Anti-inflammatory activity of methanol extract and n-hexane fraction mojabanchromanol b from Myagropsis myagroides

Aims

This study was carried out to verify the anti-inflammatory effect of methanol extract from Myagropsis myagroides (MMME) and its n-hexane fraction mojabanchromanol b.

Main methods

The murine macrophages Raw264.7 cells were used. The pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) and the expression of iNOS, COX-2, and NF-κB p65 were examined by ELISA and immunoblotting. To investigate the inhibitory effect of MMME in an animal model of inflammation, an assay to determine croton oil-induced ear edema in mice was performed.

Key findings

NO levels decreased with increasing concentration of MMME, and were inhibited up to 50%. The secretion of IL-6, TNF-α, and IL-1β was suppressed in a dose-dependent manner, especially at 50 μg/mL, inhibition activities of cytokines were over 50%. MMME also suppressed the expression of COX-2, iNOS, and NF-κB p65, suggesting that MMME could affect the expression of inflammation related cytokines and proteins through the deregulation of NF-κB. Moreover, the formation of mouse ear edema was reduced at the highest dose tested compared to that in the control, and generated similar effects compared with prednisolone at 250 mg/kg in mice ear edema evaluation test. In addition, the results in photomicrograph of mice ear tissue and mast cells also showed the same effect. After purification of fractions of MMME, it indicated that n-hexane fraction mojabanchromanol b was the most active fraction showing the inhibitory effect of IL-6 and TNF-α.

Significance

These results suggested that MMME and mojabanchromanol b may have great effects on inflammatory factors and be potential anti-inflammatory therapeutic materials.     

Synthesis, characterization and biological evaluation of some 16E-arylidene androstane derivatives as potential anticancer agents

A series of new 16E-arylidene androstane derivatives were synthesized and characterized. The new compounds were screened for their anticancer activities against the human cancer cell lines SW480, A549, HepG2 and HeLa in vitro using the MTT assay. The results of the in vitro study showed that a number of compounds have shown IC₅₀ values lower than 20 μM against the four cancer cell lines.     

Eurycomanone suppresses expression of lung cancer cell tumor markers, prohibitin, annexin 1 and endoplasmic reticulum protein 28

Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 μg/ml. The concentration that inhibited 50% of cell growth (GI₅₀) was 5.1 μg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p < 0.0001, T-test). At 8 μg/ml (GI₇₀), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p < 0.05, T-test, n = 8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 μg/ml to 15 μg/ml with a GI₅₀ of 0.58 μg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p < 0.05, T-test, n = 9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 μg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.     

Cytotoxic and antioxidant activities of diterpenes and sterols from the Vietnamese soft coral Lobophytum compactum

Two new diterpenes, lobocompactols A (1) and B (2), and five known compounds (3-7) were isolated from the methanol extract of the soft coral Lobophytum compactum using combined chromatographic methods and identified based on NMR and MS data. Each compound was evaluated for cytotoxic activity against A549 (lung) and HL-60 (acute promyelocytic leukemia) human cancer cell lines. Among them, compound 5 exhibited strong cytotoxic activity against the A549 cell line with an IC₅₀ of 4.97 ± 0.06 μM. Compounds 3, 4, and 7 showed moderate activity with IC₅₀ values of 23.03 ± 0.76, 31.13 ± 0.08, and 36.45 ± 0.01 μM, respectively. The cytotoxicity of 5 on the A549 cells was comparable to that of the positive control, mitoxantrone (MX). All compounds exhibited moderate cytotoxicity against the HL-60 cell line, with IC₅₀ values ranging from 17.80 ± 1.43 to 59.06 ± 2.31 μM. Their antioxidant activity was also measured using oxygen radical absorbance capacity method, compounds 1 and 2 exhibiting moderate peroxyl radical scavenging activity of 1.4 and 1.3 μM Trolox equivalents, respectively, at a concentration of 5 μM.     

Tumor necrosis factor alpha gene variants do not display allelic imbalance in circulating myeloid cells

Carriage of the TNF −308 A allele (rs1800629 A) has been associated with increased serum TNF-α levels, the development of sepsis syndrome, and fatal outcome, in severely traumatized patients (Menges et al., 2008 [1]). Herein, we analysed the putative allelic imbalance of TNF-α release from myeloid cells. Circulating peripheral blood cells from healthy human blood donors (n = 104) and monocyte-derived macrophages (n = 158) were analysed for their ex vivo capacity of TNF-α expression. Our findings indicate that carriage of the TNF −308 A allele is not associated with high TNF-α expression in circulating human leucocytes and monocyte-derived macrophages. Other cellular sources, e.g. tissue-resident cells like mast cells and/or tissue specific macrophages might be the cellular source of high TNF-α serum levels shortly after trauma.     

Triterpene glycosides with in vitro anti-inflammatory activity from Cyclamen repandum tubers

A new triterpene glycoside, repandoside (1) was isolated together with six known saponins (2-7) from the methanol extract of Cyclamen repandum tubers. The isolated saponins were characterized by high resolution mass spectrometry and both 1D and 2D NMR experiments. The in vitro effect of saponins on LPS-induced IL-8 and TNF-α mRNA level (by quantitative RT-PCR) and protein release (by ELISA) was evaluated in human THP-1 macrophages. We found that compounds 1 (repandoside), 2 (deglucocyclamin) and 4 (anagalloside B) at 100 μM inhibited the LPS-induced IL-8 and TNF-α expressions.     

In vitro anti-influenza virus activity of a cardiotonic glycoside from Adenium obesum (Forssk.)

Methanolic extracts of six Saudi plants were screened for their in vitro antiviral activity using influenza virus A/PR/8/34 (H1N1) and MDCK cells in an MTT assay. The results indicated that the extracts of Adeniumobesum and Tephorosianubica possessed antiviral activity (99.3 and 93.3% inhibition at the concentration of 10 μg/ml, respectively). Based on these results A. obesum was selected for further study by applying bioactivity-guided fractionation to isolate its antiviral principle. The antiviral principle was isolated from the chloroform fraction through solvent fractionation, combined open liquid chromatography and HPLC. The isolated active compound A was identified as oleandrigenin-β-d-glucosyl (1 → 4)-β-d-digitalose, on the basis of its spectral analysis (MS, 1D and 2D NMR). The isolated glycoside showed reduction of virus titre by 69.3% inhibition at concentration of 1 μg/ml (IC₅₀ = 0.86 μg/ml).     

In silico and in vitro comparative activity of novel experimental derivatives against Leishmania major and Leishmania infantum promastigotes

This in silico and in vitro comparative study was designed to evaluate the effectiveness of some biurets (K1 to K8) and glucantime against Leishmania major and Leishmania infantum promastigotes. Overall, eight experimental ligands and glucantime were docked using AutoDock 4.3 program into the active sites of Leishmania major and Leishmania infantum pteridine reductase 1, which were modeled using homology modeling programs. The colorimetric MTT assay was used to find L. major and L. infantum promastigotes viability at different concentrations of biuret derivatives in a concentration and time-dependent manner and the obtained results were expressed as 50% and 90% of inhibitory concentration (IC₅₀ and IC₉₀). In silico method showed that out of eight experimental ligands, four compounds were more active on pteridine reductase 1. K3 was the most active against L. major promastigotes with an IC₅₀ of 6.8 μM and an IC₉₀ of 40.2 μM, whereas for L. infantum promastigotes was K8 with IC₅₀ of 7.8 μM. The phenylethyl derivative (K7) showed less toxicity (IC₅₀s > 60 μM) in both Leishmania strains. Glucantime displayed less growth inhibition in concentration of about 20 μM. In silico and especially docking results in a recent study were in accordance with the in vitro activity of these compounds in presented study and compound K3, K2 and K8 showed reasonable levels of selectivity for the Leishmania pteridine reductase 1.     

The in vitro and in vivo apoptotic effects of Mahonia oiwakensis on human lung cancer cells

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis (MOE), a selected species in Taiwan, and investigated their effects on various human lung cells. We found that MOE-induced apoptotic death in human A549 non-small-cell lung carcinoma (NSCLC) cells in a dose- and time-dependent manner. Treatment with the extracts also caused an increase in the sub-G1 fraction of cells, chromosome condensation, and DNA fragmentation. The mitochondrial-mediated pathway was implicated in this MOE-induced apoptosis as evidenced by the activation of the caspase cascade, cleavage of poly (ADP-ribose) polymerase (PARP), disruption of mitochondrial membrane potential, and release of cytochrome C. A higher ratio of Bax/Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced cell apoptosis. In A549 tumor-xenografted nude mice, MOE also retarded in vivo proliferation (P < 0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P < 0.05) and increased transferase-mediated dUTP nick-end labeling (TUNEL)-positive staining (P < 0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo, suggesting that it may have therapeutic potential against human lung cancer.     

A novel allelopathic substance, 13-epi-orthosiphol N,in Orthosiphon stamineus

Orthosiphon stamineus (Java tea) has been widely used as traditional herb and several bioactive compounds against animal cells have been isolated. However, no bioactive compound against plants has been reported. Therefore, we investigated possible allelopathic properties and substances in O. stamineus. Aqueous methanol extracts of O. stamineus inhibited root and hypocotyl growth of cress (Lepidium sativum) and lettuce (Lactuca sativa) seedlings. Increasing the extract concentration increased the inhibition, which suggests that O. stamineus may have allelopathic properties. When the extract was divided into an ethyl acetate and an aqueous fraction, the ethyl acetate fraction showed the stronger inhibitory effect. Thus, the ethyl acetate phase was further purified, and the main allelopathic substance was isolated and identified as 13-epi-orthosiphol N, a novel compound, by spectral data. 13-epi-Orthosiphol N inhibited root and hypocotyl growth of cress and lettuce at concentrations greater than 10 μmol/L. The concentrations required for 50% inhibition ranged from 41 to 102 μmol/L. These results suggest that 13-epi-orthosiphol N may be an allelochemical and main contributor to the growth inhibitory effect of O. stamineus and may have potential as a template for the development of new plant control substances.     

The novel 3,4-dihydropyrimidin-2(1H)-one urea derivatives of N-aryl urea: synthesis, anti-inflammatory, antibacterial and antifungal activity evaluation

A series of novel 3,4-dihydropyrimidin-2(1H)-one urea derivatives of biological interest were prepared by sequential Bigineli’s reaction, reduction followed by reaction of resulting amines with different arylisocynates. All the synthesized (1-23) compounds were screened against the pro-inflammatory cytokines (TNF-α and IL-6) and antimicrobial activity (antibacterial and antifungal). Biological activity evaluation study reveled that among all the compounds screened, compounds 12 and 17 found to have promising anti-inflammatory activity (68-62% TNF-α and 92-86% IL-6 inhibitory activity at 10 μM). Interestingly compounds 3, 4, 5, 6, 15, 22 and 23 revealed promising antimicrobial activity at MIC of 10-30 μg/mL against selected pathogenic bacteria and fungi.     

Cytotoxic activity of secondary metabolites derived from Artemisia annua L. towards cancer cells in comparison to its designated active constituent artemisinin

Artemisia annua L. (sweet wormwood, qinhao) has traditionally been used in Chinese medicine. The isolation of artemisinin from Artemisia annua and its worldwide accepted application in malaria therapy is one of the showcase success stories of phytomedicine during the past decades. Artemisinin-type compounds are also active towards other protozoal or viral diseases as well as cancer cells in vitro and in vivo. Nowadays, Artemisia annua tea is used as a self-reliant treatment in developing countries. The unsupervised use of Artemisia annua tea has been criticized to foster the development of artemisinin resistance in malaria and cancer due to insufficient artemisinin amounts in the plant as compared to standardized tablets with isolated artemisinin or semisynthetic artemisinin derivatives. However, artemisinin is not the only bioactive compound in Artemisia annua. In the present investigation, we analyzed different Artemisia annua extracts. Dichloromethane extracts were more cytotoxic (range of IC₅₀: 1.8-14.4 μg/ml) than methanol extracts towards Trypanosoma b. brucei (TC221 cells). The range of IC₅₀ values for HeLa cancer cells was 54.1-275.5 μg/ml for dichloromethane extracts and 276.3-1540.8 μg/ml for methanol extracts. Cancer and trypanosomal cells did not reveal cross-resistance among other compounds of Artemisia annua, namely the artemisinin-related artemisitene and arteanuine B as well as the unrelated compounds, scopoletin and 1,8-cineole. This indicates that cells resistant to one compound retained sensitivity to another one. These results were also supported by microarray-based mRNA expression profiling showing that molecular determinants of sensitivity and resistance were different between artemisinin and the other phytochemicals investigated.     

Flow cytometric screening for anti-leishmanials in a human macrophage cell line

High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC₅₀’s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445 ± 0.0005 μg/ml, 0.1203 ± 0.018 mg/ml, and 26.71 μM using THP-1 cells, and 0.179 ± 0.035 μg/ml, 0.1948 ± 0.0364 mg/ml, and 13.77 ± 10.74 μM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.     

In vitro cytotoxicity of melleolide antibiotics: structural and mechanistic aspects

Melleolide sesquiterpene aryl esters are secondary products of the mushroom genus Armillaria. We compared the cytotoxicity of eleven melleolides—five thereof are new natural products—against four human cancer cell lines. Armillaridin, 4-O-methylarmillaridin, and dehydroarmillylorsellinate were most active, at IC₅₀ = 3.0, 4.1 and 5.0 μM, respectively, against Jurkat T cells for the former two compounds, and K-562 cells for the latter. Dehydroarmillylorsellinate did not inhibit respiration and RNA-synthesis of K-562 cells at 5 μM. However, replication of DNA dropped to 35% after 120 min at this concentration, and translational activity also decreased.     

Ganoderol B: A potent α-glucosidase inhibitor isolated from the fruiting body of Ganoderma lucidum

α-Glucosidase inhibitor has considerable potential as a diabetes mellitus type 2 drug because it prevents the digestion of carbohydrates. The search for the constituents reducing α-glucosidase activity led to the finding of active compounds in the fruiting body of Ganoderma lucidum. The CHCl₃ extract of the fruiting body of G. lucidum was found to show inhibitory activity on α-glucosidase in vitro. The neutral fraction, with an IC₅₀ of 88.7 μg/ml, had stronger inhibition than a positive control, acarbose, with an IC₅₀ of 336.7 μg/ml (521.5 μM). The neutral fraction was subjected to silica gel column chromatography and repeated p-HPLC to provide an active compound, (3β,24E)-lanosta-7,9(11),24-trien-3,26-diol (ganoderol B). It was found to have high α-glucosidase inhibition, with an IC₅₀ of 48.5 μg/ml (119.8 μM).     

Bio-guided isolation of potential antimicrobial and antioxidant agents from the stem bark of Trilepisium madagascariense

Aim

This study describes the activity-guided isolation of antimicrobial and antioxidant agents from Trilepisium madagascariense stem bark.

Methods

The methanol crude extract of T. madagascariense was partitioned sequentially into n-hexane, ethyl acetate, n-butanol and the residual aqueous fractions. The ethyl acetate fraction was subjected to column chromatography and the structures of isolated compounds were elucidated using GC-MS and/or NMR data by comparing with those reported in the literature. Antimicrobial activity was assayed by agar well diffusion and broth microdilution techniques on 8 bacteria and 10 yeasts. The antioxidant activity was determined by DPPH radical scavenging method.

Results

The bioassay-guided fractionation of the crude methanol extract of T. madagascariense afforded two known compounds [vanillic acid () and isoliquiritigenin ()] and two mixtures of fatty acids (n-hexane fraction and first column fraction of ethyl acetate fraction, F1). The fractionation of the crude methanol extract enhanced the antimicrobial activity. Compound 2 was generally more active than compound 1. For all the tested samples, the most sensitive microbes were Enterococcus faecalis ATCC 10541 (MIC range of 60-780 μg/ml) for bacteria and Candida guillermondi (MIC range of 0.01-190 μg/ml) for yeasts. The DPPH radical scavenging activity (RSa) of compound 2 (RSa₅₀ = 28.73 μg/ml) was comparable to that of the crude methanol extract (RSa₅₀ = 29.92 μg/ml).

Conclusion

The antimicrobial activities and the antioxidant properties of the methanol crude extract, fractions and compounds 1 and 2 from the stem bark of T. madagascariense are being reported for the first time. These results may justify the traditional use of this plant for the treatment of gastrointestinal disorders.     

Antimalarial compounds from Schefflera umbellifera

The organic extract of the leaves of Schefflera umbellifera exhibited good antimalarial activity when tested against the chloroquine-susceptible strain (D10). Bioassay-guided fractionation of the dichloromethane fraction of the dichloromethane/methanol extract yielded an active compound, betulin, which exhibited good antiplasmodial activity with an IC₅₀ value of 3.2 µg/ml. The reference compound, chloroquine gave an IC₅₀ value of 27.2 ng/ml. Two other compounds were also isolated from the dichloromethane extract namely, 7-hydroxy-6-methoxycoumarin and ent-kaur-16-en-19-oic acid. These two compounds did not exhibit any significant antiplasmodial activity.     

Evaluation of the MTT lymphocyte proliferation assay for the diagnosis of neurocysticercosis

Neurocysticercosis (NCC) is caused by the larval form of the pork tapeworm Taenia solium when lodged in the central nervous system (CNS). Clinical diagnosis of NCC is complicated due to its polymorphic manifestations with no specific signs or symptoms. A wide range of serological assays and neuroimaging modalities are used for its diagnosis. The aim of the present study was to evaluate the MTT assay for the diagnosis of NCC and to determine its sensitivity, specificity and accuracy. MTT assay was based upon the cellular reduction of the tetrazolium salt by the proliferating cells and quantification of the colored product. Total 59 patients with NCC-related active epilepsy (AE), 30 with AE other than NCC (disease controls) and 64 healthy volunteers were enrolled for the study. Lymphocytes were freshly isolated from the enrolled subjects and cultured on cyst fluid antigen coated tissue culture plates. MTT assay was performed according to the standard protocol. The mean values of proliferation index (PI) with cyst fluid antigens were 2.13 ± 0.72, 0.622 ± 0.31 and 0.71 ± 0.36 for NCC patients, disease controls and healthy volunteers respectively. PI values for NCC patients were higher than the cut-off value (mean of controls + 2 standard deviations; 1.31). The sensitivity, specificity and accuracy of the MTT assay for the diagnosis of NCC were 87.93%, 94.68% and 91.5% respectively. For single cyst infection the sensitivity of the assay was found to be 86.4%. The present study shows that MTT is an adaptable technique which can be used for diagnosis of NCC.     

Cytokine effects on cell survival and death of A549 lung carcinoma cells

PurposeIL-13, TNF-α and IL-1β have various effects on lung cancer growth and death, but the signaling pathways mediating these effects have not been extensively analyzed. Therefore, the effects of IL-13, TNF-α and IL-1β alone, and in combination with Fas, on cell viability and death as well as major signaling pathways involved in these effects were investigated in A549 lung carcinoma cells.ResultsUsing MTT and flow cytometry, IL-13, TNF-α and IL-1β pretreatment decreased Fas-induced cell death. These anti-cell death effects were attenuated by pretreatment with inhibitors of Nuclear factor-κB [NF-κB], Phoshatidylinositole-3 kinase [PI3-K], JNK, p38 and ERK1/2 pathways.Using Western blot, IL-13, TNF-α and IL-1β treated cells showed time-dependent expression of p-ERK1/2, p-p38, p-JNK, p-Akt and p-IκBα proteins, decreased IκBα protein expression, no cleavage of Caspase-3 and PARP1 proteins and no notable alterations of Fas protein. IL-13 and TNF-α treated cells showed time-dependent increase of FLIP_L expression.ConclusionIL-13, TNF-α and IL-1β attenuate the pro-cell death effects of Fas on A549 cells, at least partially, by pathways involving the NF-κB, PI3-K and MAP kinases, but not by alterations of Fas protein expression. The IL-13 and TNF-α cell survival effects may also be due to increased expression of FLIP_L protein.