Signaling Pathways Involved in the Neuroprotective Effect of Osthole: Evidence and Mechanisms.

Neurodegenerative diseases constitute a major threat to human health and are usually accompanied by progressive structural and functional loss of neurons. Abnormalities in synaptic plasticity are involved in neurodegenerative disorders. Aberrant cell signaling cascades play a predominant role in the initiation, progress as well as in the severity of these ailments. Notch signaling is a pivotal role in the maintenance of neural stem cells and also participates in neurogenesis. PI3k/Akt cascade regulates different biological processes including cell proliferation, apoptosis, and metabolism. It regulates neurotoxicity and mediates the survival of neurons. Moreover, the activated BDNF/TrkB cascade is involved in promoting the transcription of genes responsible for cell survival and neurogenesis. Despite significant progress made in delineating the underlying pathological mechanisms involved and derangements in cellular metabolic promenades implicated in these diseases, satisfactory strategies for the clinical management of these ailments are yet to be achieved. Therefore, the molecules targeting these cell signaling cascades may emerge as useful leads in developing newer management strategies. Osthole is an important ingredient of traditional Chinese medicinal plants, often found in various plants of the Apiaceae family and has been observed to target these aforementioned mediators. Until now, no review has been aimed to discuss the possible molecular signaling cascades involved in osthole-mediated neuroprotection at one platform. The current review aimed to explore the interplay of various mediators and the modulation of the different molecular signaling cascades in osthole-mediated neuroprotection. This review could open new insights into research involving diseases of neuronal origin, especially the effect on neurodegeneration, neurogenesis, and synaptic plasticity. The articles gathered to compose the current review were extracted by using the PubMed, Scopus, Science Direct, and Web of Science databases. A methodical approach was used to integrate and discuss all published original reports describing the modulation of different mediators by osthole to confer neuroprotection at one platform to provide possible molecular pathways. Based on the inclusion and exclusion criteria, 32 articles were included in the systematic review. Moreover, literature evidence was also used to construct the biosynthetic pathway of osthole. The current review reveals that osthole promotes neurogenesis and neuronal functioning via stimulation of Notch, BDNF/Trk, and P13k/Akt signaling pathways. It upregulates the expression of various proteins, such as BDNF, TrkB, CREB, Nrf-2, P13k, and Akt. Activation of Wnt by osthole, in turn, regulates downstream GSK-1ß to inhibit tau phosphorylation and ß-catenin degradation to prevent neuronal apoptosis. The activation of Wnt and inhibition of oxidative stress, Aß, and GSK-3ß mediated ß-catenin degradation by osthole might also be involved in mediating the protection against neurodegenerative diseases. Furthermore, it also inhibits neuroinflammation by suppressing MAPK/NF-κB-mediated transcription of genes involved in the generation of inflammatory cytokines and NLRP-3 inflammasomes. This review delineates the various underlying signaling pathways involved in mediating the neuroprotective effect of osthole. Modulation of Notch, BDNF/Trk, MAPK/NF-κB, and P13k/Akt signaling pathways by osthole confers protection against neurodegenerative diseases. The preclinical effects of osthole suggest that it could be a valuable molecule in inspiring the development of new drugs for the management of neurodegenerative diseases and demands clinical studies to explore its potential. An effort has been made to unify the varied mechanisms and target sites involved in the neuroprotective effect of osthole. The comprehensive description of the molecular pathways in the present work reflects its originality and thoroughness. The reviewed literature findings may be extrapolated to suggest the role of othole as a "biological response modifier" which contributes to neuroprotection through kinase modulatory, immunomodulatory, and anti-oxidative activity, which is documented even at lower doses. The current review attempts to emphasize the gaps in the existing literature which can be explored in the future.

Neuroprotective potential of formononetin, a naturally occurring isoflavone phytoestrogen.

The increased prevalence of neurological illnesses is a burgeoning challenge to the public healthcare system and presents greater financial pressure. Formononetin, an O-methylated isoflavone, has gained a lot of attention due to its neuroprotective potential explored in several investigations. Formononetin is widely found in legumes and several types of clovers including Trifolium pratense L., Astragalus membranaceus, Sophora tomentosa, etc. Formononetin modulates various endogenous mediators to confer neuroprotection. It prevents RAGE activation that results in the inhibition of neuronal damage via downregulating the level of ROS and proinflammatory cytokines. Furthermore, formononetin also increases the expression of ADAM-10, which affects the pathology of neurodegenerative disease by lowering tau phosphorylation, maintaining synaptic plasticity, and boosting hippocampus neurogenesis. Besides these, formononetin also increases the expression of antioxidants, Nrf-2, PI3K, ApoJ, and LRP1. Whereas, reduces the expression of p65-NF-κB and proinflammatory cytokines. It also inhibits the deposition of Aß and MAO-B activity. An inhibition of Aß/RAGE-induced activation of MAPK and NOX governs the protection elicited by formononetin against inflammatory and oxidative stress-induced neuronal damage. Besides this, PI3K/Akt and ER-α-mediated activation of ADAM10, ApoJ/LRP1-mediated clearance of Aß, and MAO-B inhibition-mediated preservation of dopaminergic neurons integrity are the major modulations produced by formononetin. This review covers the biosynthesis of formononetin and key molecular pathways modulated by formononetin to confer neuroprotection.

Neuroprotective potential of biochanin-A and review of the molecular mechanisms involved.

Biochanin-A is a naturally occurring plant phytoestrogen, which mimics specific the agonistic activity of estrogens. Biochanin-A is known to possess numerous activities, including neuroprotective, anti-diabetic, hepatoprotective, anti-inflammatory, antioxidant, and antimicrobial activities, along with the anticancer activity. Neuroinflammation is thought to play a pivotal pathological role in neurodegenerative disease. Sustained neuroinflammatory processes lead to progressive neuronal damage in Parkinson's and Alzheimer's disease. Activation of PI3K/Akt cascade and inhibition of MAPK signaling cascade have been observed to be responsible for conferring protection against neuroinflammation in neurodegenerative diseases. An increased oxidative stress promotes neuronal apoptosis via potentiating the TLR-4/NF-κB and inhibiting PI3K/Akt signaling mediated increase in pro-apoptotic and decreases in antiapoptotic proteins. Various authors have explored biochanin-A's neuroprotective effect by using various cell lines and animal models. Biochanin-A has been reported to mediate its neuroprotective via reducing the level of oxidants, inflammatory mediators, MAPK, TLR-4, NF-κB, NADPH oxidase, AchE, COX-2 and iNOS. Whereas, it has been observed to increase the level of anti-oxidants, along with phosphorylation of PI3K and Akt proteins. The current review has been designed to provide insights into the neuroprotective effect of biochanin-A and possible signaling pathways leading to protection against neuroinflammation and apoptosis in the central nervous system. This review will be helpful in guiding future researchers to further explore biochanin A at a mechanistic level to obtain useful lead molecules.

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.

Design, Synthesis, and Activity Evaluation of Stereoconfigured Tartarate Derivatives as Potential Anti-inflammatory Agents In Vitro and In Vivo.

Preclinical and clinical data reveal that inflammation is strongly correlated with the pathogenesis of a number of diseases including those of cancer, Alzheimer, and diabetes. The inflammatory cascade involves a multitude of cytokines ending ultimately with the activation of COX-2/LOX for the production of prostaglandins and leukotrienes. While the available inhibitors for these enzymes suffer from nonoptimal selectivity, in particular for COX-2, we present here the results of purposely designed tartarate derivatives that exhibit favorable selectivity and significant effectiveness against COX-2 and LOX. Integrated approaches of molecular simulation, organic synthesis, and biochemical/physical experiments identified 15 inhibiting COX-2 and LOX with respective IC50 4 and 7 nM. At a dose of 5 mg kg-1 to Swiss albino mice, 15 reversed algesia by 65% and inflammation by 33% in 2-3 h. We find good agreement between experiments and simulations and use the simulations to rationalize our observations.

Mechanistic interplay of various mediators involved in mediating the neuroprotective effect of daphnetin.

Daphnetin is a 7, 8 dihydroxy coumarin isolated from different medicinal plants of the Thymelaeaceae family and exhibits copious pharmacological activities including neuroprotection, anti-cancer, anti-malarial, anti-inflammatory, anti-parasitic and anti-arthritic activity. It has been proved to be an effective neuroprotective agent in several preclinical animal studies and cell line examinations. It is found to interact with different cellular mediators and signaling pathways to confer protection against neurodegeneration. The reactive oxygen species and inflammatory mediators are the major culprits of different neurodegenerative diseases. Oxidative stress activates the pro-apoptotic proteins and inhibits anti-apoptotic proteins, leading to neuronal cell death. Daphnetin restores cellular redox balance by upregulating the antioxidants level (GSH and SOD), anti-apoptotic protein (Bcl-2), as well as by reducing the levels of proinflammatory cytokines, executioner caspase-3, pro-apoptotic-Bax, and oxidative stress markers. Furthermore, activation of Nrf-2/HO-1 signaling and upregulation of HSP-70 governs the protection elicited by daphnetin against oxidative stress-induced neuronal apoptosis. Daphnetin modulated inhibition of JNK-MAPK, JAK-STAT, and TLR-4/NF-κB signaling pathways also contributed to its neuroprotective effect. The positive effects of daphnetin have been also related to its AChE, BChE, and BACE-1 inhibitory potential. The present review has been designed to explore the mechanistic interplay of various mediators in mediating the neuroprotective effects of daphnetin.

Daphnetin, a natural coumarin averts reserpine-induced fibromyalgia in mice: modulation of MAO-A.

Fibromyalgia is a common, chronic, and generalized pain syndrome that is often associated with comorbid depression. The etiology of fibromyalgia is complex; most researchers have documented that the hallmark symptoms are due to the central nervous system's abnormal functioning. Neurotransmitters such as serotonin, norepinephrine, and glutamate, have been reported to be key regulators of fibromyalgia syndrome. Daphnetin is a 7, 8 dihydroxy coumarin widely distributed in Thymelaeaceae family plants, possessing various activities such as anti-arthritic, anti-tumor, anti-malarial, and anti-parasitic. The present study was designed to explore the potential of daphnetin against reserpine-induced fibromyalgia in mice. In mice, a fibromyalgia-like state was achieved by injecting reserpine (0.5 mg/kg, s.c) continuously for 3 days. All behavioral tests were conducted on the 4th and 6th day of experimentation. Reserpine administration significantly increased the mechanical hypersensitivity in electronic von Frey (eVF) and pressure application measurement (PAM) tests. It also increased the immobility period and time to reach the platform in force swim test (FST) and Morris water maze (MWM) test, respectively. In the biochemical analysis, reserpine treatment upregulated the monoamine oxidase-A (MAO-A) activity and level of glutamate, tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), and thiobarbituric acid reactive substances (TBARS). Whereas, it decreased the level of glutathione (GSH), dopamine, serotonin, and norepinephrine. Daphnetin pretreatment attenuated the behavioral and biochemical changes induced by reserpine. Thus, the current investigation results delineate that daphnetin might exert its protective effect by inhibiting inflammatory stress and MAO-A-mediated neurotransmitter depletion and oxidative stress.

Optimization of extraction conditions of Angelica archangelica extract and activity evaluation in experimental fibromyalgia.

The plant Angelica archangelica, owing to its magnificent therapeutic effectiveness in folklore medicine system, has been regarded as an "angel plant." The current investigation was aimed to optimize extraction conditions of A. archangelica roots and to investigate in vivo role of optimized extract in fibromyalgia. Plant material (dried roots) was subjected to methanol extraction at variable temperature (40 to 60 °C) and time (12 to 36 hr) conditions as per two-factorial design strategy, and responses in terms of antioxidant activity were determined. The optimized extraction conditions were found to be temperature of 60 °C and time of 36 hr. HPLC fingerprinting indicated the presence of coumarins in extract. To induce fibromyalgia, the mice were administered reserpine at a dose of 0.5 mg/kg. Mice were orally treated with 100, 200, and 400 mg/kg extract, and magnitude of fibromyalgia was quantified. In comparison to reserpine group, the extract treatment attenuated pain as shown by significant increase in paw withdrawal threshold against mechanical stimuli (P < 0.05), improved motor ability indicated by increase in fall-off time in inclined plane test (P < 0.05), improved locomotion indicated by increased square crossings in open field test (P < 0.05), and improved cognition as shown by significant reduction in time to reach platform in Morris water maze test and passive avoidance task test (P < 0.05). Extract treatment significantly halted reserpine-induced rise in serum cytokine level (P < 0.05) and brain oxidative stress (P < 0.05). Angelica archangelica extract exerted its beneficial effects in fibromyalgia possibly through the attenuation of oxidative stress-mediated inflammatory cascade. PRACTICAL APPLICATION: Leads from natural products have become an integral part of drug designing processes and have high acceptability due to their better tolerance. The optimization of extraction conditions of plant yields better results and could reduce the processing time, thus increasing its industrial value.

Protective Effect of Esculetin, Natural Coumarin in Mice Model of Fibromyalgia: Targeting Pro-Inflammatory Cytokines and MAO-A.

Fibromyalgia is a refractory syndrome characterized by chronic wayward pain and complex co-morbid psychological trepidation. The current treatments have a limited role and proper clinical benefits are far from satisfactory. Naturally occurring coumarins such as osthole are known to have analgesic and anti-inflammatory activities. Therefore, the current investigation was designed to explore the potential of natural coumarin esculetin (2.5, 5, and 10 mg/kg) in mitigating reserpine-induced fibromyalgia in Swiss albino mice. Esculetin is a 6,7 dihydroxy-coumarin obtained from various plant sources such as Aesculus hippocastanum L, Ceratostigma willmottianum, Citrus limonia, etc. Reserpine (0.5 mg/kg/day s.c.) treatment for first 3 days, significantly altered the behavior of mice as evidenced by reduced paw withdrawal threshold in pressure application measurement (PAM) test and electronic von-Frey (eVF) test, increased immobility time in forced swim test (FST), increased latency to reach the platform in Morris water maze (MWM) test and reduced number of square crossed in the open field test (OFT). These behavioral deficits with reserpine treatment were integrated with a reduced level of serotonin (5-HT), reduced glutathione (GSH), along with an increase in monoamine oxidase-A (MAO-A) activity, pro-inflammatory cytokines (IL-1ß, TNF-α), thiobarbituric acid reactive substances (TBARS) and glutamate level. Esculetin (10 mg/kg/day i.p) treatment for 5 days, significantly abrogated reserpine induced behavioral and biochemical alterations. Whereas, no significant improvement was observed with lower doses of esculetin i.e. 2.5 and 5 mg/kg.

Role of water in cyclooxygenase catalysis and design of anti-inflammatory agents targeting two sites of the enzyme.

While designing the anti-inflammatory agents targeting cyclooxygenase-2 (COX-2), we first identified a water loop around the heme playing critical role in the enzyme catalysis. The results of molecular dynamic studies supported by the strong hydrogen-bonding equilibria of the participating atoms, radical stabilization energies, the pKa of the H-donor/acceptor sites and the cyclooxygenase activity of pertinent muCOX-2 ravelled the working of the water-peptide channel for coordinating the flow of H·/electron between the heme and Y385. Based on the working of H·/electron transfer channel between the 12.5 Å distant radical generation and the radical disposal sites, a series of molecules was designed and synthesized. Among this category of compounds, an appreciably potent anti-inflammatory agent exhibiting IC50 0.06 µM against COX-2 and reversing the formalin induced analgesia and carageenan induced inflammation in mice by 90% was identified. Further it was revealed that, justifying its bidentate design, the compound targets water loop (heme bound site) and the arachidonic acid binding pockets of COX-2.

Anti-nociceptive and anti-inflammatory effect of imperatorin: evidences for involvement of COX-2, iNOS, NFκB and inflammatory cytokines.

Aim: The objective of the current investigation was to explore the analgesic effect of naturally occurring furanocoumarin, imperatorin and the involvement of inducible cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS), NFκB and cytokines in the observed effect.Materials and methods: Anti-nociceptive effect was explored by inducing chemical hyperalgesia using acetic acid and formalin in mice. ED50 of imperatorin was calculated in acetic acid model. Modulation of cyclooxygenase and nitric oxide pathway by imperatorin was examined by stimulator/precursor challenge with substance P and L-arginine, respectively and quantification of COX-2, iNOS and NFκB expression by immunohistochemical analysis in spinal tissues. Involvement of inflammatory cytokines TNF-α and IL-1ß was investigated using LPS challenge and subsequent ELISA analysis of these inflammatory mediators in serum. Carrageenan inflicted paw edema was employed to explore the anti-inflammatory activity of imperatorin.Results: A significant reduction in the nociceptive behaviour was observed with imperatorin treatment in acetic acid and formalin test. ED50 of imperatorin was found to be 4.53 mg/kg. Pre-treatment with substance P and L-arginine significantly attenuated the anti-nociceptive activity of imperatorin in formalin test. Immunohistochemical findings revealed marked decrease in spinal COX-2, iNOS and NFκB expression. Imperatorin administration significantly reduced LPS induced rise in level of TNF-α and IL-1ß dose dependently. In carrageenan-induced paw edema test, maximum possible anti-inflammatory effect of imperatorin was evident after 240 min of carrageenan administration.Conclusion: Current investigation revealed that anti-nociceptive and anti-inflammatory potential of imperatorin is probably mediated through the attenuation of COX-2, iNOS, NFκB activity and reduction in circulatory cytokines.

Bergapten Ameliorates Vincristine-Induced Peripheral Neuropathy by Inhibition of Inflammatory Cytokines and NFκB Signaling.

Bergapten, a furanocoumarin derivative found in a variety of medicinal plants, is documented to possess anti-inflammatory activity. However, whether bergapten is useful in alleviating the symptoms as well as the progress of peripheral neuropathy is not yet studied. The current investigation has been designed to explore the effect of bergapten on vincristine-induced neuropathic pain. Rats were grouped as normal, neuropathic control (vincristine), gabapentin, and bergapten treated groups with five animals in each group. Vincristine (100 µg/kg, i.p.) was administered for 10 days with 2 days break. Gabapentin (60 mg/kg, i.p.) and bergapten (10 mg/kg i.p.) treatments were given once daily for 14 days. The animals were assessed for hyperalgesia and allodynia. After 14 days, animals were sacrificed to detect plasma pro-inflammatory cytokines (TNF α, IL-1ß), spinal cord, and sciatic nerve oxidative stress and expression of iNOS, COX-2, and NFkB in the spinal cord. There was a marked reduction in pain behaviors in the bergapten group as compared to the vincristine group. Bergapten also attenuated pro-inflammatory cytokines (TNFα and IL-1ß), oxidative stress, and expression of NFkB, COX-2, and iNOS. Overall the current study concludes that bergapten could serve as a potential lead to drug development for the treatment of neuropathic pain.

Ameliorative effect of imperatorin in chemically induced fibromyalgia: Role of NMDA/NFkB mediated downstream signaling.

Fibromyalgia (FM) is a chronic pain syndrome involving complex interplay of biogenic amines and NMDA receptor mediated hypersensitization of nociceptive pathways. Clinical management of FM is poorly addressed with only a few available therapeutic options. Coumarins are active phenolic molecules of natural origin found to have broad pharmacological activities. Current investigation explores the role of naturally occurring coumarin, imperatorin in mouse model of fibromyalgia. Administration of reserpine (0.5 mg/kg, s.c.) thrice at 24 h intervals induced behavioral and neurochemical alterations characteristic of fibromyalgia. Reserpine was found to induce allodynia quantified using electronic von Frey (e-VF) and pressure application measurement (PAM) test, depression as indicated by an increased duration of immobility in forced swim test (FST), decreased motor coordination and locomotor activity in inclined plane test (IPT) and open field test (OFT) respectively. Cognitive deficits were evident by an increased latency to locate hidden platform in Morris water maze (MWM) and passive avoidance test (PAT). Reserpine treatment was found to cause an increased anxiety as revealed by increased time spent in closed arm of the elevated plus maze (EPM). Furthermore, an up- regulation in NMDA and NFκB expression in the brain and spinal cord was observed in reserpine treated groups. Administration of imperatorin (10 mg/kg, i.p) for a period of 5 days ameliorated all behavioral deficits, biochemical changes and decreased expression of NMDA and NFκB in the brain and spinal cord of treated mice. These findings indicate an interplay of NMDA/NFκB modulation by imperatorin in the reserpine induced fibromyalgia in mice.

Possible Molecular Mediators Involved and Mechanistic Insight into Fibromyalgia and Associated Co-morbidities.

Fibromyalgia is a chronic complex syndrome of non-articulate origin characterized by musculoskeletal pain, painful tender points, sleep problems and co-morbidities including depression, migraine. The etiopathogenesis of fibromyalgia is complex, variable and remains inconclusive. The etiological factors that have been defined include stress, genetic predisposition and environmental components. As per the reports of the American College of Rheumatology (ACR) the prevalence of fibromyalgia varies from 2 to 22% among the general population with poor diagnostic features primarily pain. Fibromyalgia encompasses a spectrum of co-morbid conditions with multifarious pathogenesis. The highly prevalent manifestations of fibromyalgia include heterogeneous pain and aches. Biochemical and neurobiological elements of fibromyalgia include neurotransmitters, hypothalamic pituitary adrenal axis (HPA axis), inflammatory cytokines, monoaminergic pathway, opioid peptides, sex hormones, nerve growth factor (NGF) and local free radical insult. An imbalance in the serotonergic system is the major underlying etiological factor that has been explored most widely. Owing to complex interplay of diverse pathophysiological pathways, overlapping co-morbidities such as depression have been clinically observed. Therapeutic management of fibromyalgia involves both non pharmacological and pharmacological measures. The current review presents various dysregulations and their association with symptoms of fibromyalgia along with their underlying neurobiological aspects.

Osthole ameliorates neurogenic and inflammatory hyperalgesia by modulation of iNOS, COX-2, and inflammatory cytokines in mice.

BACKGROUND: Osthole is a bioactive component reported in medicinal plants such as Angelica pubescens and Cnidium monnieri, known for analgesic activity. However, the toxicity, median effective dose (ED50), and dual modulation of nitric oxide and cyclooxygenase pathways along with inflammatory cytokines of osthole are yet to be determined. METHODS: The animals (mice) were assessed for general behaviour and mortality in varying doses (50, 300, and 2000 mg kg-1) of osthole for acute toxicity over 14 days. The analgesic activity was investigated using acetic acid and formalin-induced hyperalgesia, and anti-inflammatory activity was explored in carrageenan-induced paw oedema. ED50 of osthole was calculated using Design Expert software. Involvement of nitric oxide and cyclooxygenase pathways was investigated by agonist challenges with L-arginine and substance P, respectively. The expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was determined in spinal sections by immunohistochemical analysis. Lipopolysaccharide (LPS) challenge was used to assess in vivo effect on inflammatory cytokines (TNFα and IL-6). RESULTS: Acute toxicity studies revealed no behavioural abnormality or mortality on osthole treatment and unremarkable histological findings. Osthole was found to significantly decrease acetic acid and formalin-induced hyperalgesia (ED50 = 5.43 mg kg-1) and carrageenan-induced paw oedema with no toxicity symptoms. Osthole produced a marked decrease in iNOS and COX-2 expression as well as TNFα and IL-6. The findings corroborate to modulation of iNOS and COX-2 and inflammatory cytokines by osthole. This study provides promising insights and prospects for application of osthole in pain management.

Synergy of Physico-chemical and Biological Experiments for Developing a Cyclooxygenase-2 Inhibitor.

The physiological consequences of COX-2 overexpression in the development of cancer, diabetes and neurodegenerative diseases have made this enzyme a promising therapeutic target. Herein, COX-2 active site was analyzed and new molecules were designed. We identified a highly potent molecule (S)-3a with IC50 value and the selectivity for COX-2 0.6 nM and 1666, respectively. The MTD of (S)-3a was 2000 mg kg-1 and its pharmacokinetic studies in rat showed t1/2 7.5 h. This compound reversed acetic acid induced analgesia and carragennan induced inflammation by 50% and 25% in rat when used at a dose 10 mg kg-1. Mechanistically, it was found that compound (S)-3a inhibits COX-2. Overall, the combination of physico-chemical and biological experiments facilitated the development of a new lead molecule to anti-inflammatory drug.

Indolyl-isoxazolidines attenuate LPS-stimulated pro-inflammatory cytokines and increase survival in a mouse model of sepsis: Identification of potent lead.

A library of indolyl-isoxazolidines (6-9) has been synthesized by regio- and stereoselective microwave irradiated 1,3-dipolar cycloadditions of C-(3-indolyl)-N-phenylnitrone (2') with variedly substituted dipolarophiles (3'-5') and screened for their anti-inflammatory activities through inhibition of pro-inflammatory cytokines such as TNF-α and IL-6. Amongst the evaluated compounds (6-9), bicyclic isoxazolidine (9a) was found to exhibit significant inhibitory potential against LPS induced human IL-6 and TNF-α in THP-1 cells. Compound 9a was further assessed for in vivo analgesic and anti-inflammatory activities via acetic acid induced writhing and carrageenan induced paw edema models in mice, respectively. The results showed that compound possesses potent anti-inflammatory-analgesic activity comparable to indomethacin and did not show toxicity up to a 2000 mg kg-1 dose as evidenced by histopathological studies. Consequently, the most active compound 9a was also evaluated against LPS-induced septic death and exhibited a significant protection in in vivo mouse model. Taken all together, the results suggest that the compound 9a is able to attenuate pro-inflammatory cytokines such as IL-6 and TNF-α; accelerate resolution of inflammation, and also increased survival rate of septic mice. Therefore, these "lead" isoxazolidines can be used as promising candidate for further analgesic/anti-inflammatory drug design and development.

TNF-α and IL-6 inhibitors: Conjugates of N-substituted indole and aminophenylmorpholin-3-one as anti-inflammatory agents.

The conjugates obtained by the combination of indole and aminophenyl morpholinone were screened for TNF-α and IL-6 inhibition in microglial cells. Compound 4 was found to be the most potent anti-inflammatory agent as it reduced LPS induced level of inflammatory cytokines TNF-α and IL-6 by 71% and 53%, respectively. A significant decrease in NO and MMPs release from BV2 cells in culture pretreated with this compound as well as inhibition of nuclear translocation of NF-κB and AP-1 was observed. 75% inhibition of acetic acid induced algesia in swiss albino mice was noticed in the presence of compound 4. Experimental data and molecular docking studies indicate that the compounds are targeting TNF-α, iNOS and IL-6.

Rationally designed benzopyran fused isoxazolidines and derived ß2,3,3-amino alcohols as potent analgesics: Synthesis, biological evaluation and molecular docking analysis.

Based on structure activity analysis of morphine related opiates, we have synthesized some novel benzopyran fused isoxazolidines (2a-e) and derived conformationally constrained ß2,3,3-amino alcohols (3a-e), which were evaluated in vivo for their anti-nociceptive activity through acetic acid induced writhing test (peripheral) and formalin induced algesia (central). Results showed that, compound 2a possesses significant opioid agonist activity. Further, molecular docking analysis reveals that compound 2a binds to δ-opioid receptor (DOR) with comparatively better D-score than to µ (MOR) and κ (KOR) receptors. Compound 2a did not show any toxicity up to a 2000 mg kg-1 dose.

Rational Design of Small Peptides for Optimal Inhibition of Cyclooxygenase-2: Development of a Highly Effective Anti-Inflammatory Agent.

Among the small peptides 2-31, (H)Gly-Gly-Phe-Leu(OMe) (30) reduced prostaglandin production of COX-2 with an IC50 of 60 nM relative to 6000 nM for COX-1. The 5 mg kg(-1) dose of compound 30 rescued albino mice by 80% from capsaicin-induced paw licking and recovered it by 60% from carrageenan-induced inflammation. The mode of action of compound 30 for targeting COX-2, iNOS, and VGSC was investigated by using substance P, l-arginine, and veratrine, respectively, as biomarkers. The interactions of 30 with COX-2 were supported by isothermal calorimetry experiments showing a Ka of 6.10 ± 1.10 × 10(4) M(-1) and ΔG of -100.3 kJ mol(-1) in comparison to a Ka 0.41 × 10(3) ± 0.09 M(-1) and ΔG of -19.2 ± 0.06 kJ mol(-1) for COX-1. Moreover, compound 30 did not show toxicity up to a 2000 mg kg(-1) dose. Hence, we suggest peptide 30 as a highly potent and promising candidate for further development into an anti-inflammatory drug.