| کلیدواژههای انگلیسی مقاله |
Traumatic brain injury, Phytotherapy, Molecular docking simulation, Oxidative stress, Superoxide dismutase, What&,rsquo s Known Traumatic brain injury (TBI) is a major health issue with limited therapeutic options. Natural compounds with anti-inflammatory and neuroprotective properties have shown potential in TBI treatment. Certain bioactive compounds, including fatty acids and silane derivatives, possess pharmacokinetic properties that enable blood-brain barrier penetration and neuroprotection. What&,rsquo s New This study identifies nanoherbal Paraboea leuserensis as a novel therapeutic candidate for TBI. Molecular docking confirms strong interactions with ERK2, CCR2, and JNK3, suggesting neuroprotective effects. In vivo tests show increased SOD activity and reduced MDA levels, highlighting its antioxidative and anti-inflammatory potential. Findings support its development for neural recovery and pharmaceutical applications. IntroductionTraumatic brain injury (TBI) is a significant global public health issue and is among the leading causes of mortality and disability, affecting an estimated 64&,ndash 74 million individuals annually. 1, TBI is a common neurological injury with a high morbidity rate. Its impact on mental health is increasingly recognized as a major consequence, potentially affecting the quality of life and daily functioning. 2, The etiology of TBI includes various factors, such as falls, traffic accidents, sports-related injuries, and physical violence, including blast injuries. The mechanisms of injury are influenced by factors such as geographic region, socioeconomic status, age, and sex. 3, Post-TBI symptoms may encompass physical manifestations, such as nausea, dizziness, and blurred vision, as well as cognitive and emotional disturbances, such as executive dysfunction, depression, and anxiety. 4, The pathological mechanisms of TBI encompass both primary and secondary damage. Secondary damage, which occurs post-trauma, involves the activation of various cellular signaling pathways, including the inflammatory response. Cellular mechanosensors compromised by high pressure can initiate ongoing injury, particularly in cases of repeated trauma. 5, Following injury, a complex interaction transpires between pro-inflammatory and anti-inflammatory signaling pathways involving extracellular signal-regulated kinase 2 (ERK2), c-Jun N-terminal kinase 3 (JNK3), and C-C chemokine receptor type 2 (CCR2). ERK2 and JNK3 are recognized for their contributions to the production of pro-inflammatory cytokines, thereby exacerbating neuroinflammation, whereas CCR2 is instrumental in recruiting monocytes and microglia to the injury site, further aggravating neuronal damage. 6, Although JNK3 is frequently associated with apoptosis, under certain conditions, it can also promote neuronal survival, indicating a dual role in the pathophysiology of TBI. 7, , 8, Consequently, interest in the ERK2, CCR2, and JNK3 signaling pathways in TBI therapy is increasing because of their potential to address inflammation and neurodegeneration. ERK2 and JNK3 regulate inflammatory responses and neuronal apoptosis, whereas CCR2 mediates the recruitment of immune cells, rendering them strategic targets for anti-inflammatory interventions. 9, , 10, The administration of corticosteroids, such as methylprednisolone, is a common therapeutic strategy for managing inflammation following TBI. Methylprednisolone acetate, a 6-methyl derivative of prednisolone, is classified as a synthetic glucocorticoid with significant anti-inflammatory effects. This compound is characterized by its appearance as a white crystalline powder, which is insoluble in water but soluble in organic solvents, such as alcohol, chloroform, and methanol. It has a molecular formula of C22H30O5 and a molecular weight of 416.51 Da. Methylprednisolone is extensively used to treat various inflammatory conditions, including multiple myeloma, rheumatic disorders, respiratory diseases, kidney diseases, ocular issues, hematological disorders, neoplastic diseases, nervous system disorders, gastrointestinal illnesses, endocrine dysfunction, and dermatological problems. 11, In the context of TBI, methylprednisolone is employed to suppress secondary inflammatory responses, mitigate cerebral edema, and stabilize cell membranes and pro-inflammatory cytokine production. However, the administration of high doses following TBI may exacerbate apoptosis in the hypothalamic and pituitary regions, potentially leading to critical illness-related corticosteroid insufficiency (CIRCI) and increasing the risk of mortality during the acute phase. 12, , 13, The limitations of methylprednisolone efficacy, coupled with its potential adverse effects, have prompted the pursuit of safer and more natural therapeutic alternatives. Prior research has investigated the application of medicinal plants in the treatment of TBI, with Aloe vera and cinnamon emerging as notable candidates due to their demonstrated antioxidant and anti-inflammatory properties, as well as their protective effects on brain tissue in animal models. 14, , 15, These findings further substantiate the potential of herbal plants as complementary therapeutic options for managing TBI.One potential candidate is Paraboea leuserensis, an endemic plant from the Leuser ecosystem found in the provinces of Aceh and North Sumatra, including the regions of Dairi, Karo, and Langkat (Indonesia). This plant belongs to the Gesneriaceae family, which comprises approximately 3,500 species from 147&,ndash 150 genera distributed across tropical and subtropical regions. 16, Among the Karo people, Paraboea leuserensis is known as &,ldquo Gagatan Harimau&,rdquo and has been traditionally used as a remedy for stomach ailments and as a stamina booster. 17, , 18, Pharmacologically, Paraboea is recognized for its anti-inflammatory and antioxidant properties, primarily due to its key bioactive constituents, including flavonoids such as myricetin, myricitrin, quercetin, kaempferol, and ellagic acid. These compounds are widely recognized for their neuroprotective and immunomodulatory effects, 19, making the exploration of Paraboea leuserensis as a natural anti-inflammatory agent for TBI therapy highly relevant.Therefore, this study aimed to evaluate the therapeutic potential of nanoherbal compounds from Paraboea leuserensis using in silico and in vivo approaches in a rat model of TBI. An in silico approach is employed to assess the interaction of the compounds with key target proteins involved in post-TBI inflammation and neurodegeneration, namely ERK2, JNK3, and CCR2, using molecular docking methods. An in vivo approach is used to evaluate the biological effects of the compounds on oxidative stress biomarkers, such as MDA levels and SOD activity. Materials and Methods Plant Collection and Nanoherbal Preparation Paraboea leuserensis B.L. Burtt leaves were collected from Timbang Lawan Village, Pancur Batu Subdistrict, Deli Serdang Regency, North Sumatra Province. Plant identification was conducted by the Herbarium Bogoriense BRIN Cibinong (No. B-1216/II.6.2/IR.01.02/6/2023). The collected leaves were then air-dried and ground using a blender. Subsequently, the leaves were processed into nanoparticles using a Planetary Ball Mill (PBM, Changsha Tianchuang Powder Technology Co., Ltd., China) at Nanotech Indonesia. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis Bioactive compounds from Paraboea leuserensis in 96% ethanol were identified using GC-MS (Shimadzu QP 2010S, Kyoto, Japan), employing Wiley/NIST library software for data analysis. The analysis was conducted with an Rtx-5 ms column measuring 30 m. The injector and detector temperatures were maintained at 250 &,deg C, while the operating temperature ranged from 50 to 300 &,deg C. The column temperature was programmed to increase from 50 &,deg C to 120 &,deg C at a rate of 4 &,deg C per min, held for one min, and then raised from 120 &,deg C to 300 &,deg C at a rate of 6 &,deg C per min, held for 5 min, resulting in a total retention time of 80 min. Helium was used as the carrier gas, with a mass-to-charge ratio range of 50-500 AMU. Electron ionization was performed at 70 eV. 20, In Silico Experiments Bioactive Compounds from Paraboea leuserensis, GC-MS analysis (nanoherbal methanol extract) identified seven bioactive compounds from Paraboea leuserensis in this study. The identification of these compounds was confirmed using the PubChem database (https,//pubchem.ncbi.nlm.nih.gov, National Center for Biotechnology Information, USA) to obtain compound names and their corresponding PubChem CID numbers. The identified compounds included silane (CAS) (PubChem CID, 7918), hexadecanoic acid (CAS) (PubChem CID, 985), 9-Octadecenoic acid (Z) (CAS) (PubChem CID, 445639), and 9-Octadecen-12-ynoic acid methyl ester (CAS) (PubChem CID, 5363161). In addition, three other compounds comprising 3-hydroxymethyl-5-(4-nitroimidazol-l-yl) isoxazole, 3-hexadecyloxycarbonyl-5-(2-hydroxyethyl)-4-methylimidazolium ion, and high-oleic safflower oil (CAS) were also detected, although no corresponding PubChem CID records were available for these compounds. Drug-Likeness and Pharmacokinetic Prediction, The compounds of the nanoherbal Paraboea leuserensis used in this study were obtained from the PubChem database (https,//pubchem.ncbi.nlm.nih.gov,) in the form of two-dimensional (2D) structures (.sdf format) and Simplified Molecular Input Line Entry System (SMILES) notations. Drug-likeness was evaluated using the SwissADME web server (http,//www.swissadme.ch, Swiss Institute of Bioinformatics, Switzerland) based on Lipinski&,rsquo s Rule of Five, which considers molecular weight (&,le 500 Da), lipophilicity (LogP&,le 5), hydrogen bond donors (&,le 5), and hydrogen bond acceptors (&,le 10) as essential parameters for predicting oral bioavailability. 21, Pharmacokinetic properties, including absorption, distribution, metabolism, and excretion (ADME), were predicted using the pkCSM web server (http,//biosig.lab.uq.edu.au/pkcsm/prediction, University of Queensland, Australia). The absorption parameters were assessed based on human intestinal absorption (HIA) and Caco-2 cell permeability, while the distribution properties were evaluated through predictions of blood-brain barrier (BBB) permeability. Metabolism potential was analyzed by determining the interaction of each compound with cytochrome P450 2D6 (CYP2D6), identifying whether the compound acts as a substrate or inhibitor. The excretion potential was assessed using the substrate affinity for organic cation transporter 2 (OCT2). Additionally, aqueous solubility (LogS) was predicted using the Estimated Solubility (ESOL) model to estimate the water solubility of the compounds. The Brain or Intestinal Estimated Permeation Methods (BOILED-Egg model) was also applied to visualize the probability of HIA and BBB permeability, providing a comprehensive prediction of the pharmacokinetic behavior of these nanoherbal compounds.Screening of the Biological Activities of Compounds Using the Prediction of Activity Spectra for Substances (PASS) Online Test, The biological activities of compounds derived from the Paraboea leuserensis nanoherbal extract were predicted utilizing the Prediction of Activity Spectra of Substances (PASS) online tool, accessible via the Way2drug webserver (http,//way2drug.com/PassOnline/, Institute of Biomedical Chemistry, Russia). Initially, the compound names were entered into the PubChem database (http,//pubchem.ncbi.nlm.nih.gov,) to retrieve the corresponding SMILES structures. These SMILES were subsequently uploaded to the PASS server to predict the potential biological activities. PASS analysis yielded two critical parameters for each compound, Pa (probability of activity) and Pi (probability of inactivity). A compound is deemed to possess potential biological activity when its Pa value surpasses its Pi value. According to the PASS prediction criteria, the Pa is categorized into three levels, Pa&,gt 0.7 indicates a high likelihood of biological activity, 0.5&,lt Pa&,lt 0.7 suggests moderate activity, and Pa&,lt 0.5 indicates a low probability of activity. 22, , 23, Molecular Docking Molecular docking was executed using BIOVIA Discovery Studio software (BIOVIA, Dassault Syst&,egrave mes, France) for ligand preparation, where ligand structures were initially downloaded from the PubChem database (http,//pubchem.ncbi.nlm.nih.gov,) and saved in SDF format for further analysis. The ligands underwent energy minimization utilizing Open Babel software integrated with PyRx v.0.8 (The Scripps Research Institute, USA), which included not only the phytochemical compounds from Paraboea leuserensis but also native ligands and appropriate standard drugs. 23, The three-dimensional structures of the target proteins ERK2 (PDB ID, 5NHJ), CCR2 (PDB ID, 6GPS), and JNK3 (PDB ID, 7KSK) were obtained from the Protein Data Bank (PDB https,//www.rcsb.org/, Research Collaboratory for Structural Bioinformatics, USA) and validated using X-ray diffraction methods, with water molecules removed from the protein structures via PYMOL software (Schr&,ouml dinger, LLC, USA). Molecular docking and visualization were performed using AutoDock Vina integrated within PyRx v0.8, employing a targeted docking approach with an exhaustiveness setting of 8 to predict the optimal binding poses of the protein-ligand complexes. The grid box parameters (center coordinates in X, Y, Z and box dimensions in &,Aring ) were defined as follows, ERK2 (center, &,ndash 6.6501, 8.7536, 33.8196 size, 56.2779&,times 46.5995&,times 92.6204 &,Aring ), CCR2 (center, 5.4914, &,ndash 7.2525, &,ndash 14.7086 size, 36.0682&,times 26.1593&,times 39.3003 &,Aring ), and JNK3 (center, 8.8896, 8.7637, 13.7096 size, 43.9146&,times 38.8612&,times 43.7974 &,Aring ). In Vivo Treatment Experimental Animals, This study utilized 30 male Wistar rats (200&,ndash 250 g, 10&,ndash 15 weeks old) sourced from the Biology Laboratory of Universitas Sumatera Utara (USU), Medan, Indonesia. The rats underwent a two-week acclimatization period in the Animal Physiology Laboratory, Biology Study Program, USU, under controlled conditions (12-hour light/dark cycle, 35&,ndash 60% humidity) in plastic cages (40&,times 30 cm) sterilized by radiation. The animals were provided ad libitum access to water, corn, and standard pellets. The study was approved by the Health Research Ethics Committee of USU Medan (No. 01019/KEPH-FMIPA/2023). The rats were randomly assigned to six groups (n=5/group), G0 (negative control), G+ (subjected to TBI induced by a 50 g weight drop from a height of 2 m), MP (TBI+methylprednisolone 10 mg/Kg BW), PL100 (TBI+nanoherbal Paraboea leuserensis 100 mg/Kg BW), PL200 (TBI+nanoherbal Paraboea leuserensis 200 mg/Kg B), and PL300 (TBI+nanoherbal Paraboea leuserensis 300 mg/Kg BW). The test drugs and compounds were administered orally once daily for 30 consecutive days. SOD and MDA Analysis, At the end of the experimental period, the rats were euthanized by intramuscular injection of a lethal dose of euthanasia solution into the thigh muscle, a method that may require a slightly longer onset compared to intravenous administration. Following euthanasia, blood samples were collected via cardiac puncture, allowed to clot at room temperature, and centrifuged at 3000 rpm for 15 min to obtain serum for biochemical analyses. The levels of SOD and MDA were measured using commercial ELISA kits (Sigma-Aldrich, USA) according to the manufacturer&,rsquo s protocols to assess antioxidant capacity and lipid peroxidation, respectively. Statistical Analysis Data are expressed as mean&,plusmn SEM. Statistical analyses were conducted using one-way ANOVA, followed by Tukey&,rsquo s post-hoc test, using GraphPad Prism version 10.0.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was determined at p-values of P&,lt 0.05 (*), P&,lt 0.01 (**), P&,lt 0.001 (***), and P&,lt 0.0001 (****).Results GC-MS Analysis The results of the GC-MS analysis showed the presence of several compounds with varying retention times and concentrations, as shown in table 1,, along with the chromatogram in figure 1,. The compounds detected included silane (CAS) with a percentage of 24.94%, hexadecanoic acid (CAS) at 26.49%, 9-octadecenoic acid (Z) at 37.54%, 9-octadecene-12-ynoic acid, methyl ester 1.73%, and hi-oleic sunflower oil 0.98%. NoCompounds nameMolecular formulaMolecular weightRetention Time (min)Concentration (%)SmilesPubChem ID13-Hydroxymethyl-5-(4-nitroimidazol-l-yl)isoxazolidine--1.6807.891--2Silane (CAS)C4H6O3102.093.06624.943CC(=O)OC(=O)C791833-Hexadecyloxycarbonyl-5-(2-hydroxyethyl)-4-methylimidazolium ion--25.9280.466--4Hexadecanoic acid (CAS)C16H32O2256.4232.74426.488CCCCCCCCCCCCCCCC(=O)O98559-Octadecenoic (Z)-C18H34O2282.538.00337.541CCCCCCCC/C=CCCCCCCCC(=O)O44563969-Octadecen-12-ynoic acid, methyl ester (CAS)C19H32O2278.451.1351.732CCCCCC#CC/C=C/CCCCCCCC(=O)OC53631617Hi-oleic safflower oil (CAS)--53.4150.979--Table 1.Identified compounds of the nanoherbal Paraboea leuserensisFigure 1. This figure shows the Gas Chromatography&,ndash Mass Spectrometry (GC-MS) chromatogram of the nanoherbal Paraboea leuserensis. Drug-Likeness Prediction and Pharmacokinetics Prediction Based on the analysis using Lipinski&,rsquo s rule, several compounds evaluated in this study demonstrate potential for use as drugs despite violations of specific parameters. The results are summarized in table 2,. Silane (CAS) does not violate Lipinski&,rsquo s rule, indicating potential for oral bioavailability. The physicochemical properties of this compound align with characteristics typically associated with compounds easily absorbed by the body. Hexadecanoic acid (palmitic acid) exhibits high lipophilicity, which can affect its water solubility and may reduce oral bioavailability. However, palmitic acid is still commonly used in pharmaceutical formulations, particularly topical and cosmetic preparations. Additionally, 9-octadecenoic acid (Z)- or oleic acid is widely used in drug formulations, even though its logP value exceeds Lipinski&,rsquo s threshold. Lastly, 9-octadecen-12-ynoic acid, methyl ester (CAS), also demonstrates high lipophilicity.The results of the pharmacokinetic evaluation of Paraboea leuserensis nanoherbal compounds, shown in table 2,, indicate that all compounds have high HIA values, ranging from 91.82% to 100%. Silane had the highest absorption value (100%) with very acceptable solubility and a Caco-2 permeability of 1.18, indicating a high ability to pass through the intestinal membrane. Hexadecanoic acid has an absorption value of 92%, Caco-2 permeability of 1.1558, and is moderately soluble. The compound 9-octadecen-12-ynoic acid methyl ester showed an absorption value of 93.63%, the highest Caco-2 permeability of 1.583, and moderate solubility, indicating very acceptable potential for membrane absorption and transport. Meanwhile, 9-octadecenoic acid had the lowest absorption value of 91.82% but remained high, with a Caco-2 permeability of 1.563 and moderate solubility. None of these compounds acted as CYP2D6 inhibitors or OCT2 substrates, indicating a stable renal metabolism and elimination profile and minimal risk of drug interactions. Based on the prediction of compound distribution using the BOILED-Egg model shown in figure 2,, silane and 9-octadecenoic acid are in the white area, indicating proper intestinal absorption but are unable to penetrate the BBB. In contrast, hexadecanoic acid and 9-octadecen-12-ynoic acid methyl ester are located in the yellow area, indicating their ability to cross the BBB and potentially reach the central nervous system (CNS). Based on the pharmacokinetic parameters in table 2, and the predictive distribution in figure 2,, hexadecanoic acid and 9-octadecen-12-ynoic acid methyl ester were considered the best candidates because they had the best absorption profile, permeability, and BBB penetration ability.NoCompound nameLipinskiViolation Pharmacokinetic characteristicsMWMlogP &,le 4.15NorO &,le 10NHorOH &,le 5Human Intestinal AbsorptionCaco-2 permeabilityBBB permeabilityCYP2D6 substrateCYP2D6 inhibitorOCT2 substrateLogS (ESOL)1Silane (CAS)102.090.31030100%1.18-0.281 (No)NoNoNoVery soluble2Hexadecanoic acid (CAS)256.424.1912192%1.1558-0.111 (Yes)NoNoNoModerately soluble39-Octadecenoic (Z)-282.54.5712191.82%1.563-0.168 (No)NoNoNoModerately soluble49-Octadecen-12-ynoic acid, methyl ester (CAS)278.44.4712193.63%1.583-0.09 (Yes)NoNoNoModerately solubleMW, Molecular weight MlogP, Moriguchi logarithm of partition coefficient NorO, Number of nitrogen NHorOH, Number of NH or OH Caco-2 permeability, Caco-2 cell monolayer permeability BBB permeability, Blood-brain barrier permeability CYP2D6, Cytochrome P450 2D6 OCT2 substrate, Organic cation transporter 2 substrate LogS, Logarithm of solubility |