Antinociceptive and anti-inflammatory potential, and chemical characterization of the dichloromethane fraction of Solanum lycocarpum (Solanaceae) ripe fruits by LC-DAD-MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Solanum lycocarpum A. St. Hil. (Solanaceae) is a species from the Brazilian Cerrado, exhibiting several medicinal properties, being used by the population in the treatment of ulcers, bronchitis, asthma and hepatitis, which involve inflammatory processes. AIM OF THIS STUDY: This study aimed to chemically characterize the dichloromethane fraction (DCM), as well as verify its antinociceptive, anti-inflammatory and antioxidant potential. MATERIALS AND METHODS: The DCM fraction was obtained by partitioning the ethanol extract. The chemical constituents of the DCM fraction were characterized by LC-DAD-MS. The DPPH and FRAP assays were used to evaluate the antioxidant potential. The carrageenan-induced paw edema model was used to assess the anti-inflammatory effects, and the inflammatory infiltrate was evaluated by qualitative and quantitative histological analyses. The antinociceptive action of the DCM fraction was evaluated by acetic acid-induced abdominal writhing test, formalin-induced nociception and hot-plate test. RESULTS: Steroidal alkaloids solasonine, solasodine and solamargine, as well as the alkaloid peiminine/imperialine and caffeoylquinic acids, were annotated in DCM fraction by LC-DAD-MS. The DCM fraction showed antioxidative action in the in vitro DPPH and FRAP tests, as well as an anti-inflammatory effect for the three evaluated doses of 30, 100 and 300 mg/kg in the fourth and sixth hours after the administration of carrageenan. The histological analyses evidenced considerably reduction in leukocyte migration and the number of polymorphonuclear leukocytes. The study also demonstrated antinociceptive activity for the DCM fraction, which reduced abdominal writhing at three concentrations evaluated, as well as a decrease in paw licking in the formalin-induced nociception test both in the neurogenic phase and the inflammatory phase, with greater effectiveness compared to the anti-inflammatory indomethacin. The DCM fraction also increased the latency time of the animals in the hot plate test 60 min after treatment, although it did not seem to involve the opioidergic system. CONCLUSION: This work evidenced that the dichloromethane fraction of S. lycocarpum fruit possesses antinociceptive and anti-inflammatory potential, which supports its use in folk medicine for management inflammatory conditions.

Impact of Eucalyptus maculata Hook resin exudate constituents on reducing COX-2 gene expression: In-vivo anti-inflammatory, molecular docking and dynamics studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Eucalyptus maculata Hook from the Myrtaceae family is a native Australian plant that is frequently cultivated in Egypt. Many Eucalyptus species, including E. maculata, were widely used by the Dharawal, the indigenous Australian people, for their anti-inflammatory properties. AIM OF THE STUDY: The purpose of this study was to determine the anti-inflammatory activity of the ethanol extract of E. maculata resin exudate, its methylene chloride and n-butanol fractions, as well as the isolated compounds. MATERIALS AND METHODS: the ethanol extract was partitioned by methylene chloride, and n-butanol saturated with water. The fractions were chromatographed to isolate pure compounds. In-vivo anti-inflammatory activity of the ethanol extract, the fractions at a dose of 200 mg/kg, and the isolated compounds (20 mg/kg) was estimated using carrageenan-induced rat paws edema method against indomethacin (20 mg/kg). The activity was supported by histopathological and biochemical parameters. RESULTS: Three isolated compounds were identified as aromadendrin (C1), 7-O-methyl aromadendrin (C2), and naringenin (C3). Our findings demonstrated that the tested fractions significantly reduced the paw edema starting from the 3rd to the 5th hour as compared to the positive control, compounds C2 and C3 showed the greatest significant reduction in paw edema. The ethanol extract, fractions, C2, and C3 demonstrated an anti-inflammatory potential through reducing the levels of TNF-α, IL-6, and PGE2, as well as COX-2 protein expression compared to the negative control. These results were supported by molecular docking, which revealed that the isolated compounds had high affinity to target COX-1 and COX-2 active sites with docking scores ranging from -7.3 to -9.6 kcal mol-1 when compared to ibubrofen (-7.8 and -7.4 kcal mol-1, respectively). Molecular dynamics simulations were also performed and confirmed the docking results. CONCLUSION: The results supported the traditional anti-inflammatory potency of E. maculata Hook, and the biochemical mechanisms underlying this activity were highlighted, opening up new paths for the development of potent herbal anti-inflammatory medicine. Finally, our findings revealed that E. maculata resin constituents could be considered as promising anti-inflammatory drug candidates.

Glycolipid-enriched fraction of Osmanthus fragrans inhibits LPS-induced expression of inflammatory genes, COX-2, E-selectin, and Interleukin-8.

ETHNOPHARMACOLOGICAL RELEVANCE: Osmanthus fragrans Lour. is a small ornamental tree native to the Southeastern parts of China. It is mainly cultivated because of its characteristic fragrance, and used in the food and perfume industry. Besides, its flowers are used in traditional Chinese medicine to treat a variety of diseases including those related to inflammation. AIM OF THE STUDY: The aim of the study was to investigate in more detail the anti-inflammatory properties of O. fragrans flowers, and to characterize their active principles and mechanisms of action. MATERIALS AND METHODS: O. fragrans flowers were successively extracted with n-hexane, dichloromethane and methanol. The extracts were further fractionated by chromatographic separation. COX-2 mRNA expression in PMA-differentiated, LPS-stimulated THP-1 cells was used as lead assay for activity-guided fractionation. The most potent fraction was chemically analyzed by LC-HRMS. The pharmacological activity was also evaluated in other inflammation-related in-vitro models, such as analysis of IL-8 secretion and E-selectin expression in HUVECtert cells and selective inhibition of COX-isoenzymes. RESULTS: n-Hexane and dichloromethane extracts of O. fragrans flowers significantly inhibited COX-2 (PTGS2) mRNA expression. Additionally, both extracts inhibited COX-2 enzyme activity, whereas COX-1 enzyme activity was affected to a significantly lower extent. Fractionation of the extracts led to a highly active, glycolipid-containing fraction. In total, 10 glycolipids were tentatively annotated by LC-HRMS. This fraction also inhibited LPS-induced COX-2 mRNA expression, IL-8 secretion and E-selectin expression. The effects were limited to LPS-induced inflammation and not observed when inflammatory genes were induced by TNF-α, IL-1ß or FSL-1. Since all these inducers of inflammation act via different receptors, it is likely that the fraction interferes with the binding of LPS to the TLR4-receptor, which mediates pro-inflammatory effects of LPS. CONCLUSION: Taken together, the results demonstrate the anti-inflammatory potential of O. fragrans flower extracts in general, and of the glycolipid-enriched fraction in particular. The effects of glycolipid-enriched fraction are potentially mediated via the inhibition of the TLR4 receptor complex.

The dichloromethane extract of Callicarpa longissima rich in diterpenoid phenols exerts anti-inflammatory effect via inhibiting the TLR4/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Callicarpa longissima is a typical Yao ethnomedicine that has been used to treat arthritis in China. Our previous study found that the dichloromethane extract (DCME) of C. longissima showed anti-inflammatory activity in vitro. However, the anti-inflammatory mechanism and detailed chemical composition of DCME remain unclear, which lead to the original interest of this study. AIM OF THE STUDY: The study aimed to evaluate the anti-inflammatory properties of the DCME from C. longissima and further explore the accurate chemical components responsible for this active extract. MATERIALS AND METHODS: The anti-inflammatory activity of DCME in vivo was tested with carrageenan-induced mice paw edema model. Its anti-inflammatory mechanism was explored with LPS-stimulated RAW264.7 macrophages model. The compounds in DCME were isolated by repeated column chromatography and their structures were identified on the basis of nuclear magnetic resonance spectroscopy. The anti-inflammatory activities of the isolates in vitro were also tested by suppressing releases of inflammatory mediators (NO, IL-6 and TNF-α) in RAW264.7 macrophages model. In addition, the molecular docking analysis, which evaluated the potential interaction between the compounds and Toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB), was performed. RESULTS: DCME effectively alleviated the mice paw edema induced by carrageenan. In LPS-stimulated RAW264.7 cells, DCME significantly decreased the production of interleukin (IL)-6 and tumor necrosis factor α (TNF-α) via inhibiting their mRNA transcription, down-regulated the expression of TLR4 and myeloid differentiation factor 88, inhibited the phosphorylation of alpha inhibitor of NF-κB (IκBα), NF-κB p65, and degradation of IκBα. Twelve diterpenoid phenols were identified from DCME, and they not only showed different inhibitory effects on the production of NO, IL-6 and TNF-α in LPS-stimulated RAW264.7 cells, but also could bind to TLR4 and NF-κB as analyzed by molecular docking. CONCLUSIONS: Taken together, DCME from C. longissima could inhibit inflammatory response both in vitro and in vivo, which is mainly attributed to the synergistic effect of abundant diterpenoid phenols through inhibiting the TLR4/NF-κB signaling pathway, and might be a promising agent for the treatment of inflammatory diseases.

Anti-inflammatory potential of dichloromethane leaf extracts of Eucalyptus globulus (Labill) and Senna didymobotrya (Fresenius) in mice.

BACKGROUND: Inflammation is an immune response characterized by swelling, redness, pain and heat. Inflammation is mainly managed using conventional medicines that are associated with many side effects. Plant-based remedies are considerably better alternative therapies for they have fewer side effects. OBJECTIVE: This study aimed at determining the anti-inflammatory potential of dichloromethane (DCM) leaf extracts of Eucalyptus globulus and Senna didymobotrya in mice. METHODS: Fresh leaves of these plants were harvested from Embu County, Kenya. Quantitative phytochemical analysis was done using Gas Chromatography-Mass Spectrometry (GC-MS). Anti-inflammatory test comprised nine groups of five animals each: normal, negative, positive controls and 6 experimental groups. Inflammation was induced with Carrageenan. One hour post-treatment, the different groups were intraperitoneally administered with the reference drug, diclofenac, 3% DMSO and six DCM leaf extracts at doses of 25, 50, 100, 150, 200 and 250mg/kgbw. RESULTS: GC-MS results revealed α-phellandrene, camphene, terpinolene, and limonene among others. Anti-inflammatory effects showed that extract doses of 100,150,200 and 250mg/kg bw significantly reduced the inflamed paw. Doses of 200 and 250mg/kgbw in both plants were more potent and compared with diclofenac. E. globulus extract dose of 250mg kg bw reduced inflamed paw in the 1st, 2nd, 3rd and 4th hours, by 2.27,6.52,9.09 and 10.90% respectively while S.didymobotrya at similar dose ranges, inflamed paw reduced by 2.41, 5.43, 8.31 and 9.05% respectively. CONCLUSION: E. globulus and S. didymobotrya have potent anti-inflammatory activities, attributed to their constituent phytochemicals. This study confirms the traditional use of these plants in treating inflammation.

A mixtures approach to solvent exposures and amyotrophic lateral sclerosis: a population-based study in Denmark.

Studies of occupational solvent exposures and amyotrophic lateral sclerosis (ALS) have been conflicting. We conducted a population-based case-control study of mixed occupational solvent exposures and ALS. Using the Danish National Patient Registry, we identified ALS cases in Denmark from 1982 to 2013, and matched them to 100 controls based on sex and birth year. We estimated cumulative exposures to solvents (benzene, methylene chloride, toluene, trichloroethylene, perchloroethylene, and 1,1,1-trichloroethane) via job exposure matrices and applied them to occupational history from the Danish Pension Fund. Sex-stratified conditional logistic regression analyses revealed higher adjusted odds of ALS for men with exposure to benzene (aOR = 1.20; 95% CI 1.02, 1.41) and methylene chloride (aOR = 1.23; 95% CI 1.07, 1.42). We used weighted quantile sum regression to explore combined solvent exposures and risk of ALS in exposed subjects and found increased odds of 26 to 28% in all exposure lag periods for every one-unit increase in the mixture index in men. Weights of methylene chloride predominated the mixture index in all lag periods. Our study suggests an increased risk of ALS in men exposed to multiple solvents, with the greatest influence being from methylene chloride. These findings highlight the need to utilize mixtures analysis when considering co-occurring exposures.

Dietary quercetin abrogates hepatorenal oxidative damage associated with dichloromethane exposure in rats.

Exposure to dichloromethane (DCM), a commonly used chlorinated solvent in industrial settings and for the production of many household products, reportedly elicits detrimental effects in animals and humans. The present study investigated the protective role of dietary quercetin on DCM-induced hepatorenal damage in rats. Experimental rats were orally administered with DCM (150 mg/kg) and 30 min later with quercetin at 10, 20 and 40 mg/kg or none for 7 consecutive days. The results indicated that DCM-mediated significant (p<0.05) increases in serum alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase and alkaline phosphatase activities as well as urea and creatinine levels were dose-dependently normalized to the control values in rats co-treated with quercetin. Further, quercetin co-treatment ameliorated DCM-mediated decrease in the hepatic and renal activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase as well as glutathione level in the treated rats. Moreover, quercetin co-treatment markedly reduced lipid peroxidation level and protected against histological changes in liver and kidney of the treated rats. Taken together, quercetin abrogated hepatorenal oxidative damage in DCM-treated rats via improvement of antioxidant status and suppression of oxidative damage.

Methylene Chloride.

Methylene chloride is an industrial solvent used in commercial paint strippers and degreasing agents. This chemical is widely used in consumer products, yet without appropriate protections, exposure may lead to death. Already banned in some countries, the Environmental Protection Agency (EPA) recently proposed limiting its use in the United States.

Exposure to ambient dichloromethane in pregnancy and infancy from industrial sources and childhood cancers in California.

BACKGROUND: The incidence of childhood cancers has been increasing and environmental exposure to air toxics has been suggested as a possible risk factor. This study aims to explore ambient exposure to dichloromethane (methylene chloride). METHODS: We frequency matched by birth year approximately 20 cancer-free controls identified from birth records to all childhood cancers ages 0-5 in the California Cancer Registry diagnosed from 1988 to 2012; i.e. 13,636 cases and a total of 270,673 controls. Information on industrial releases of dichloromethane within 3km of birth addresses was retrieved from mandatory industry reports to the EPA's Toxics Release Inventory (TRI). We derived exposure to dichloromethane within close vicinity of birth residences using several modeling techniques including unconditional logistic regression models with multiple buffer distances, inverse distance weighting, and quadratic decay models. RESULTS: We observed elevated risks for germ cell tumors [Odds Ratio (OR): 1.52, 95% Confidence Interval (CI) 1.11, 2.08], particularly teratomas (OR: 2.08, 95% CI 1.38-3.13), and possible increased risk for acute myeloid leukemias (AML) (OR: 1.64, 95% CI 1.15-2.32 in the quadratic decay model). Risk estimates were similar in magnitude whether releases occurred in pregnancy or the child's first year of life. CONCLUSION: Our findings suggest that exposure to industrial dichloromethane releases may be a risk factor for childhood germ cell tumors, teratomas, and possibly AML.

Outcomes after resection of occupational cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma caused by exposure to 1,2-dichloropropane and/or dichloromethane is recognized as occupational cholangiocarcinoma. The aim of this study was to investigate the outcomes after resection of occupational cholangiocarcinoma to establish a treatment strategy for this disease. METHODS: Clinicopathological findings and outcomes after surgical intervention in 20 patients with occupational cholangiocarcinoma were investigated. RESULTS: Of 20 the patients, curative resection was performed in 16 patients. Three patients underwent radiation at the stump of the bile ducts. Adjuvant chemotherapy was performed in 12 patients. Biliary intraepithelial neoplasia, intraductal papillary neoplasm of the bile duct, and/or chronic bile duct injury was detected in most subjects. Intraabdominal infection developed after surgery in nine patients. Cholangiocarcinoma recurred in 12 of the 20 patients. The recurrent tumors in five patients developed at a different part of the bile duct from the primary tumor and a second resection was performed in four of these five patients. CONCLUSIONS: The incidence of postoperative complications including intraabdominal infection was high in patients with occupational cholangiocarcinoma. Multicentric recurrence occurred not infrequently after surgery because the bile ducts had a high potential for the development of carcinoma. The aggressive treatment including second resection for the multicentric recurrence appeared to be effective.

Primary Sjögren's syndrome and occupational risk factors: A case-control study.

OBJECTIVES: A case-control study was carried out to investigate the relation between primary Sjögren's syndrome (pSS) and occupational exposure. METHODS: One hundred seventy five cases of pSS followed up into the internal medicine departments of three French university hospitals from 2010 to 2013 were included. For each case, two age and gender matched controls were selected during the same period in the same departments. Occupational exposure was assessed retrospectively by industrial hygienists and occupational practitioners. Exposure to occupational factors such as organic solvents or silica was investigated using semiquantitative estimates of exposure. An exposure score was calculated for each subject based on probability, intensity, daily frequency, and duration of exposure for each period of employment. The final cumulative exposure score was obtained, taking into account all periods of employment. RESULTS: Significant associations with pSS were observed for dichloromethane (OR 9.28, 95%CI 2.60-33.03), perchlorethylene (OR 2.64, 95%CI 1.20-5.77) chlorinated solvents (OR 2.95, 95%CI 1.77-4.93), benzene (OR 3.30, 95%CI 1.07-10.26), toluene (OR 4.18 95%CI 1.41-12.43), white spirit (OR 3.60, 95%CI 1.39-9.33), aromatic solvents (OR 3.03, 95%CI 1.41-6.50) and any types of solvents (OR 2.76, 95%CI 1.70-4.47). Risk of pSS was significantly associated with a high cumulative exposure score of occupational exposure to toluene (OR 4.69, 95%CI 1.42-15.45), white spirit (OR 3.30, 95%CI 1.07-10.26), aromatic solvents (OR 2.50, 95%CI 1.06-5.91) and any types of solvents (OR 2.25, 95%CI 1.20-4.22). CONCLUSION: This work suggests the influence of occupational risk factors in the occurrence of pSS.

Exposure to chlorinated solvents and lung cancer: results of the ICARE study.

OBJECTIVE: To investigate the role of occupational exposure to chlorinated solvents in lung cancer aetiology. METHODS: ICARE (Investigation of occupational and environmental CAuses of REspiratory cancers) is a French, multicentre, population-based, case-control study. Information on the lifelong work history of 2926 cases and 3555 controls was collected using standardised questionnaires. Occupational exposures were assessed using job-exposure matrices for five chlorinated solvents. Solvents were studied separately and in combinations. ORs were computed using unconditional logistic regression models adjusted for classic risk factors, including a history of cigarette smoking and exposure to asbestos. Adjustment for socioeconomic status (SES) was also made. RESULTS: After adjustment for exposure to asbestos, we observed a positive, statistically significant association with lung cancer for men and women exposed to a combination of perchloroethylene (PCE), trichloroethylene and dichloromethane (DCM). Further adjustment for SES slightly decreased this association. In contrast, no statistically significant associations were found for other solvent combinations. CONCLUSIONS: These results suggest that exposure to PCE may constitute a risk factor for lung cancer, especially among women, who seem to have a higher prevalence of exposure than men.

Chemical exposure levels in printing workers with cholangiocarcinoma.

OBJECTIVE: This study aimed to identify chemicals used by printing workers with cholangiocarcinoma, as well as the levels of exposure to the chemicals. METHODS: Information necessary to identify chemicals used by printing workers with cholangiocarcinoma and to estimate chemical exposure concentrations was obtained from the Ministry of Health, Labour and Welfare, Japan. Working environment concentrations of the chemicals in the printing rooms were estimated using a well-mixed model, and exposure concentrations during the ink removal operation were estimated using a near-field and far-field model. Shift time- weighted averages (TWA) of exposure concentrations were also calculated. RESULTS: Two workers from each of three small printing plants examined suffered from cholangiocarcinoma, and all six of these workers had been exposed to 1,2-dichloropropane (1,2-DCP) for 10-16 years. The estimated working environment concentrations of 1,2-DCP in the printing rooms were 17-180 ppm and estimated exposure concentrations during the ink removal operation were 150-620 ppm. Shift TWA values were estimated to be 62-240 ppm. Four of the six workers had also been exposed to dichloromethane (DCM) at estimated working environment concentrations of 0-98 ppm and estimated exposure concentrations during the ink removal operation of 0-560 ppm. Shift TWA values were estimated to be 0-180 ppm. Other chlorinated organic solvents (1,1,1-trichloroethane, 1,1-dichloro-1-fluoroethane) and petroleum solvents (gasoline, naphtha, mineral spirit, mineral oil, kerosene) were also used in the ink removal operation. CONCLUSIONS: All six printing workers with cholangiocarcinoma were exposed to very high levels of 1,2-DCP for a long term.

[Cholangiocarcinoma developing in printing company workers: a new type of occupational cancer].

The incidence of cholangiocarcinoma among the past or present workers in the department of offset color proof-printing at a printing company in Osaka was extremely high. The workers were relatively young and were exposed to several chemicals including organic solvents such as dichloromethane and 1,2-dichloropropane. Although the exact cause of cholangiocarcinoma in the patients remain unknown, it is likely that the development of cholangiocarcinoma was triggered during exposure to these chemicals. Some chemicals can act as environmental factors that lead to the development of cholangiocarcinoma. Therefore, we believe that cholangiocarcinoma is a new type of occupational cancer.

Occupational exposure to methylene chloride and risk of cancer: a meta-analysis.

METHODS: We searched MEDLINE and EMBASE for epidemiologic studies on occupational exposure to methylene chloride and risk of cancer. Estimates of study-specific odds ratios (ORs) were calculated using inverse-variance-weighted fixed-effects models and random-effects models. Statistical tests for heterogeneity were applied. RESULTS: We summarized data from five cohort studies and 13 case-control studies. The pooled OR for multiple myeloma was (OR 2.04; 95 % CI 1.31-3.17) in relation to occupational exposure to methylene chloride but not for non-Hodgkin's lymphoma, leukemia, breast, bronchus, trachea and lung, brain and other CNS, biliary passages and liver, prostate, pancreas, and rectum. Furthermore, we focused on specific outcomes for non-Hodgkin's lymphoma and multiple myeloma because of exposure misclassification. The pooling OR for non-Hodgkin's lymphoma and multiple myeloma was 1.42 (95 % CI 1.10-1.83) with moderate degree of heterogeneity among the studies (I (2) = 26.9 %, p = 0.205). CONCLUSIONS: We found an excess risk of multiple myeloma. The non-Hodgkin's lymphoma and leukemia that have shown weak effects should be investigated further.

Vestibular and balance findings in nonsymptomatic workers exposed to styrene and dichloromethane.

OBJECTIVE: The aim of our study was to assess the vestibular and balance system in non-symptomatic workers exposed to styrene and dichloromethane at the workplace. DESIGN: Subjects underwent videonystagmography including saccades, smooth pursuit (SP), optokinetic test (OKN), gaze nystagmus assessment, bithermal caloric test, and static posturography. STUDY SAMPLE: Study groups included 74 workers in plastics manufacturing, aged 40 (SD 8) years, exposed to styrene and dichloromethane, and the reference group of 49 non-exposed subjects, aged 36 (SD 10) years. RESULTS: More than 60% of exposed and non-symptomatic workers revealed abnormal results of vestibular tests. Saccadic latency elongation (p = 0.0098), lower gain in SP (p = 0.0037) and OKN (p = 0.0000) were more common in the exposed group, as well as lower reactivity (p = 0.0337) and mean slow phase velocity of caloric nystagmus. Static posturography revealed higher sway velocities in the test with eyes closed, on foam and worse results of three from five limit of stability tests. No relationship between chemicals exposure and vestibular and balance test results was found. CONCLUSIONS: In principle, our findings indicate the possibility of high-level deficits in the central part of vestibular system. Lower vestibular reactivity may suggest that bilateral vestibular hypofunction might also be the possible consequence of solvent exposure.