Low-frequency electroacupuncture exerts antinociceptive effects through activation of POMC neural circuit induced endorphinergic input to the periaqueductal gray from the arcuate nucleus.

It has been widely recognized that electroacupuncture (EA) inducing the release of ß-endorphin represents a crucial mechanism of EA analgesia. The arcuate nucleus (ARC) in the hypothalamus is a vital component of the endogenous opioid peptide system. Serving as an integration center, the periaqueductal gray (PAG) receives neural fiber projections from the frontal cortex, insular cortex, and ARC. However, the specific mechanisms how EA facilitates the release of ß-endorphin within the ARC, eliciting analgesic effects are yet to be elucidated. In this study, we conducted in vivo and in vitro experiments by transcriptomics, microdialysis, photogenetics, chemical genetics, and calcium imaging, combined with transgenic animals. Firstly, we detected 2 Hz EA at the Zusanli (ST36) increased the level of ß-endorphin and transcriptional level of proopiomelanocortin (POMC). Our transcriptomics profiling demonstrated that 2 Hz EA at the ST36 modulates the expression of c-Fos and Jun B in ARC brain nuclear cluster, and the transcriptional regulation of 2 Hz EA mainly occur in POMC neurons by Immunofluorescence staining verification. Meaning while, 2 Hz EA specifically activated the cAMP-PKA-CREB signaling pathway in ARC which mediating the c-Fos and Jun B transcription, and 2 Hz EA analgesia is dependent on the activation of cAMP-PKA-CREB signaling pathway in ARC. In order to investigate how the ß-endorphin produced in ARC transfer to integration center PAG, transneuronal tracing technology was used to observe the 2 Hz EA promoted the neural projection from ARC to PAG compared to 100 Hz EA and sham mice. Inhibited PAGGABA neurons, the transfer of ß-endorphin from the ARC nucleus to the PAG nucleus through the ARCPOMC-PAGGABA neural circuit. Furthermore, by manipulating the excitability of POMC neurons from ARCPOMC to PAGGABA using inhibitory chemogenetics and optogenetics, we found that this inhibition significantly reduced transfer of ß-endorphin from the ARC nucleus to the PAG nucleus and the effectiveness of 2 Hz EA analgesia in neurological POMC cyclization recombination enzyme (Cre) mice and C57BL/6J mice, which indicates that the transfer of ß-endorphin depends on the activation of POMC neurons prefect from ARCPOMC to PAGGABA. These findings contribute to our understanding of the neural circuitry underlying the EA pain-relieving effects and maybe provide valuable insights for optimizing EA stimulation parameters in clinical pain treatment using the in vivo dynamic visual investigating the central analgesic mechanism.

Novel Triazole-Containing "Dipeptides": Synthesis, Molecular Docking and Analgesic Activity Studies.

Dipeptides of a new structure based on ß-triazolalanines and (L)-α-amino acids were synthesized and optimal conditions were developed that ensure both chemical and optical purity of the final products. Molecular docking was carried out and possible intermolecular interactions of dipeptides with potential targets were established. Based on these studies, the analgesic property of chosen dipeptides was studied and it was found that some compounds possess revealed antinociceptive activity in the tail-flick test.

Glycosylated SARs Cov 2 interaction with plant lectins.

Lectins are non-immune carbohydrate-binding proteins/glycoproteins that are found everywhere in nature, from bacteria to human cells. They have also been a valuable biological tool for the purification and subsequent characterisation of glycoproteins due to their carbohydrate binding recognition capacity. Antinociceptive, antiulcer, anti-inflammatory activities and immune modulatory properties have been discovered in several plant lectins, with these qualities varying depending on the lectin carbohydrate-binding site. The Coronavirus of 2019 (COVID-19) is a respiratory disease that has swept the globe, killing millions and infecting millions more. Despite the availability of COVID-19 vaccinations and the vaccination of a huge portion of the world's population, viral infection rates continue to rise, causing major concern. Part of the reason for the vaccine's ineffectiveness has been attributed to repeated mutations in the virus's epitope determinant elements. The surface of the Coronavirus envelope is heavily glycosylated, with approximately sixty N-linked oligomannose, composite, and hybrid glycans covering the core of Man3GlcNAc2Asn. Some O-linked glycans have also been discovered. Many of these glyco-chains have also been subjected to multiple mutations, with only a few remaining conserved. As a result, numerous plant lectins with specificity for these viral envelope sugars have been discovered to interact preferentially with them and are being investigated as a potential future tool to combat coronaviruses such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by preventing viral attachment to the host. The review will discuss the possible applications of plant lectins as anti-coronaviruses including SARS-CoV-2, antinociceptive, anti-inflammation and its immune modulating effect.

Recent progress on anti-nociceptive effects of carbon monoxide releasing molecule-2 (CORM-2).

The role of carbon monoxide (CO) has evolved albeit controversial disputes on its toxicity. This biological gasotransmitter participates in the endogenous regulation of neurotransmitters and neuropeptides released in the nervous system. Exogenous CO gas inhalation at a lower concentration has been the subject of investigations, which have revealed its biological homeostatic mechanisms and protective effects against many pathological conditions. This therapeutic procedure of CO is, however, limited due to its immediate release, which favours haemoglobin at a high affinity with the subsequent generation of toxic carboxyhaemoglobin in tissues. In order to address this problem, carbon monoxide releasing molecule-2 (CORM-2) or also known as tricarbonyldichlororuthenium II dimer is developed to liberate a controlled amount of CO in the biological systems. In this review, we examine several potential mechanisms exerted by this therapeutic compound to produce the anti-nociceptive effect that has been demonstrated in previous studies. This review could shed light on the role of CORM-2 to reduce pain, especially in cases of chronic and neuropathic pain.

Hydroxytakakiamide and Other Constituents from a Marine Sponge-Associated Fungus Aspergillus fischeri MMERU23, and Antinociceptive Activity of Ergosterol Acetate, Acetylaszonalenin and Helvolic Acid.

An unreported prenylated indole derivative hydroxytakakiamide (4) was isolated, together with the previously described ergosterol (1), ergosterol acetate (2), and (3R)-3-(1H-indol-3-ylmethyl)-3, 4-dihydro-1H-1,4-benzodiazepine-2,5-dione (3), from the column fractions of the crude ethyl acetate extract of the culture of a marine sponge-associated fungus, Aspergillus fischeri MMERU 23. The structure of 4 was elucidated by the interpretation of 1D and 2D NMR spectral data and high-resolution mass spectrum. The absolute configuration of the stereogenic carbon in 3 was proposed to be the same as those of the co-occurring congeners on the basis of their biogenetic consideration and was supported by the comparison of its sign of optical rotation with those of its steroisomers. The crude ethyl acetate extract and 2 were evaluated, together with acetylaszonalenin (5) and helvolic acid (6), which were previously isolated from the same extract, for the in vivo antinociceptive activity in the mice model. The crude ethyl acetate extract exhibited antinociceptive activity in the acetic acid-induced writhing and formalin tests, while 2, 5, and 6 displayed the effects in the late phase of the formalin test. On the other hand, neither the crude ethyl acetate extract nor 2, 5, and 6 affected the motor performance of mice in both open-field and rotarod tests. Additionally, docking studies of 2, 5, and 6 were performed with 5-lipoxygenase (5-LOX) and phosphodiesterase (PDE) enzymes, PDE4 and PDE7, which are directly related to pain and inflammatory processes. Molecular docking showed that 6 has low affinity energy to PDE4 and PDE7 targets while retaining high affinity to 5-LOX. On the other hand, while 2 did not display any hydrogen bond interactions in any of its complexes, it achieved overall better energy values than 6 on the three antinociceptive targets. On the other hand, 5 has the best energy profile of all the docked compounds and was able to reproduce the crystallographic interactions of the 5-LOX complex.

Three new antinociceptive diterpenoids from the fruits of Rhododendron molle.

Investigation of the fruits of Rhododendron molle G. Don led to the isolation of three new grayanane-type diterpenoids, rhodomolleins LIV-LVI (1-3). The structures and absolute configurations of new compounds were fully elucidated by spectroscopic analysis and single-crystal X-ray diffraction, including HRESIMS, 1 D and 2 D NMR data. Compounds 1-3 were evaluated for analgesic activities utilizing an acetic acid-induced writhing test in mice. Compound 1 showed a significant antinociceptive effect with writhe inhibition rates of 72.9% and 100% at doses of 6 mg/kg and 20 mg/kg in mice, respectively. The binding mode of 1 to N-ethylmaleimide-sensitive factor (NSF, PDB: 6IP2) was explored by molecular docking, indicating the presence of hydrogen bond interactions which account for its analgesic activity.

Antinociceptive iridoid glycosides from the aerial parts of Paederia foetida.

Two previously undescribed iridoid glycosides, 6'-O-trans-feruloyl-(4S,6R)-3,4-dihydro-3ß-ethoxypaederoside (1) and 6'-O-trans-caffeoyl-(4S,6R)-3,4-dihydro-2'-O-3α-paederoside (2), were isolated from the 90% EtOH extract of the air dried aerial parts of Paederia Foetida. Structural elucidation of all the compounds was performed by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy. The two isolated iridoid glycosides were tested in vivo for their antinociceptive properties. As a result, 2 showed potent antinociceptive effect and its ID50 value (53.4 µmol/kg) was 2-fold less than those of the positive control drugs aspirin and acetaminophen.

Analgesic Peptides: From Natural Diversity to Rational Design.

Pain affects one-third of the global population and is a significant public health issue. The use of opioid drugs, which are the strongest painkillers, is associated with several side effects, such as tolerance, addiction, overdose, and even death. An increasing demand for novel, safer analgesic agents is a driving force for exploring natural sources of bioactive peptides with antinociceptive activity. Since the G protein-coupled receptors (GPCRs) play a crucial role in pain modulation, the discovery of new peptide ligands for GPCRs is a significant challenge for novel drug development. The aim of this review is to present peptides of human and animal origin with antinociceptive potential and to show the possibilities of their modification, as well as the design of novel structures. The study presents the current knowledge on structure-activity relationship in the design of peptide-based biomimetic compounds, the modification strategies directed at increasing the antinociceptive activity, and improvement of metabolic stability and pharmacodynamic profile. The procedures employed in prolonged drug delivery of emerging compounds are also discussed. The work summarizes the conditions leading to the development of potential morphine replacements.

Highly potent anti-inflammatory, analgesic and antioxidant activities of 3,5-disubstituted tetrahydro-2H-1,3,5-thiadiazine thiones.

Four series of tetrahydro-2H-1,3,5-thiadiazine-2-thiones (series A and B including two novel enantiopure isomers), tetrahydro-2H-1,3,5-thiadiazine-6-thiones (series C) and N-3 ester derivatives of tetrahydro-2H-1,3,5-thiadiazine-6-thiones (series D) were synthesized and evaluated for their anti-inflammatory, analgesic and anti-oxidant activities. These THTT analogues specially series D were first time examined for their in vitro anti-inflammatory, in vivo analgesic and anti-oxidant activities. Among them lipophilic compounds (series B and D) were found to be highly active for anti-inflammatory evaluation with IC50 values between 5.1-16.9 and 4.1-32.4 µM, respectively when compared with the standard drug ibuprofen IC50 = 11.2 µM. The structure-activity relationship exposed the importance of lipophilic substituents especially ester and n-propyl group for inhibition of inflammation. The molecular docking studies demonstrated that all the active analogues of THTT have notable binding relations with Arg120 of the active sites of COX-1 enzyme either through CS moiety of the THTT nucleus or with COO attached at N-3 of THTT nucleus. In vivo analgesic activity of the selected THTT compounds 14, 17, 18, 19 (series B) and 28 (series D) were also carried out by acetic acid-induced writhing procedure. The compound 28 showed significant anti-nociceptive/analgesic activity at the oral dose of 5 mg/kg body weight with the percent protection (32.05 %) when compared with standard indomethacin at 10 mg/kg (48.83 %). Additionally, these compounds demonstrated the moderate level of antioxidant potential with IC50 values in the range of 60.9 to 93.6 µM (standard butylated hyroxyanisole; IC50 = 44.2 µM). These results indicated that this class of heterocyclic compounds may be a template specially to design better anti-inflammatory and analgesic agents.

Truxillic acid monoamides as fatty acid binding protein 5 inhibitors.

Fatty acid binding proteins (FABPs) are intracellular chaperones that deliver bioactive lipids to cytosolic enzymes and nuclear receptors, thereby regulating diverse biological functions. FABP5 is a member of the FABP family that mediates endocannabinoid transport and inactivation, with pharmacological or genetic FABP5 inhibition conferring antinociceptive effects. Consequently, FABP5 inhibitors have emerged as promising analgesics and demonstrate antinociceptive activity in models of pain. Recently developed FABP5 inhibitors based upon the α-truxillic acid monoester (TAME) scaffold demonstrate high affinities for FABP5 but are commonly accompanied by reduced selectivity against related FABPs, notably FABP3 that is expressed in the heart, highlighting the need to identify additional scaffolds that afford enhanced selectivity while maintaining FABP5 potency. Here, we describe the synthesis and biological evaluation of truxillic acid monoamides (TAMADs) as potent, selective, and efficacious FABP5 inhibitors. Combining in silico molecular docking and in vitro binding assay approaches, our findings demonstrate that TAMADs exhibit exceptional selectivity against FABP3 and several compounds attain high FABP5 affinities. Examination of antinociceptive activity revealed that TAMADs and their corresponding TAMEs demonstrate comparable efficacy and temporal activity profiles in vivo. These results position TAMAD as a suitable scaffold for the development of FABP5 inhibitors with diminished FABP3 cross-reactivity.

Blocking the major inflammatory pathways by newly synthesized thiadiazine derivatives via in-vivo, in-vitro and in-silico mechanism.

A series of new thiadiazine derivatives including 2-(5-alkyl/aryl-6-thioxo-1,3,5-thiadiazinan-3-yl) propanoic acids (a) and 4-methyl-2-(5-alkyl/aryl-6-thioxo-1,3,5-thiadiazinan-3-yl) pentanoic acids (b) were synthesized by reacting primary alkyl/aryl amines with CS2, followed by reaction with formaldehyde and amino acids. The chemical structures of synthesized compounds were confirmed by 13C- NMR and 1H- NMR techniques. The inhibitory potential of major inflammatory enzymes, COX-2 and 5-LOX was examined. Moreover, anti-nociceptive and anti-inflammatory activities were evaluated in the in vivo thermally induced nociceptive, and carrageenan induced paw edema models in mice. The in-vitro results reflect that these compounds exhibited concentration dependent inhibition of COX-2 and 5-LOX. The tested compounds at 50 mg/kg showed significant effect on thermally induced pain, and reduced latency time (seconds) as compared to the vehicle treated animals. Moreover, tested compounds exhibited percent inhibition of paw edema in the carrageenan induced paw edema model in mice. Furthermore, the binding modes of the most active COX-2 and 5-LOX inhibitors were determined through computational methods. The computational study reflects that the docked compounds have high binding affinities for COX-2 and 5-LOX enzymes, which leads to inhibition of these enzymes.

New N- and C-modified RGD-hemorphins as potential biomedical application on Ti-surface materials: synthesis, characterization and antinociceptive activity.

This manuscript presented the synthesis and characterization of two new N- and C-modified analogues of VV-hemorphin-7 containing RGD (Arg-Gly-Asp) residues as potential nociceptive agents and bioactive materials. It has been shown that the addition of one or two RGD sequences to natural VV-hemorphin-7 increases its effect on acute nociception, but the reduction of the inflammatory phase depends on the concentration of the peptide. The structure-property relationship of the new peptide derivatives was highlighted by electrochemical and FT-IR methods of analysis. Because of the proven bone-structural bonds of hydroxyapatite, the simultaneous deposition of peptide/hydroxyapatite on the surface of a titanium surface was investigated. The deposition was performed in a medium of gelatin solution containing dissolved amounts of peptide and hydroxyapatite using ultrasound. SEM-EDS analyzes confirmed the presence of a layer of the studied system.

An overview of the effect of medicinal herbs on pain.

This paper is typically intended to carefully collect and properly review the antinociceptive activities of medicinal plants. In this review article, by searching keywords of medicinal plants, pain, herbal medicine, antinociceptive, phytotherapy in databases of Web of Science, Scopus, Google Scholar, Springer, Wiley, Proquest, PubMed, Nature, Magiran, Emerald, SID, ISI, and some other indexing cites, or traditional books, desired articles were obtained until 2021. The title of medicinal plants was searched diligently in Persian and English. Ultimately, 270 articles were studied. The findings possibly indicated that several medicinal plants are among the most valuable plants that have antinociceptive activities. There efficiently are various antinociceptive compounds in medicinal plants. The antinociceptive activity of these specific compounds may be through their peculiar effects on the opioid system, cholinergic pathways, and stimulation of GABA receptors, with the peripheral and central antinociceptive mechanism. Antiinflammatory processes, inhibition of the synthesis, and the release of arachidonic acid, prostaglandins, phospholipase, nitric oxide, and cyclooxygenase-2 have been reported as analgesic mechanisms of some herbs. In a reasonable conclusion, our review thoughtfully provides a comprehensive summary of present data from some scientific studies on the common herbs with antinociceptive and antiinflammatory activities.

Antinociceptive diterpenoid alkaloids from the roots of Aconitum austroyunnanense.

A phytochemical investigation on the roots of Aconitum austroyunnanense afforded three undescribed aconitine-type C19-diterpenoid alkaloids, austroyunnanines A-C (1-3). Structural elucidation of all the compounds were performed by spectral methods such as 1 D and 2 D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy. The isolated alkaloids were tested in vivo for their antinociceptive properties. Consequently, austroyunnanine B (2) exhibited significant antinociceptive effect and its ID50 value (48.0 µmol/kg) was 2-fold less than those of the positive control drugs aspirin and acetaminophen.

Antinociceptive C19-diterpenoid alkaloids from the roots of Aconitum nagarum.

Three new C19-diterpenoid alkaloids, nagarumines A-C (1-3), together two known alkaloids, deoxyaconitine (4) and N-deethyldeoxyaconitine (5), were isolated from the roots of Aconitum nagarum. The structures of the new compounds were elucidated by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, as well as high resolution mass spectrometry. The in vivo pharmacological studies revealed that nagarumine C (3) possessed comparable antinociceptive activity (ED50 = 76.0 µmol/kg) with the positive control drugs aspirin and acetaminophen.

Hyaluronic Acid-Based Nanosystems for CD44 Mediated Anti-Inflammatory and Antinociceptive Activity.

The nervous and immune systems go hand in hand in causing inflammation and pain. However, the two are not mutually exclusive. While some diseases cause inflammation, others are caused by it. Macrophages play an important role in modulating inflammation to trigger neuropathic pain. Hyaluronic acid (HA) is a naturally occurring glycosaminoglycan that has a well-known ability to bind with the cluster of differentiation 44 (CD44) receptor on classically activated M1 macrophages. Resolving inflammation by varying the molecular weight of HA is a debated concept. HA-based drug delivery nanosystems such as nanohydrogels and nanoemulsions, targeting macrophages can be used to relieve pain and inflammation by loading antinociceptive drugs and enhancing the effect of anti-inflammatory drugs. This review will discuss the ongoing research on HA-based drug delivery nanosystems regarding their antinociceptive and anti-inflammatory effects.

ß-Lactam TRPM8 Antagonists Derived from Phe-Phenylalaninol Conjugates: Structure-Activity Relationships and Antiallodynic Activity.

The protein transient receptor potential melastatin type 8 (TRPM8), a non-selective, calcium (Ca2+)-permeable ion channel is implicated in several pathological conditions, including neuropathic pain states. In our previous research endeavors, we have identified ß-lactam derivatives with high hydrophobic character that exhibit potent and selective TRPM8 antagonist activity. This work describes the synthesis of novel derivatives featuring C-terminal amides and diversely substituted N'-terminal monobenzyl groups in an attempt to increase the total polar surface area (TPSA) in this family of compounds. The primary goal was to assess the influence of these substituents on the inhibition of menthol-induced cellular Ca2+ entry, thereby establishing critical structure-activity relationships. While the substitution of the tert-butyl ester by isobutyl amide moieties improved the antagonist activity, none of the N'-monobencyl derivatives, regardless of the substituent on the phenyl ring, achieved the activity of the model dibenzyl compound. The antagonist potency of the most effective compounds was subsequently verified using Patch-Clamp electrophysiology experiments. Furthermore, we evaluated the selectivity of one of these compounds against other members of the transient receptor potential (TRP) ion channel family and some receptors connected to peripheral pain pathways. This compound demonstrated specificity for TRPM8 channels. To better comprehend the potential mode of interaction, we conducted docking experiments to uncover plausible binding sites on the functionally active tetrameric protein. While the four main populated poses are located by the pore zone, a similar location to that described for the N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist cannot be discarded. Finally, in vivo experiments, involving a couple of selected compounds, revealed significant antinociceptive activity within a mice model of cold allodynia induced by oxaliplatin (OXA).

Modulation of inflammatory mediators involved in the antinociceptive and anti-inflammatory effects of a new thioamide derivative: thiocyanoacetamide.

Inflammatory pain is part of the body's defense mechanism and plays an important role in the healing process. Although some drugs are efficient and intensively used for their potent anti-inflammatory properties, they present problematic side effects. The aim of this study was to evaluate the anti-nociceptive effect of the thiocyanoacetamide (Thm) compared to paracetamol (Para), dexamethasone (Dex) and morphine (Morph) and to study inflammatory mediators on models of acute inflammatory pain in rats using the formalin injection test in the hind paw of rats as chemical stimulus. The obtained results showed significant modulation of pain by Thm pretreatment with a maximum at an effective dose (10 mg/kg) proved by the absence of licking and biting of the affected paw during the early and late phases of inflammation. This effect was comparable to Dex at 10 mg/kg, Para at 400 mg/kg and less than Morph at 5 mg/kg pretreatment doses. The study of anti-inflammatory targets showed that Thm pretreatment maintained plasma serotonin release at normal level compared to the negative control group (T-) and corrected the decrease in the plasma level of prostaglandins after inflammatory induction with no variation in the level of histamine in different groups. The evaluation of inflammation mediators demonstrated that the pretreatment with Thm induced the decrease in the amount of both IL-1 Beta and TNF alpha in plasma and the increase in their amount in the tissue of the injection site. The Thm has been promoted as an anti-nociceptive drug that induces modulation of inflammatory mediators.

Involvement of GABAergic and Serotonergic Systems in the Antinociceptive Effect of Jegosaponin A Isolated from Styrax japonicus.

The antinociceptive activity of the flower extracts of Styrax japonicus was confirmed in our previous study. However, the key compound for analgesia has not been distinguished, and the corresponding mechanism is obscure. In this study, the active compound was isolated from the flower by multiple chromatographic techniques and structurally illustrated using spectroscopic methods and referring to the related literature. The antinociceptive activity of the compound and the underlying mechanisms were investigated using animal tests. The active compound was determined to be jegosaponin A (JA), which showed significant antinociceptive responses. JA was also shown to possess sedative and anxiolytic activities but no anti-inflammatory effect, implying the association of the antinociceptive effects with the sedative and anxiolytic activities. Further antagonists and calcium ionophore tests showed that the antinociceptive effect of JA was blocked by flumazenil (FM, antagonist for GABA-A receptor) and reversed by WAY100635 (WAY, antagonist for 5-HT1A receptor). Contents of 5-HT and its metabolite (5-HIAA) increased significantly in the hippocampus and striatum tissues after JA administration. The results indicated that the antinociceptive effect of JA was regulated by the neurotransmitter system, especially GABAergic and serotonergic systems.

Exploring the possible mechanism involved in the anti-nociceptive effect of ß-sitosterol: modulation of oxidative stress, nitric oxide and IL-6.

Β-sitosterol is a phytosterol, documented to possess various activities including protection against inflammation, diabetes and Alzheimer's disease. The current investigation was designed to explore the analgesic potential of ß-sitosterol and the possible molecular mechanism involved in the observed effect. ß-sitosterol was administered at varying doses of 10, 20, and 40 mg/kg before subjecting the mice to acetic acid and formalin challenges. The number of writhings in acetic acid and the number of flinchings and foot tappings were quantified in the formalin test. For mechanistic studies, substance P (cyclooxygenase-2 (COX-2) stimulator) and L-Nitro arginine methyl ester (L-NAME) (nitric oxide synthetases (NOS) inhibitor) and L-arginine (nitric oxide precursor) were administered before ß-sitosterol treatment. ß-sitosterol (10, 20, 40 mg/kg) treatment significantly reduced acetic acid-induced writhings and ameliorated the formalin-induced inflammatory phase dose-dependently. Whereas, 40 mg/kg dose of ß-sitosterol abrogated the formalin-induced neurogenic phase. Substance-P abrogated the effect of ß-sitosterol in both neurogenic and inflammatory phases. Whereas, L-arginine only abrogated the inflammatory phase. In biochemical analysis, ß-sitosterol treatment reduced the level of interleukin-6 (IL-6), thiobarbituric acid reactive substances (TBARS) and increased the level of reduced glutathione (GSH). Furthermore, L-arginine and substance-P abrogated the GSH increasing and TBARS lowering effect of ß-sitosterol (40 mg/kg). Overall, the current study delineated that ß-sitosterol may induce an anti-nociceptive effect via inhibiting the IL-6, oxidative stress, cyclo-oxygenase and nitric oxide.