Investigation of the chemical structure and analgesic and anti-inflammatory properties of polysaccharides that constitute the dietary fibers of soursop (Annona muricata) fruit.

Soursop fruits are widely used in the folk medicine to treat a variety of health conditions. Once the chemical structure of dietary fibers from fruits is closely related to its biological functions in the human body, we aimed to explore structural features and biological activity of dietary fibers from soursop. Polysaccharides that constitute the soluble and insoluble fibers were extracted and further analyzed using monosaccharide composition, methylation, molecular weight determination and 13C NMR data. Soursop soluble fibers (SWa fraction) were characterized as having type II arabinogalactan and a highly methyl esterified homogalacturonan, while non-cellulosic insoluble fibers (SSKa fraction) were mainly composed by a pectic arabinan, a xylan-xyloglucan complex and a glucuronoxylan. The oral pre-treatment with SWa and SSKa promoted antinociception in mice writhing test, reducing the number of pain-like behaviors (in 84.2 % and 46.9 %, respectively, at 10 mg/kg) and peritoneal leucocyte migration (55.4 % and 59.1 %, at 10 mg/kg), effects possibly associated with the pectins present in fruit pulp extractions. SWa also significantly inhibited the plasmatic extravasation of Evans blue dye in 39.6 % at 10 mg/kg. This paper describes for the first time the structural features of soursop dietary fibers that may be of biological significance in future.

Neoline, fuziline, songorine and 10-OH mesaconitine are potential quality markers of Fuzi: In vitro and in vivo explorations as well as pharmacokinetics, efficacy and toxicity evaluations.

ETHNOPHARMACOLOGICAL RELEVANCE: Fuzi, the lateral roots of Aconitum carmichaelii Debx, plays an irreplaceable role in treating Yang deficiency and cold coagulation syndromes. However, Fuzi has a narrow margin of safety since its pharmacological constituents, Aconitum alkaloids, have potential cardiotoxicity and neurotoxicity. The current quality markers (Q-markers) for the control of Fuzi's efficacy and toxicity are 3 monoester-diterpenoid alkaloids, namely, benzoylaconine (BAC), benzoylhypaconine and benzoylmesaconine (BMA) and 3 diester-diterpenoid alkaloids, namely, aconitine (AC), hypaconitine and mesaconitine (MA). However, mounting evidence indicates that the current 6 Q-markers may not be efficacy- or toxicity-specific enough for Fuzi. AIM OF THE STUDY: The aim of this study was to explore and evaluate efficacy- or toxicity-specific potential quality markers (PQ-markers) of Fuzi. MATERIALS AND METHODS: PQ-markers were explored by analyzing 30 medicinal samples and alkaloids exposed in mouse. Pharmacokinetics of PQ-markers on C57BL/6J mice were determined. Anti-inflammatory effects of PQ-markers were evaluated by λ-carrageenan-induced paw edema model and lipopolysaccharide-induced RAW264.7 cell inflammatory model, while analgesic effects were assessed by acetic acid-induced pain model and Hargreaves test. Cardiotoxicity and neurotoxicity of PQ-markers were assessed by histological and biochemical analyses, while acute toxicity was evaluated by modified Kirschner method. RESULTS: After in vitro and in vivo explorations, 7 PQ-markers, namely, neoline (NE), fuziline (FE), songorine (SE), 10-OH mesaconitine (10-OH MA), talatizamine, isotalatizidine and 16ß-OH cardiopetalline, were found. In the herbal medicines, NE, FE, SE and 10-OH MA were found in greater abundance than many other alkaloids. Specifically, the amounts of NE, FE and SE in the Fuzi samples were all far higher than that of BAC, and the contents of 10-OH MA in 56.67% of the samples were higher than that of AC. In mouse plasma and tissues, NE, FE, SE, talatizamine, isotalatizidine and 16ß-OH cardiopetalline had higher contents than the other alkaloids, including the 6 current Q-markers. The pharmacokinetics, efficacy and toxicity of NE, FE, SE and 10-OH MA were further evaluated. The average oral bioavailabilities of NE (63.82%), FE (18.14%) and SE (49.51%) were higher than that of BMA (3.05%). Additionally, NE, FE and SE produced dose-dependent anti-inflammatory and analgesic effects, and their actions were greater than those of BMA. Concurrently, the toxicities of NE, FE and SE were lower than those of BMA, since no cardiotoxicity or neurotoxicity was found in mice after NE, FE and SE treatment, while BMA treatment notably increased the creatine kinase activity and matrix metalloproteinase 9 level in mice. The average oral bioavailability of 10-OH MA (7.02%) was higher than that of MA (1.88%). The median lethal dose (LD50) of 10-OH MA in mice (0.11 mg/kg) after intravenous injection was close to that of MA (0.13 mg/kg). Moreover, 10-OH MA produced significant cardiotoxicity and neurotoxicity, and notable anti-inflammatory and analgesic effects that were comparable to those of MA. CONCLUSIONS: Seven PQ-markers of Fuzi were found after in vitro and in vivo explorations. Among them, NE, FE and SE were found to be more efficacy-specific than BMA, and 10-OH MA was as toxicity-specific as MA.

Isolation, Structural Characterization, and Biological Activity of the Two Acidic Polysaccharides from the Fruits of the Elaeagnus angustifolia Linnaeus.

Elaeagnus angustifolia Linnaeus is a medicinal plant and its fruit has pharmacological activity such as antiinflammatory, antiedema, antinociceptive, and muscle relaxant functions, etc. Two acidic homogeneous polysaccharides (EAP-H-a1 and EAP-H-a2) were isolated from the fruits of Elaeagnus angustifolia L. through DEAE-52 and Sephadex G-75 column chromatography, and the physicochemical, structural properties, and biological activities of the polysaccharides were investigated. Both EAP-H-a1 and EAP-H-a2 were composed of Rha, Ara, Xyl, Glc, and Gal with the molar ratios of 13.7:20.5:23.3:8.8:33.4 and 24.8:19.7:8.2:8.4:38.6, respectively, and with the molecular weights of 705.796 kDa and 439.852 kDa, respectively. The results obtained from Fourier transform infrared spectroscopy (FTIR) confirmed the polysaccharide nature of the isolated substances. Congo red assay confirmed the existence of a triple-helix structure. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis revealed that EAP-H-a1 and EAP-H-a2 had irregular fibrous, filament-like surfaces; and both had crystalline and amorphous structures. Bioactivity analysis showed that the crude polysaccharide, EAP-H-a1, and EAP-H-a2 had clear DPPH and ABTS free radical scavenging activity, and could promote the secretion of NO and the phagocytic activities of RAW 264.7 and THP cells, which showed clear antioxidant and immuno-regulatory activity. These results indicated that Elaeagnus angustifolia L fruit acidic polysaccharides may have potential value in the pharmaceutical and functional food industries.

Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds.

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (ß-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

Betulinic Acid-Azaprostanoid Hybrids: Synthesis and Pharmacological Evaluation as Anti-inflammatory Agents.

BACKGROUND: Prevention and treatment of chronic inflammatory diseases require effective and low-toxic medicines. Molecular hybridization is an effective strategy to enhance the biological activity of new compounds. Triterpenoid scaffolds are in the focus of attention owing to their anti-inflammatory, antiviral, antiproliferative, and immunomodulatory activities. Heteroprostanoids have different pleiotropic effects in acute and chronic inflammatory processes. OBJECTIVE: The study aimed to develop structurally new and low toxic anti-inflammatory agents via hybridization of betulinic acid with azaprostanoic acids. METHODS: A series of betulinic acid-azaprostanoid hybrids was synthesized. The synthetic pathway included the transformation of betulin via Jones' oxidation into betulonic acid, reductive amination of the latter and coupling obtained by 3ß-amino-3-deoxybetulinic acid with the 7- or 13-azaprostanoic acids and their homo analogues. The hybrids 1-9 were investigated in vivo on histamine-, formalin- and concanavalin A-induced mouse paw edema models and two models of pain - the acetic acid-induced abdominal writhing and the hotplate test. The hybrids were in vitro evaluated for cytotoxic activity on cancer (MCF7, U- 87 MG) and non-cancer humane cell lines. RESULTS: In the immunogenic inflammation model, the substances showed a pronounced anti-inflammatory effect, which was comparable to that of indomethacin. In the models of the exudative inflammation, none of the compounds displayed a statistically significant effect. The hybrids produced weak or moderate analgesic effects. All the agents revealed low cytotoxicity on human immortalized fibroblasts and cancer cell lines compared with 3ß- amino-3-deoxybetulinic acid and doxorubicin. CONCLUSION: The results indicate that the principal anti-inflammatory effect of hybrids is substantially provided with the triterpenoid scaffold and in some cases with the azaprostanoid scaffold, but the latter makes a significant contribution to reducing the toxicity of hybrids. Hybrid 1 is of interest as a potent low toxic agent against immune-mediated inflammation.

A strategy for qualitative and quantitative profiling of Angelicae Pubescentis Radix and detection of its analgesic and anti-inflammatory components by spectrum-effect relationship and multivariate statistical analysis.

This study established a spectrum-effect relationship method for screening and quantifying the analgesic and anti-inflammatory active ingredients in Angelicae Pubescentis Radix (AP) by ultra-high-performance liquid chromatography-quadrupole mass spectrometry detector analysis (UPLC-QDA). First, the fingerprint of AP was established to determine the common peaks. Next, six batches of AP samples, with significant differences, were selected for evaluation of pharmacological activity. Subsequently, the spectrum-effect relationship was used to screen the active ingredients. Finally, the screened ingredients were quantified using UPLC-QDA. In total, 21 common peaks were identified and four effective compounds (bergapten, columbianetin acetate, osthole and isoimperatorin) were selected using the gray relational analysis and partial least squares regression analysis. Quantitative analysis showed that the content of the four effective compounds was the highest in a randomly selected batch, S7 (Hubei). To our knowledge, this is the first attempt that evaluated the quality and spectrum-effect relationship of AP by quantitative analysis and chemometrics. This study identified the key pharmacologically active components of AP and thereby improved the quality evaluation system of AP. This method has broad application prospects for screening effective components and will be helpful in establishing more reliable, scientific and reasonable quality standards for AP and other traditional Chinese medicines.

Validation of the Key Active Ingredients and Anti-Inflammatory and Analgesic Effects of Shenjin Huoxue Mixture Against Osteoarthritis by Integrating Network Pharmacology Approach and Thin-Layer Chromatography Analysis.

BACKGROUND: Shenjin Huoxue Mixture (SHM), a classic traditional herb mixture has shown significant clinical efficacy against osteoarthritis (OA). Our previous experimental study has confirmed its anti-inflammatory and analgesic effect on acute soft tissue injury in rats, with the compound of glycyrrhizinate in SHM identified and the content of paeoniflorin in SHM determined by high-performance liquid chromatography (HPLC). However, the components and its pharmacological mechanisms of SHM against OA have not been systematically elucidated yet. Thus this study aimed to predict the key active ingredients and potential pharmacological mechanisms of SHM in the treatment of OA by network pharmacology approach and thin-layer chromatography (TLC) validation. METHODS: The active ingredients of SHM and their targets, as well as OA-related targets, were identified from databases. The key active ingredients were defined and ranked by the number of articles retrieved in PubMed using the keyword "(the active ingredients [Title/Abstract]) AND Osteoarthritis[Title/Abstract] ", and validated partially by TLC. The pharmacological mechanisms of SHM against OA were displayed by GO term and Reactome pathway enrichment analysis with Discovery Studio 3.0 software docking to testing the reliability. RESULTS: Finally, 16 key active ingredients were identified and ranked, including quercetin validated through TLC. Inflammatory response, IL-6 signaling pathway and toll-like receptor (TLR) cascades pathway were predicted as the main pharmacological mechanisms of SHM against OA. Especially, 12 out of 16 key active ingredients, including validated quercetin, were well docked to IL-6 proteins. CONCLUSION: Our results confirmed the anti-inflammatory and analgesic effect of SHM against OA through multiple components, multiple targets and multiple pathways, which revealed the theoretical basis of SHM against OA and may provide a new drug option for treating OA.

Combined Liquid Chromatography-mass Spectrometry and Next-generation DNA Sequencing Detection of Adulterants and Contaminants in Analgesic and Anti-inflammatory Herbal Medicines.

INTRODUCTION: Methods for assessing the quality of herbal medicine preparations have advanced significantly in recent years in conjunction with increases in herbal medicine use and reports of adulteration and contamination. OBJECTIVE: This study examined the quality of analgesic and anti-inflammatory herbal medicine preparations available on the Australian market by detecting the presence of listed ingredients, adulterants and contaminants. METHODS: Forty-nine analgesic and anti-inflammatory herbal medicine preparations were randomly sourced from Australian capital cities. They were audited using a dual approach of liquid chromatography-mass spectrometry (LC-MS) combined with next-generation DNA sequencing. Once screened, a comparison of listed ingredients with verified ingredients was conducted to determine the accuracy of labelling, and the extent of adulteration and contamination. RESULTS: Twenty-six of 49 (53%) herbal medicines were adulterated or contaminated with undeclared ingredients. LC-MS revealed the presence of pharmaceutical adulterants including atropine and ephedrine. DNA sequencing uncovered concerning levels of herbal substitution, adulteration and contamination, including the use of fillers (alfalfa, wheat and soy), as well as pharmacologically relevant species (Centella asiatica, Panax ginseng, Bupleurum and Passiflora). Pig/boar and bird DNA was found in some preparations, inferring substandard manufacturing practices. Of the 26 contaminated samples, 19 (73%) were manufactured in Australia, and 7 (27%) were imported from other countries (6 from China, 1 from New Zealand). In 23 of 49 (47%) herbal medicine samples, no biological ingredients were detected at all. These were predominantly pain and anti-inflammatory preparations such as glucosamine and eicosapentaenoic and docosahexaenoic acids found in krill and fish oils, so DNA would not be expected to survive the manufacturing process. CONCLUSION: The high level of contamination and substitution of herbal medicine preparations sourced from Australian dispensaries supports the need for more stringent pharmacovigilance measures in Australia and abroad.

A biochemometrics strategy combining quantitative determination, bioactivity evaluation and relationship analysis for identification of analgesic alkaloids of raw and vinegar-processed Corydalis turtschaninovii.

A biochemometrics strategy combining quantitative determination, bioactivity evaluation, and relationship analysis was proposed for identification of analgesic components of herbs. First, a robust liquid chromatography tandem mass spectrometry method was developed for simultaneous determination of nine major alkaloids in crude and vinegar-processed Corydalis turtschaninovii. Nine alkaloids were separated on a BEH C18 column with a mobile phase consisting of acetonitrile and water spiked with 0.1% formic acid and then detected by multiple reactions monitoring in the positive ion mode. Nitidine chloride was employed as the internal standard. The method displayed good linearity and the precisions of intra-day and inter-day were all within 3.0%. The recovery rates of each alkaloid ranged from 97.1 to 102.9%. The method was successfully applied for quantitative analysis of nine alkaloids in ten batches of crude and vinegar-processed Corydalis turtschaninovii. Second, the analgesic effects of crude and vinegar-processed Corydalis turtschaninovii were evaluated in mice. Third, principle component analysis, canonical correlation analysis, and partial least squares regression were used to analysis the relationship between the contents of nine major alkaloids and the analgesic effect of different crude and vinegar-processed samples. Tetrahydropalmatine, coptisine, and dehydrocorydaline have a close positive correlation with the analgesic effect.

Araticum (Annona crassiflora Mart.) as a source of nutrients and bioactive compounds for food and non-food purposes: A comprehensive review.

Araticum (Annona crassiflora Mart.) is a fruitful tree native to the Brazilian Cerrado biome that holds high nutritional, functional and economic potential. This plant has been used since ancient times by folk medicine for the treatment of several pathological conditions. There has been increasing interest in the development of pulp-based food products as well as the by-products utilization to obtain value-added ingredients. Understanding the chemical composition and biological activities of different botanical parts of Annona crassiflora Mart. provides a basis to support future researches and applications. In this context, this paper carries out an exhaustive review of the scientific literature, on the main phytochemicals of different botanical parts of Annona crassiflora Mart. (fruit, leaves, stem and root) and their biological activities, assessing their potential uses for several industrial segments. Annona crassiflora Mart. fruits and especially their by-products (peel and seeds) and leaves have been shown a wide range of bioactive compounds such as phenolic compounds, alkaloids, annonaceous acetogenins, tocols, carotenoids, phytosterols, dietary fiber, vitamins, minerals and essential oils. These compounds contribute to various biological activities, including antioxidant, hepatoprotective, anti-inflammatory, antitumoral, analgesic, antidiabetic, skin healing, antidiarrhoeic, antimicrobial, antiparasitic, insecticide and herbicide activities of Annona crassiflora Mart. extracts. Therefore, these findings demonstrate that Annona crassiflora Mart. fruit, by-products and leaves can be excellent candidates to be used as functional foods and/or sources for obtaining bioactive compounds for the food, cosmetics and pharmaceutical applications.

Ailanthus altissima (Miller) Swingle seed oil: chromatographic characterization by GC-FID and HS-SPME-GC-MS, physicochemical parameters, and pharmacological bioactivities.

This study aimed to identify the physicochemical and the chemical properties of Ailanthus altissima (Miller) Swingle seed oil and to evaluate its in vitro antioxidant and antibacterial activities and in vivo analgesic and anti-inflammatory activities. The fatty acids' composition was determined using GC-FID. The oil was screened for antioxidant activity by DPPH test. The analgesic and anti-inflammatory activities were determined using the acetic acid writhing test in mice and the carrageenan-induced paw edema assay in rats, respectively. Volatile compounds were characterized by HS-SPME-GC-MS. A. altissima produces seeds which yielded 17.32% of oil. The seed oil was characterized by a saponification number of 192.6 mg KOH∙g of oil, a peroxide value of 11.4 meq O2∙kg of oil, a K232 of 4.04, a K270 of 1.24, and a phosphorus content of 126.2 ppm. The main fatty acids identified were palmitic (3.06%), stearic (1.56%), oleic (38.35%), and linoleic acids ones (55.76%). The main aroma compounds sampled in the headspace were carbonyl derivatives. The oil presents an important antioxidant activity (IC50 = 24.57 µg/mL) and a modest antimicrobial activity. The seed oil at 1 g/kg showed high analgesic (91.31%) and anti-inflammatory effects (85.17%). The presence of high levels of unsaturated fatty acids and the noteworthy antioxidant capacity of the seed oil can hypothesize its use as an analgesic and anti-inflammatory agent.

Development and Validation of Analytical Method for Simultaneous Estimation of Active Constituents in a Polyherbal Ointment by Gas Chromatography.

Background: The simultaneous, quantitative determination of all active ingredients present in the analgesic formulation (Dazzle ointment) requires an ideal and novel method by which these phytoconstituents can be separated with the highest resolution without any interference from one another. Objective: The present work was conducted to develop and validate a quantitative method for the simultaneous estimation of all five phytoconstituents present in a polyherbal analgesic ointment by GC. Methods: α-Pinene, 1,8-cineole, camphor, menthol, and methyl salicylate present in the ingredients of the ointment were analyzed and quantified by GC using a crosslinked 5% phenyl polydimethylsiloxane capillary column, nitrogen as a carrier gas, and a flame-ionization detector. Aniline was used as the internal standard. Method validation was also performed in order to demonstrate its selectivity, linearity, accuracy, precision, LOD, LOQ, and robustness. Results: The calibration curves of all five marker compounds showed good linear correlation coefficients (r² >0.998) within the tested ranges. The precision of the method was tested by carrying out intra- and interday analyses of the same sample. RSD values were observed to be <1.00%. The accuracy of the method, determined by performing recovery studies, was found to be between 99.25 and 101.39%. The developed method was also demonstrated to be robust (RSD <1.29%) by making small but deliberate variations in method parameters. Conclusions: The developed GC method is simple, precise, and accurate, it and can be used for the rapid quality control testing of the polyherbal formulation. Highlights: The developed GC method will assist in the standardization of polyherbal analgesic formulation consists of α-pinene, 1,8-cineole, camphor, menthol, and methyl salicylate as active constituents.

Sphenodesme involucrata var. paniculata (C.B. Clarke) Munir.: Chemical characterization, anti-nociceptive and anti-inflammatory activities of methanol extract of leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Sphenodesme involucrata var. paniculata (C. B. Clarke) Munir is native as well as endemic to South India. Its leaves are used in folklore medicine to treat pain and rheumatism. OBJECTIVE: This study was aimed to investigate the chemical characterization, anti-nociceptive and mode of action underlying the anti-inflammatory effects of methanol extract of S. involucrata leaves (MESi). METHODS: Phytoconstituents of MESi was analyzed using colorimetric and liquid chromatography-mass spectrometry (LC-MS) methods, and the oral acute toxicity was evaluated in mice up to 2000 mg/kg. The anti-nociceptive effect was evaluated in hot plate and writhing tests; whereas the anti-inflammatory effect was investigated using carrageenan, cotton pellet and lipopolysaccharide (LPS)-induced peritonitis models at doses of 100, 200 and 400 mg/kg. Additionally nitric oxide (NO) and inflammatory cytokines levels were also evaluated. RESULTS: MESi exhibited the high content of phenolics and flavonoids as well as compounds like austricine, benzylglucosinolate, gossypin, justicidin B and cirsimarin were detected in LC-MS. In the acute toxicity study, oral administration of MESi did not cause any toxic effect and mortality up to 2000 mg/kg body weight in mice. In the anti-nociceptive tests, MESi augmented the latency period at higher dose (400 mg/kg), on the other hand attenuated writhings at the dose of 400 mg/kg by 87.87% (p < 0.001). In the carrageenan induced paw oedema MESi significantly inhibited the oedema formation at dose 400 mg/kg by 32.1%; besides, anti-inflammatory effect was registered in the cotton pellets-induced inflammation model at doses 200 and 400 mg/kg by 27.09% (p < 0.001) and 35.47% (p < 0.001) respectively. On the other hand, MESi appreciably reduced leukocyte, neutrophils infiltration, nitric oxide, TNF-α and IL-1ß levels and increased the IL-10 level in the (LPS)-induced peritonitis model. CONCLUSION: The results conclude that MESi has no acute toxic effect and it demonstrated potent anti-nociceptive and anti-inflammatory activities. Its anti-nociceptive activities are probably mediated through peripheral and central mechanisms. The anti-inflammatory effect of MESi involved the inhibition of neutrophils migration and the modulation of Th1 and Th2 cytokines, besides the attenuation of production of PGE2 and NO. LC-MS analysis revealed the predominant presence of the austricine, benzylglucosinolate, gossypin, justicidin B and cirsimarin compounds, which are possibly involved in the anti-nociceptive and anti-inflammatory effects of MESi. The current study provided supportive evidence for the folklore use of S. involucrata in the treatment of pain and inflammatory conditions.

Pharmacological and toxicological study of a chemical-standardized ethanol extract of the branches and leaves from Eysenhardtia polystachya (Ortega) Sarg. (Fabaceae).

Eysenhardtia polystachya is used for the empirical treatment of cancer, infections, diarrhea, inflammation, and pain. This study identified, using GC-MS, the main chemical components in an ethanol extract of E. polystachya branches and leaves (EPE) and tested its cytotoxic, antimicrobial, anti-diarrheal, anti-inflammatory, and antinociceptive effects. The in vitro and in vivo toxicity of EPE was evaluated using the comet assay in human peripheral blood mononuclear cells (PBMC) and the acute toxicity test in mice, respectively. The cytotoxic and the antimicrobial effects were performed using the MTT assay and the minimum inhibitory concentration (MIC) test, respectively. The levels of pro-inflammatory mediators in LPS-stimulated macrophages were measured to evaluate the in vitro anti-inflammatory effects of EPE. The antidiarrheal (castor oil test, small intestine transit, and castor oil-induced enteropooling), and anti-inflammatory activities (TPA and carrageenan) of EPE were also performed. The antinociceptive actions of EPE were carried out with the following tests: acetic acid, formalin, and hot plate. The hypnotic and locomotor effects were analyzed using pentobarbital and a rotarod system, respectively. The main component in EPE was D-pinitol (26.93%). The antidiarrheal and antinociceptive effects of D-pinitol were also evaluated. EPE showed low in vitro toxicity (DNA damage in PBMC at concentrations higher than 200 µg/ml), and low in vivo toxicity (LD50 > 2000 mg/kg i.p. and p.o.). Furthermore, EPE lacked cytotoxic activity (IC50 > 300 µg/ml) on human cancer cells, but showed good antimicrobial effects in E. coli (MIC=1.56 µg/ml) and S. aureus (MIC = 0.78 µg/ml). In multi-drug resistant microorganisms, EPE showed MIC> 100 µg/ml. EPE exerted in vitro anti-inflammatory effects, mainly, by the decrease in the production of H2O2 (IC50 = 43.9 ±â€¯3.8 µg/ml), and IL-6 (73.3 ±â€¯6.9 µg/ml). EPE (ED50 =7.5 ±â€¯0.9 mg/kg) and D-pinitol (ED50 = 0.1 ±â€¯0.03 mg/kg) showed antidiarrheal activity, and antinociceptive effects in the acetic acid test with ED50 = 117 ±â€¯14.5 mg/kg for EPE and 33 ±â€¯3.2 mg/kg for D-pinitol. EPE showed also antinociceptive activity in the phase 2 of the formalin test (ED50 = 48.9 ±â€¯3.9 mg/kg), without inducing hypnotic effects or altering the locomotor activity in mice. The results here presented corroborate the folk medicinal use of Eysenhardtia polystachya in the treatment of infections, diarrhea, inflammation, and pain. D-pinitol, the main metabolite of EPE, showed antinociceptive and antidiarrheal effects with similar potency compared to standard drugs.

Neuropharmacological effects of essential oil from the leaves of Croton conduplicatus Kunth and possible mechanisms of action involved.

ETHNOPHARMACOLOGICAL RELEVANCE: Croton conduplicatus Kunth (Euphorbiaceae) is a Brazilian aromatic medicinal plant, widely known as "quebra-faca". In folk medicine, its leaves and stem-barks are used as a natural analgesic for the treatment of headaches. AIM OF THE STUDY: In this study, we describe for the first time the neuropharmacological potential of the essential oil obtained from the leaves of Croton conduplicatus (EO) in experimental models of pain, anxiety and insomnia. The mechanisms of action involved in these activities were also investigated. MATERIAL AND METHODS: Different experimental models were used to evaluate the antinociceptive (acetic acid, formalin-induced nociception and hot plate tests), anxiolytic (elevated plus maze and hole board tests) and sedative (thiopental-induced sleeping time) effects of EO in mice. EO was evaluated in three different doses (25, 50 and 100 mg/kg, i.p.) and compared with positive and negative controls in all experimental protocols. When appropriate, animals were pretreated with pharmacological antagonists (naloxone, atropine and flumazenil) in order to evaluate the mechanisms of action involved. A docking study also was performed to identify possible targets involved. RESULTS: EO (25, 50 and 100 mg/kg, i.p.) demonstrated a significant antinociceptive activity in all experimental models. Pretreatment with naloxone or atropine reversed the antinociceptive response (p < 0.05), suggesting the involvement of opioid and muscarinic receptors, respectively. A docking study was performed with the major components identified in EO (1,8 cineole - 21.42%, spathulenol - 15.47%, p-cymene - 12.41% and caryophyllene oxide - 12.15%), demonstrating favorable interaction profile with different subtypes of muscarinic (M2, M3 and M4) and opioids (delta and mu) receptors. EO also showed anxiolytic (mainly at doses of 25 and 50 mg/kg, i.p.) and sedative (only at the dose of 100 mg/kg, i.p.) effects in mice. These pharmacological responses were reversed by flumazenil (p < 0.05), indicating possible involvement of GABAA receptors. CONCLUSION: Our findings support the traditional use of this plant as a natural analgesic and suggest that EO is a multi-target natural product, presenting not only antinociceptive effect but also anxiolytic and sedative activities depending on the dose used.

Screening of analgesic and anti-inflammatory active component in Fructus Alpiniae zerumbet based on spectrum-effect relationship and GC-MS.

Fructus Alpiniae zerumbet is widely used in Guizhou province as a miao folk herb with anti-inflammatory, analgesic, protection against cardiovascular diseases, antihypertension and antioxidant activities. To further investigate the chemical material basis, the spectrum-effect relationship was established using gray relational analysis between the chromatographic fingerprint and its bioactivities. Herein, the fingerprints of essential oils from Fructus Alpiniae zerumbet (EOFAZ) from various sources were determined by gas chromatography mass spectrometry, and the analgesic and anti-inflammatory bioactivities were investigated using the mouse model of acetic acid-induced writhing test and dimethylbenzene-induced mouse ear edema test. Finally, 17 common peaks were identified from nine batches of A. zerumbet, by comparison with the standard mass spectra in Nist2005, Wiley275 library. Meanwhile, the results showed significant analgesic and anti-inflammatory effects in all of the different sources of EOFAZ. In particularly, peak 1 (α-pipene), peak 3 (ß-pinene), peak 9 (camphor) and peak 16 (α-cadinol) might be the main bioactive ingredients for analgesic and anti-inflammatory activities. The model of the spectrum-effect relationships of EOFAZ was successfully discovered, which provided a novel platform for finding the bioactive components, a theoretical foundation for its further study and helping to establish quality control of Fructus A. zerumbet.

Antinociceptive and anti-inflammatory activities of the Jatropha isabellei dichloromethane fraction and isolation and quantitative determination of jatrophone by UFLC-DAD.

CONTEXT: Jatropha isabellei Müll. Arg. (Euphorbiaceae) has been used in the traditional medicine to treat arthritis. OBJECTIVE: To evaluate the anti-inflammatory and antinociceptive activities of the dichloromethane fraction (DFJi) from underground parts of J. isabellei, and to develop an analytical method to quantify the diterpene jatrophone. MATERIALS AND METHODS: Anti-inflammatory and antinociceptive activities of the DFji were determined by an acute arthritis model through assessment of the paw elevation time (PET) and articular diameter (AD) of Wistar rats treated orally (50, 100 or 200 mg/kg in a single-dose), and intravenously (0.1, 1, 10, 25 or 50 mg/kg in a bolus administration). The isolation of jatrophone from the DFji was carried out and confirmed by spectroscopic techniques. A UFLC-DAD method was developed and validated. RESULTS: When orally administered, the highest dose (200 mg/kg) of DFJi was able to significantly reduce the PET to 24.8 ± 1.4 s (p < 0.01), when compared with the control group (33.7 ± 1.8 s). The administration of the intravenous dose of 10 mg/kg reduced the PET to 14.8 ± 0.3 s (p < 0.001). The oral and intravenous administration of the DFJi at dose of 200 and 10 mg/kg significantly prevented the formation of edema, reducing the AD in 25.3% and 32.5% (p < 0.01), respectively. The UFLC-DAD method allowed the quantification of jatrophone, which was found to be around 90 µg/mg of fraction. DISCUSSION AND CONCLUSION: The DFJi displayed antinociceptive and antiedematogenic activities, representing a promising plant product for the arthritis treatment.

Sesquiterpene lactone! a promising antioxidant, anticancer and moderate antinociceptive agent from Artemisia macrocephala jacquem.

BACKGROUND: Sesquiterpene lactones (STLs) make a diverse and huge group of bio-active constituents that have been isolated from several plant families. However, the greatest numbers are present in Asteraceae family having more than 3000 different reported structures. Recently several researchers have reported that STLs have significant antioxidant and anticancer potentials. METHODS: To investigate the antioxidant, anticancer and antinociceptive potentials of STLs, gravity column chromatography technique was used for isolation from the biologically rich chloroform fraction of Artemisia macrocephala Jacquem. The antioxidant activity of the isolated STLs was determined by DPPH and ABTS free radical scavenging activity, anticancer activity was determined on 3 T3, HeLa and MCF-7 cells by MTT assay while the antinociceptive activity was determined through acetic acid induced writhings, tail immersion method and formalin induced nociception method. RESULTS: The results showed that the STLs of Artemisia macrocephala possesses promising antioxidant activity and also it decreased the viability of 3 T3, HeLa and MCF-7 cells and mild to moderate antinociceptive activity. CONCLUSION: Sesquiterpenes lactones (STLs) are widely present in numerous genera of the family Asteraceae (compositae). They are described as the active constituents used in traditional medicine for the treatment of various diseases. The present study reveals the significant potentials of STL and may be used as an alternative for the management of cancer. Anyhow, the isolated compound is having no prominent antinociceptive potentials.

Essential oil composition and antinociceptive activity of Thymus capitatus.

CONTEXT: The essential oil (EO) from Thymus capitatus Hoff. et Link. (Lamiaceae) has been traditionally used for its medicinal properties, such as anti-inflammatory, analgesic, antioxidant and antimicrobial properties. OBJECTIVE: Characterize the constituents from T. capitatus EO and further evaluate the antinociceptive activity by in vivo and in vitro procedures. MATERIALS AND METHODS: Gas chromatography-mass spectrometry was used to identify and quantify the constituents of the T. capitatus EO. The antinociceptive activity was evaluated in vivo by the glutamate-induced nociception model in male Swiss mice (25 g), at doses of 3, 6 and 12 mg/kg, 1 h before evaluation of the licking time response (0-15 min). The mechanism of T. capitatus EO (1-500 µg/mL) on the isolated nerve excitability of Wistar rat (300 g) was assessed by the single sucrose technique. RESULTS AND DISCUSSION: The EO of T. capitatus presented 33 components, mainly monoterpenes and sesquiterpenes, carvacrol (ca. 80%) was its major constituent. T. capitatus EO induced antinociception in orally treated mice (3, 6, and 12 mg/kg) reducing the licking time from control (100.3 ± 11.9 s) to 84.8 ± 12.2, 62.7.6 ± 9.9, and 41.5 ± 12.7 s, respectively (n = 8; p < 0.05). Additionally, we have demonstrated that T. capitatus EO (500 µg/mL) decreased the compound action potential amplitude (VCAP) of about 80.0 ± 4.3% from control recordings (n = 4; p < 0.05). Such activity was presumably mediated through a voltage-gated Na+ channels. CONCLUSIONS: The present study demonstrated the antinociceptive activity of Thymus capitatus essential oil, which acts via peripheral nervous excitability blockade.

Antinociceptive effect of methanol extract of Dalbergia sissoo leaves in mice.

BACKGROUND: Dalbergia sissoo DC. (Family: Fabaceae) is a medium to large deciduous tree, is locally called "shishu" in Bangladesh. It is used to treat sore throats, dysentery, syphilis, bronchitis, inflammations, infections, hernia, skin diseases, and gonorrhea. This study evaluated the antinociceptive effect of the methanol extract of D. sissoo leaves (MEDS) in mice. METHODS: The extract was assessed for antinociceptive activity using chemical and heat induced pain models such as hot plate, tail immersion, acetic acid-induced writhing, formalin, glutamate, and cinnamaldehyde test models in mice at the doses of 100, 200, and 400 mg/kg (p.o.) respectively. Morphine sulphate (5 mg/kg, i.p.) and diclofenac sodium (10 mg/kg, i.p.) were used as reference analgesic drugs. To confirm the possible involvement of opioid receptor in the central antinociceptive effect of MEDS, naloxone was used to antagonize the effect. RESULTS: MEDS demonstrated potent and dose-dependent antinociceptive activity in all the chemical and heat induced mice models (p < 0.001). The findings of this study indicate that the involvement of both peripheral and central antinociceptive mechanisms. The use of naloxone verified the association of opioid receptors in the central antinociceptive effect. CONCLUSIONS: This study indicated the peripheral and central antinociceptive activity of the leaves of D. sissoo. These results support the traditional use of this plant in different painful conditions.