Anti-inflammatory activity of Eupatorium perfoliatum L. extracts, eupafolin, and dimeric guaianolide via iNOS inhibitory activity and modulation of inflammation-related cytokines and chemokines

Ethnopharmacological relevance

Eupatorium perfoliatum L. has been used traditionally for the treatment of fever, malaria and inflammation-associated diseases. Nowadays it is mostly used as immune activating remedy. The following study was performed to evaluate extracts with different polarity and defined lead-compounds from the herbal material on potential in vitro activities concerning immune cell activation, phagocytosis, and inflammation-related processes.

Materials and methods

MeOH-, EtOH-, and DCM extracts, beside several subfractions and isolated polysaccharides, sesquiterpene lactones and flavonoids were prepared and characterized analytically from the aerial parts of E. perfoliatum. Immunological activity was tested within lymphocyte transformation test on PBMC, test on enhancement of phagocytosis and of NO-production by murine RAW 264.7 macrophages. Anti-inflammatory effects were assessed from LPS-stimulated RAW 264.7 cells by NO/iNOS quantification, gene array, real-time PCR and ELISA.

Results

No stimulatory activity was found within lymphocyte transformation test, for phagocytic activity and NO formation in macrophages. MeOH-, EtOH- and DCM extracts showed anti-inflammatory activity against LPS-stimulated macrophages by inhibition of NO release (IC₅₀ > 100, 89, 19 μg/mL resp.) with eupafolin and a dimeric guaianolide having prominent NO inhibiting activity (IC₅₀ 6 resp. 16 μM). Anti-inflammatory activity was found on gene and protein level by significant down-regulation of cytokines CSF-3, IL-1α, IL-1β, and chemokines CCL2, CCL22 and CXCL10. Also TNF was down-regulated moderately (−17%).

Conclusions

Although the postulated immunostimulating properties of E. perfoliatum have not been confirmed, the anti-inflammatory effects can be seen as a verification of the traditional use against inflammatory diseases.     

顶空气相色谱法测定注射用胸腺法新中5种有机溶剂残留量

目的 建立同时测定注射用胸腺法新中乙腈、二氯甲烷、醋酸乙酯、苯和苯甲醚5种有机溶剂残留量的顶空气相色谱法。方法 采用Agilent DB-624（30 m×0.53 mm×3 μm）毛细管色谱柱;火焰离子化检测器;进样口温度200℃;检测器温度250℃;载气为氮气;载气体积流量为2.0 mL·min^-1;分流比为10：1;升温程序：起始温度40℃,保持6 min,以8℃·min^-1的速率升温至90℃,保持2 min,再以20℃·min^-1的速率升温至200℃,保持5 min;采用顶空进样方式,顶空加热箱温度80℃,样品瓶平衡时间30 min。进行系统适用性、检测限（LOD）与定量限（LOQ）、线性关系和范围、加样回收率、精密度、稳定性、耐用性考察。结果 注射用胸腺法新中5种残留溶剂在各自线性浓度范围内与峰面积线性关系良好;平均加样回收率在95.7%~106.3%;精密度、稳定性、耐用性均符合要求。5批胸腺法新中均未检出5种有机溶剂。结论 建立的顶空气相色谱法操作简单、灵敏度和准确度高、重现性和耐用性好,可用于注射用胸腺法新中5种有机溶剂残留量的测定。     

Intrahepatic cholangiocarcinoma in a worker at an offset color proof‐printing company: An autopsy case report

A 40‐year‐old Japanese man visited our hospital after test results indicated elevated hepatobiliary enzymes. He had worked at a printing plant for 8 years and been exposed to organic solvents, including 1,2‐dichloropropane (1,2‐DCP) and dichloromethane (DCM). Abdominal computed tomography (CT) showed an intrahepatic tumor with dilation of the intrahepatic bile duct. He was diagnosed with intrahepatic cholangiocarcinoma. He had no known risk factors for cholangiocarcinoma. Extended left hepatectomy with lymph node dissection was performed and the tumor was histologically diagnosed as well‐differentiated adenocarcinoma. A histological examination also showed biliary intraepithelial preneoplastic lesions in non‐cancerous liver areas. Two years after surgery, the patient developed jaundice, esophageal varices and ascites. A CT examination showed liver cirrhosis without recurrence of the cholangiocarcinoma. Although a liver transplantation was planned as a therapeutic option for his liver cirrhosis, his liver failure progressed rapidly and he died before transplantation could be performed. At autopsy, fibrosis was found in the whole liver, especially in the wall of the bile duct and periductal area suggesting chronic bile duct injury due to exposure to organic solvents. Taken together, the current case may suggest that exposure to organic solvents, including 1,2‐DCP and DCM, is a risk factor for cholangiocarcinoma. Identifying risk factors for cholangiocarcinoma will help identify the mechanism and help prevent development of the disease.     

GC/GCMS analysis of the petroleum ether and dichloromethane extracts of M oringa oleifera roots

ObjectiveTo explore the phytochemical constituents from petroleum ether and dichloromethane extracts of Moringa oleifera (M. oleifera) roots using GC/GC-MS.MethodsA total of 5.11 kg fresh and undried crushed root of M. oleifera were cut into small pieces and extracted with petroleum ether and dichloromethane (20 L each) at room temperature for 2 d. The concentrated extracts were subjected to their GC-MS analysis.ResultsThe GC-MS analysis of the petroleum ether and dichloromethane extracts of M. oleifera roots, which showed promising biological activities, has resulted in the identification 102 compounds. These constituents belong to 15 classes of compounds including hydrocarbons, fatty acids, esters, alcohols, isothiocyanate, thiocyanate, pyrazine, aromatics, alkamides, cyanides, steroids, halocompounds, urea and N-hydroxyimine derivatives, unsaturated alkenamides, alkyne and indole. GC/GC-MS studies on petroleum ether extract of the roots revealed that it contained 39 compounds, belonging to nine classes. Cyclooctasulfur S8 has been isolated as a pure compound from the extract. The major compounds identified from petroleum ether extract were trans-13-docosene (37.9%), nonacosane (32.6%), cycloartenol (28.6%) nonadecanoic acid (13.9%) and cyclooctasulfur S8 (13.9%). Dichloromethane extract of the roots was composed of 63 compounds of which nasimizinol (58.8%) along with oleic acid (46.5%), N-benzyl-N-(7-cyanato heptanamide (38.3%), N-benzyl-N-(1-chlorononyl) amide (30.3%), bis [3-benzyl prop-2-ene]-1-one (19.5%) and N, N-dibenzyl-2-ene pent 1, 5-diamide (11.6%) were the main constituents.ConclusionsThis study helps to predict the formula and structure of active molecules which can be used as drugs. This result also enhances the traditional usage of M. oleifera which possesses a number of bioactive compounds.     

Survey of human milk for volatile N-nitrosamines and the influence of diet on their formation

Human milk was analysed for volatile N-nitrosamines and the influence of diet on their possible presence and formation was assessed. 175 samples were obtained from 16 different nursing women. Analysis of samples collected at random, to ascertain baseline levels, indicated that 76.5% of the human milk samples contained less than 0.2 ppb N-nitrosodimethylamine (minimum level of reliable measurement). No other volatile nitrosamines were detected. Expressed milk was collected from volunteers over a 6-hr period after eating meals which included bacon (a source of performed nitrosamines) and at times a vegetable high in nitrate. Eating a meal containing bacon did not result in increased nitrosamine levels in milk. In certain individuals, eating a meal of bacon and a vegetable high in nitrate occasionally resulted in higher levels of N-nitrosodimethylamine in their milk.     

Free radical scavenging activity from leaves of Acacia nilotica (L.) Wild. ex Delile,an Indian medicinal tree

The present study compares the two extraction methods and evaluates the free radical scavenging activity of Acacia nilotica. Results indicated that the sequential extraction method was effective in concentrating the active principles in the ethanol extract as compared to the maceration method in DPPH assay. Based on the results, free radical scavenging property of the extracts obtained from sequential extraction method was analyzed in different assays to find out the possible antioxidant mechanism. Our results indicate that ethanol extract rich in phenolic and flavonoid contents had potent antioxidant activity and were significant in comparison with all the positive controls used in this study. The possible antioxidant mechanism of the ethanol extract can be due to its hydrogen or electron donating and direct free radical scavenging properties. Hence, the ethanol extract represents a source of potential antioxidants that could be used in pharmaceutical and food preparations.     

Ethnopharmacological study of plants from Pondoland used against diarrhoea

Ethnopharmacological relevance

Waterborne diseases such as diarrhoea are common world wide, including in Bizana, South Africa where the majority of rural dwellers depend largely on water from unprotected sources. The people from Bizana use medicinal plants as their first line of health care to cure and prevent diarrhoea.

Aim of the study

To record and document plants used for the treatment of diarrhoea in Bizana, to evaluate antibacterial and anti-inflammatory activities of selected plant extracts as well as to perform genotoxicity testing of evaluated plants.

Materials and methods

An ethnobotanical approach was used to select plants used for treating diarrhoea in Bizana for pharmacological assays using questionnaires. Nine plants were selected for bioassays based on their frequency index and the fact that they have never been evaluated against diarrhoea causing-microorganisms. The petroleum ether (PE), dichloromethane (DCM), 70% ethanol (EtOH), and water extracts were evaluated for antibacterial (Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and Shigella flexneri) activity using the microdilution technique, their ability to inhibit COX-1 and COX-2 enzymes. Genotoxicity was evaluated using the Salmonella microsome assay.

Results

This study revealed that 34 plant species belonging to 27 families are used for the treatment of diarrhoea in Bizana. The extracts showed good inhibitory activity with MIC values ranging from 0.39 to 12.5 mg/ml. The best activity was exhibited by DCM extracts of Rapanea melanophloeos, and EtOH extracts of Ficus craterostoma and Maesa lanceolata with MIC values of 0.098 mg/ml. The inhibitory activity against COX-1 enzyme was higher than COX-2, with 19 plant extracts for the former and 7 for the latter. All the tested plant extracts were not mutagenic at all concentrations tested against all tester strains of bacteria.

Conclusion

In view of the fact that the plants were selected based on their ethnobotanical usage for treating diarrhoea, the activities reported here goes a long way in validating the plants for traditional use.     

In vitro antimicrobial synergism within plant extract combinations from three South African medicinal bulbs

Ethnopharmacological relevance

Tulbaghia violacea, Hypoxis hemerocallidea and Merwilla plumbea are used in South African traditional medicine for the treatment of some infectious diseases and other ailments.

Aim of the study

The study aimed at investigating the antimicrobial efficacies of independent and various within-plant extract combinations of three medicinal bulbs to understand the possible pharmacological interactions.

Materials and methods

Bulb and leaf extracts of the three medicinal plants, independently and in combinations, were comparatively assessed for antimicrobial activity against two Gram-positive and two Gram-negative bacteria and Candida albicans using the microdilution method. The fractional inhibitory concentration indices (FIC) for two extract combinations were determined.

Results

At least one extract combination in each plant sample demonstrated good antimicrobial activity against all the test organisms. The efficacies of the various extract combinations in each plant sample varied, with the strongest synergistic effect exhibited by the proportional extract yield combination of PE and DCM extracts in Merwilla plumbea bulb sample against Staphylococcus aureus (FIC index of 0.1). Most extract combinations demonstrated either a synergistic, additive or indifferent interaction effect against the test bacteria with only a few exhibiting antagonistic effects.

Conclusion

The observed antimicrobial efficacy and synergistic interactions indicate the beneficial aspects of combination chemotherapy of medicinal plant extracts in the treatment of infectious diseases.     

The antimicrobial,antioxidative, anti-inflammatory activity and cytotoxicity of different fractions of four South African Bauhinia species used traditionally to treat diarrhoea

Ethnopharmacological importance

Many Bauhinia species, including those indigenous to South Africa, are used in traditional medicine across the world for treating ailments such as gastrointestinal tract (GIT) disorders, diabetes, infectious diseases and inflammation.

Aims

Several relevant aspects of different fractions of leaf extracts of Bauhinia bowkeri (BAB), Bauhinia galpinii (BAG), Bauhinia petersiana (BAP), and Bauhinia variegata (BAV) used in South African traditional medicine to alleviate diarrhoea related symptoms were evaluated.

Materials and Methods

The antioxidative activities of the extracts were determined using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS⁺) radical scavenging and ferric reducing antioxidant power (FRAP) methods. In vitro antimicrobial activities of the extracts were determined against bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis) and clinical isolates of the opportunistic fungal strains (Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans) using a serial dilution microplate method. The polyphenolic contents were quantified using standard methods, and anti-inflammatory activities of the crude extracts were determined using the cyclooxygenase and soybean 15-lipoxygenase enzyme inhibitory assays. The safety of the extracts was evaluated by determining the cytotoxicity against Vero cell lines.

Results

The acidified 70% acetone crude extract and their fractions had good antiradical potency against the DPPH and ABTS radicals. The methanol soluble portions of the butanol fractions were more potent (EC₅₀ ranges from 0.64±0.05 to 1.51±0.07 and 0.88±0.18 to 1.49±0.09 μg/ml against DPPH and ABTS radical respectively) compared to the standard, trolox and ascorbic acid (EC₅₀ ranges from 1.47±0.24 to 1.70±0.27 μg/ml) for both DPPH and ABTS. The crude extracts contained variable quantities of phenolic content. The crude extracts and their fractions had weak to good antimicrobial activities, inhibiting the growth of the organisms at concentrations ranging from 39 to 2500 μg/ml. The BAG crude extract and its fractions were the most active against the fungi (MICs ranging from 39 to 625 μg/ml) while the BAB extract and its fractions were the least active with the MICs ranging between 39 and 2500 μg/ml. Aspergillus fumigatus was the least susceptible fungus while Cryptococcus neoformans was the most susceptible.The phenolic-rich crude extracts of BAB, BAG, and BAP had moderate to good dose-dependent cyclooxygenase-1 enzyme inhibitory activity with inhibitions between 22.8% and 71.4%. The extracts were however, inactive against cyclooxygenase-2. The extracts had some level of cytotoxicity towards Vero cell lines, reducing cell viability to less than 10% at concentrations more than 50 μg/ml.

Conclusion

The biological activities observed in Bauhinia species provide a scientific basis for the use of the plants in traditional medicines to treat diseases with multi-factorial pathogenesis such as diarrhoea, with each aspect of activity contributing to the ultimate therapeutic benefit of the plants. However, the use of the phenolic-rich extracts of these plants to treat diarrhoea or any other ailments in traditional medicine needs to be monitored closely because of potential toxic effects and selective inhibition of COX-1 with the associated GIT injury.     

Proanthocyanidin encapsulation for sustained bioactivity in dentin bioadhesion: A two-year study

ObjectivesTo determine the long-term effect on the stability of dentin-resin interfaces after the addition of polylactide (PLA) capsules containing proanthocyanidin (PAC) to adhesive resin.MethodsSub-micron (SM) and micron (M) size capsules containing PACs were produced using a combination of emulsification and solvent evaporation techniques and characterized. Human dentin surfaces (n = 8) were etched (35% glycolic acid) and primed (15% enriched Vitis vinifera extract solution - VV_e), followed by the application of an experimental adhesive containing 0 (control), 1.5 wt% of SM or M PAC-filled PLA capsules light cured for 40 s. A crown was built using commercial composite. After 24 h-immersion (37 °C) in simulated body fluid, specimens were serially sectioned into resin-dentin beams. Microtensile bond strength (TBS), micro-permeability and fracture pattern were assessed immediately and after 1 and 2 years. Data were statistically analyzed using two-way ANOVA and post-hoc test (α = 0.05).ResultsPolydisperse capsules were manufactured with average diameter of 0.36 µm and 1.08 µm for SM and M, respectively. The addition of capsules did not affect TBS (p = 0.889). After 2 years, TBS significantly decreased in SM (p = 0.006), whereas M showed similar initial values (p = 0.291). Overall, less micro-permeability was found in M than the control and SM group (p < 0.001). After 2 years, fractured surfaces from capsule-containing groups failed within the adhesive layer while control fractured at the bottom of the hybrid layer.SignificanceThe addition of PAC-filled PLA microcapsules in a dental adhesive did not affect the bond strength while increased and sustained the protection against micro-permeability in the interface, likely due to release of PACs.