIndomethacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eALA114, TYR31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eALA114, TYR31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.324661810613943%\" rowspan=\"18\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1PXX (COX-2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.735691987513007%\" valign=\"top\"\u003e\n \u003cp\u003eTryptamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.781477627471384%\" valign=\"top\"\u003e\n \u003cp\u003e-6.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.57544224765869%\" valign=\"top\"\u003e\n \u003cp\u003eLEU390, ALA199, ALA202, GLN203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.667013527575442%\" valign=\"top\"\u003e\n \u003cp\u003eGLN203, ALA199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.915712799167533%\" valign=\"top\"\u003e\n \u003cp\u003eLEU390, ALA202\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003ep-Coumaric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eASN382, HIS388, TRP387, ALA202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eASN382\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eHIS388, TRP387, ALA202\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eGallic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eARG469, ILE124, ALA151, ASP125, THR149, THR129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eALA151, THR149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eARG469, ILE124, ASP125, THR129\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eCaffeic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eTRP387, HIS386, TYR148, TYR385, ASN382, THR206, HIS207\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eTYR148, ASN382, THR206, TRP387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eHIS386, TYR385, HIS207\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eFerulic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-6.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eHIS388, TRP387, ASN382, ALA202, ALA199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eASN382, ALA199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eHIS388, TRP387, ALA202\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eTryptophan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eARG120, TRP1, VAL523, GLY526\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eGLY526\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eARG120, VAL523\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eApigenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-9.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eARG44, CYS47, ARG469, LYS468, GLU465, PRO153, LEU152\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eCYS47, GLU465\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eARG44, ARG469, LYS468, PRO153, LEU152\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eKaempferol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-8.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eLYS468, ARG469, ARG44, GLN42, CYS47, LEU152, PRO153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eGLN42, LYS468\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eARG469, ARG44, CYS47, LEU152, PRO153\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003e(-)-Catechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-8.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eASN39, CYS37, ASN34, ALA156, PRO154, PRO153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eCYS37, ASN39, ASN34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eALA156, PRO154, PRO153\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eHesperetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-9.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eARG469, ARG44, CYS47, CYS36, PRO153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eCYS47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eARG469, ARG44, CYS36, PRO153\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eRosmarinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-9.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eARG44, GLY45, CYS47, CYS36, ASN39, PRO153, LEU152, CYS37, ALA156, VAL155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eARG44, GLY45, CYS47, ASN39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eCYS36, PRO153, LEU152, CYS37, ALA156, VAL155\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eChlorogenic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-9.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eARG44, GLY45, CYS41, ASN34, ALA156, GLU465, PRO153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eASN34, GLY45, ARG44, GLU465\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eCYS41, ALA156, PRO153\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eSitosterols\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-8.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eARG469, ARG44, TYR122, SER471, LYS79, LEU80, LYS83, LEU82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eARG469\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eARG44, TYR122, SER471, LYS79, LEU80, LYS83, LEU82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eQuercitrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eHIS386, PHE210, LYS211, THR212, ARG222, GLN289, GLU290, VAL291, ILE274\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eGLN289, ARG222, THR212, LYS211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eHIS386, PHE210, GLU290, VAL291, ILE274\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eNaringin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-10.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eSER1518, GLU46, CYS47, CYS36, ASN39, GLN461, ALA156, PRO154, GLY135, GLN1327, TYR130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eGLU46, PRO154, CYS47, ASN39, GLN461, CYS36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eSER1518, ALA156, GLY135, GLN1327, TYR130\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eRutin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-10.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eTYR130, GLY45, CYS41, CYS47, MET48, CYS36, SER49, ALA156, TYR136, HIS133, GLN1327\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eHIS133, SER49, CYS47, CYS36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eTYR130, GLY45, CYS41, MET48, ALA156, TYR136, GLN1327\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eVerbascoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-10.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eMET48, CYS47, CYS36, ASN39, GLU465, GLN461, LEU152, ALA156, HIS133, GLN1327\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003eHIS133, ASN39, GLU465, GLN461, CYS47, ALA156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eMET48, CYS36, LEU152, GLN1327\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.982973893303065%\" valign=\"top\"\u003e\n \u003cp\u003eIndomethacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.669693530079455%\" valign=\"top\"\u003e\n \u003cp\u003e-8.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.0794551645857%\" valign=\"top\"\u003e\n \u003cp\u003eCYS1036, CYS1047, PRO1153, TYR1130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.45289443813848%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.814982973893304%\" valign=\"top\"\u003e\n \u003cp\u003eCYS1036, CYS1047, PRO1153, TYR1130\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Tecomastans, Anti-oxidant, Complete Freund’s Adjuvant, Cytokines, GC-MS, LC-MS, in-silico.","lastPublishedDoi":"10.21203/rs.3.rs-4224044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4224044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eTecoma stans\u003c/em\u003e (L.) Juss.exKunth (Bignoniaceae) is mainly found in tropical and subtropical regions of Africa and Asia. The leaves, flowers, roots, and bark are used to treat various aliments includes, skin infections, kidney problems, intestinal disorders, jaundice, toothaches, joint pain and repair cracked bones, antidotes for snake, scorpion, and rat bites. The aim of the study is to assess the anti-arthritic properties of \u003cem\u003eT. stans\u003c/em\u003eleaf using Complete Freund's adjuvant (CFA)-induced rat model. The ethanol extract of \u003cem\u003eT. stans\u003c/em\u003eleaf (ETSL) was taken for Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography-Mass Spectrometry (LC-MS) analysis for the identification of potential bioactive. The in vitro antioxidant and anti-arthritic activity was studied at concentrations of 25, 50, 100, 200, 400, and 500 μg/ml. In vivo anti-arthritic activity was carried out by administering CFA (0.1 ml) into the sub-plantar surface of the right hind paw. The experimental animals were treated with indomethacin (10 mg/kg) and ETSL (250, 500 mg/kg) once a daily for fourteen days. The arthritic parameters such as paw thickness, arthritic index, arthritic score, body weight, organ weight, and hematological and biochemical parameters were evaluated. Pro-inflammatory cytokines; tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, anti-inflammatory cytokines; IL-4 and IL-10 and inflammatory mediator cyclooxygenase-2 (COX-2) were examined in blood serum. In vivo antioxidants parameters; superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and lipid peroxidation (LPO) was carried out in liver and joint. Radiological and histopathological analysis of joint was performed.A computational molecular docking investigation of the phytoconstituents was conducted against COX-2, IL-1β, IL-6, and TNF-α receptors by utilizing AutoDock 4.2 and BIOVIA-Discovery Studio Visualizer software. The in vitro result showed concentration dependent antioxidant activity with highest percentage of inhibition at 500 µg/ml. The in vivo result demonstrated significant restoration of arthritic parameters, hematological and biochemical indices and oxidative stress in CFA-induced rat which was further supported by radiological histological examination at ETSL 500 mg/kg. In addition, there was significant (p\u0026lt;0.05) reduction in pro-inflammatory cytokines, inflammatory mediators and up-regulation of anti-inflammatory cytokines was observed in the treated group. Verbascoside was found to exhibit better biding affinities -10.4, -7.4, -7 and -6.2 kcal/mol against COX-2, IL-1β, TNF-α, and IL-6 respectively, confirmed through in silico study. The observed outcome suggests that ETSL at a dosage of 500 mg/kg demonstrated notable anti-arthritic effects by suppressing pro-inflammatory cytokines and oxidative stress biomarkers. This effect could potentially be attributed to the presence of bioactive verbascoside identified in the LC-MS analysis.\u003c/p\u003e","manuscriptTitle":"Protective effect of Tecomastans (L.) Juss.exKunth in CFA-induced arthritic rat model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-12 08:50:49","doi":"10.21203/rs.3.rs-4224044/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"71fedb25-0c2e-4c07-8612-20e631f88016","owner":[],"postedDate":"April 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-29T20:18:46+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-12 08:50:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4224044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4224044","identity":"rs-4224044","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}