Synthetic polyploidization induces enhanced phytochemical profile and biological activities in Thymus vulgaris L. essential oil.

Essential oil from Thymus vulgaris L. has valuable therapeutic potential that is highly desired in pharmaceutical, food, and cosmetic industries. Considering these advantages and the rising market demand, induced polyploids were obtained using oryzalin to enhance essential oil yield. However, their therapeutic values were unexplored. So, this study aims to assess the phytochemical content, and antimicrobial, antioxidant, and anti-inflammatory activities of tetraploid and diploid thyme essential oils. Induced tetraploids had 41.11% higher essential oil yield with enhanced thymol and γ-terpinene content than diploid. Tetraploids exhibited higher antibacterial activity against all tested microorganisms. Similarly, in DPPH radical scavenging assay tetraploid essential oil was more potent with half-maximal inhibitory doses (IC50) of 180.03 µg/mL (40.05 µg TE/mg) than diploid with IC50 > 512 µg/mL (12.68 µg TE/mg). Tetraploids exhibited more effective inhibition of in vitro catalytic activity of pro-inflammatory enzyme cyclooxygenase-2 (COX-2) than diploids at 50 µg/mL concentration. Furthermore, molecular docking revealed higher binding affinity of thymol and γ-terpinene towards tested protein receptors, which explained enhanced bioactivity of tetraploid essential oil. In conclusion, these results suggest that synthetic polyploidization using oryzalin could effectively enhance the quality and quantity of secondary metabolites and can develop more efficient essential oil-based commercial products using this induced genotype.

Antioxidant and Anti-Inflammatory Activity of Five Medicinal Mushrooms of the Genus Pleurotus.

Within the group of higher fungi, edible medicinal mushrooms have a long history of being used as food and in folk medicine. These species contain biologically active substances with many potential beneficial effects on human health. The Pleurotus genus is representative of medicinal mushrooms because Pleurotus ostreatus is one of the most commonly cultivated culinary mushrooms. In our study, we focused on lesser-known species in the genus Pleurotus and measured their antioxidant and anti-inflammatory activity. We prepared extracts of the mushrooms and analyzed them using HPLC-HRMS, GC-MS, and 1H-NMR. Significant differences in biological activities were found among the Pleurotus spp. extracts. A MeOH extract of P. flabellatus was the most active as a radical scavenger with the highest ORAC, while a chloroform extract had significant anti-inflammatory COX-2 activity. The 80% MeOH extract of P. flabellatus contained the highest amounts of ergosterol, ergothioneine, and mannitol. The 80% MeOH extract of P. ostreatus Florida was the most active in the NF-κB inhibition assay and had the highest content of ß-glucans (43.3% by dry weight). Given the antioxidant and anti-inflammatory properties of P. flabellatus, the potential therapeutic usefulness of this species is worth evaluating through in-depth investigations and confirmation by clinical trials.

Prenylated Stilbenoids Affect Inflammation by Inhibiting the NF-κB/AP-1 Signaling Pathway and Cyclooxygenases and Lipoxygenase.

Stilbenoids are important components of foods (e.g., peanuts, grapes, various edible berries), beverages (wine, white tea), and medicinal plants. Many publications have described the anti-inflammatory potential of stilbenoids, including the widely known trans-resveratrol and its analogues. However, comparatively little information is available regarding the activity of their prenylated derivatives. One new prenylated stilbenoid (2) was isolated from Artocarpus altilis and characterized structurally based on 1D and 2D NMR analysis and HRMS. Three other prenylated stilbenoids were prepared synthetically (9-11). Their antiphlogistic potential was determined by testing them together with known natural prenylated stilbenoids from Macaranga siamensis and Artocarpus heterophyllus in both cell-free and cell assays. The inhibition of 5-lipoxygenase (5-LOX) was also shown by simulated molecular docking for the most active stilbenoids in order to elucidate the mode of interaction between these compounds and the enzyme. Their effects on the pro-inflammatory nuclear factor-κB (NF-κB) and the activator protein 1 (AP-1) signaling pathway were also analyzed. The THP1-XBlue-MD2-CD14 cell line was used as a model for determining their anti-inflammatory potential, and lipopolysaccharide (LPS) stimulation of Toll-like receptor 4 induced a signaling cascade leading to the activation of NF-κB/AP-1. The ability of prenylated stilbenoids to attenuate the production of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) was further evaluated using LPS-stimulated THP-1 macrophages.

Parallel in vitro and in silico investigations into anti-inflammatory effects of non-prenylated stilbenoids.

Stilbenoids represent a large group of bioactive compounds, which occur in food and medicinal plants. Twenty-five stilbenoids were screened in vitro for their ability to inhibit COX-1, COX-2 and 5-LOX. Piceatannol and pinostilbene showed activity comparable to the zileuton and ibuprofen, respectively. The anti-inflammatory potential of stilbenoids was further evaluated using THP-1 human monocytic leukemia cell line. Tests of the cytotoxicity on the THP-1 and HCT116 cell lines showed very low toxic effects. The tested stilbenoids were evaluated for their ability to attenuate the LPS-stimulated activation of NF-κB/AP-1. Most of the tested substances reduced the activity of NF-κB/AP-1 and later attenuated the expression of TNF-α. The effects of selected stilbenoids were further investigated on inflammatory signaling pathways. Non-prenylated stilbenoids regulated attenuation of NF-ĸB/AP-1 activity upstream by inhibiting the phosphorylation of MAPKs. A docking study used to in silico analyze the tested compounds confirmed their interaction with NF-ĸB, COX-2 and 5-LOX.

In Vitro Digestibility of Aluminum from Hibiscus sabdariffa Hot Watery Infusion and Its Concentration in Urine of Healthy Individuals.

Increased ingestion of aluminum (Al) can lead to its accumulation in the human body, especially in people with kidney problems. Al is also associated with several nervous diseases and its negative influence on embryo development during pregnancy has been proven in animal models. Hibiscus sabdariffa L. petals are widely used alone or in fruit tea formulas, which are recommended for drinking during pregnancy instead of tea. Its petals can contain similar and even higher amounts of Al as tea, which is a known Al accumulator. Our research investigated whether the regular intake of H. sabdariffa infusion leads to increased burden of Al. Sixteen days of ingestion of H. sabdariffa infusion (c Al = 0.5 mg.L-1) led to increased but unbalanced levels (15-86 µg L-1) of Al in urine compared to a period when the infusion was not ingested. The highest amounts of Al excreted were observed every third day during the ingestion. Mild health problems, such as nausea and dizziness (which could be related to plant properties) were reported by more sensitive volunteers.Our results suggest that the tea infusion from H. sabdariffa petals increases body burden of Al and, therefore, sensitive individuals as pregnant women and people with kidney problems should be cautious with excessive consumption of hibiscus infusion or fruit teas containing this plant. However, further study including more individuals is needed to fully confirm our preliminary results.

Prospective performance evaluation of selected common virtual screening tools. Case study: Cyclooxygenase (COX) 1 and 2.

Computational methods can be applied in drug development for the identification of novel lead candidates, but also for the prediction of pharmacokinetic properties and potential adverse effects, thereby aiding to prioritize and identify the most promising compounds. In principle, several techniques are available for this purpose, however, which one is the most suitable for a specific research objective still requires further investigation. Within this study, the performance of several programs, representing common virtual screening methods, was compared in a prospective manner. First, we selected top-ranked virtual screening hits from the three methods pharmacophore modeling, shape-based modeling, and docking. For comparison, these hits were then additionally predicted by external pharmacophore- and 2D similarity-based bioactivity profiling tools. Subsequently, the biological activities of the selected hits were assessed in vitro, which allowed for evaluating and comparing the prospective performance of the applied tools. Although all methods performed well, considerable differences were observed concerning hit rates, true positive and true negative hits, and hitlist composition. Our results suggest that a rational selection of the applied method represents a powerful strategy to maximize the success of a research project, tightly linked to its aims. We employed cyclooxygenase as application example, however, the focus of this study lied on highlighting the differences in the virtual screening tool performances and not in the identification of novel COX-inhibitors.

Pharmacophore modeling for COX-1 and -2 inhibitors with LigandScout in comparison to Discovery Studio.

BACKGROUND: Pharmacophore modeling has become an integrated tool in drug discovery. However, no prospective study compares the performance of the available software. METHODS: The two widely used pharmacophore modeling and screening software programs Discovery Studio and LigandScout were used to generate, validate, and prospectively apply COX-1 and -2 models. Selected virtual hits were tested in cell-free enzymatic assays. The correct retrieval of active compounds was compared. RESULTS: In the enzymatic testing, 10.5% of the tested hits for COX-2 and 6.6% of the predicted compounds for COX-1 were active. To directly compare the two models, both based on the same PDB entry, were selected for virtual screening. The two programs yielded vastly different hit lists, but both predicted active compounds. CONCLUSION: To obtain a comprehensive selection of active compounds, more than one program should be used for modeling.

In vitro anti-proliferative and anti-inflammatory activity of leaf and fruit extracts from Vaccinium bracteatum Thunb.

The aim of this study was to evaluate in vitro anti-proliferative (tested on MCF-7, MDA-MB-231, and MCF-10A cell lines) and anti-inflammatory (evaluated as inhibition of prostaglandin E2 synthesis catalyzed by cyclooxygenase-2) effect of various extracts from Vaccinium bracteatum leaves and fruits. The highest anti-proliferative effect possessed leaf dichloromethane extract with IC50 values ranging from 93 to 198 µg/mL. In the case of cyclooxygenase-2 inhibition, n-hexane, dichloromethane, and ethanol fruit extracts showed the best activity with IC50 values = 2.0, 5.4, and 12.7 µg/mL, respectively. These results indicate that V. bracteatum leaves and fruits could be useful source of anti-cancer and anti-inflammatory compounds.

Inhibition of in vitro leukotriene B4 biosynthesis in human neutrophil granulocytes and docking studies of natural quinones.

Quinones are compounds frequently contained in medicinal plants used for the treatment of inflammatory diseases. Therefore, the impact of plant-derived quinones on the arachidonic acid metabolic pathway is worthy of investigation. In this study, twenty-three quinone compounds of plant origin were tested in vitro for their potential to inhibit leukotriene B4 (LTB4) biosynthesis in activated human neutrophil granulocytes with 5-lipoxygenase (5-LOX) activity. The benzoquinones primin (3) and thymohydroquinone (4) (IC50 = 4.0 and 4.1 microM, respectively) showed activity comparable with the reference inhibitor zileuton (1C50 = 4.1 microM). Moderate activity was observed for the benzoquinone thymoquinone (2) (1C50 = 18.2 microM) and the naphthoquinone shikonin (1) (IC50 = 24.3 microM). The anthraquinone emodin and the naphthoquinone plumbagin (5) displayed only weak activities (IC50 > 50 microM). The binding modes of the active compounds were further evaluated in silico by molecular docking to the human 5-LOX crystal structure. This process supports the biological data and suggested that, although the redox potential is responsible for the quinone's activity on multiple targets, in the case of 5-LOX the molecular structure plays a vital role in the inhibition. The obtained results suggest primin as a promising compound for the development of dual COX-2/5-LOX inhibitors.

Evaluation of anti-inflammatory activity of selected legumes from Pakistan: in vitro inhibition of cyclooxygenase-2.

Crude methanolic extracts of selected legumes namely, black gram (Vigna mungo L.), green gram (Vigna radiata (L.) R. Wilczek ), soybean (Glycine max (L.) Merr.) and lentil (Lens culinaris Medik.) were investigated for anti-inflammatory effects, using COX-2 producing PGE(2) inhibitory assay. Percentage inhibition observed was 73.93, 79.84, 92.17 and 74.47 for black gram, green gram, soybean and lentil respectively at 20µg/ml extract concentration. The 100µg/ml concentration showed increase in the percent inhibition except for soybean. This is the first report on COX-2 inhibitory potential of food legumes.

Platelet aggregation and anti-inflammatory effects of garden pea, Desi chickpea and Kabuli chickpea.

Inflammation is the natural body defense mechanism for the removal of injurious agents, necrosed cells and tissues from the body. This study was aimed to evaluate the anti-inflammatory and platelet aggregation effects of three medicinal plants of Pakistan. Methanolic extract of garden pea inhibited arachidonic acid (AA)-induced platelet aggregation (IC50 = 35 microg/mL) and platelet activating factor (PAF)-induced platelet aggregation (IC50 = 38 microg/mL) in a dose dependent fashion. Methanolic extract of Desi chickpea inhibited arachidonic acid (AA) induced platelet aggregation (IC50 value = AA = 46 microg/mL) in dose dependent fashion while was found not active against PAF-induced platelet aggregation. Methanolic extract of Kabuli chickpea was found not active against both arachidonic acid (AA)-induced platelet aggregation and PAF-induced platelet aggregation. The best potential to inhibit in vitro COX-2 activity showed garden pea (Pisum sativum: the synthesis of PGE2 reduced by 92% in comparison with untreated control wells) followed by Desi chickpea (Cicer arietinum var; 87% inhibition) and Kabuli chickpea extracts (Cicer arietinum var: 65% inhibition). All extracts were tested at concentration 20 microg/mL. in COX-2 assay. The results indicate that if the same were happening in vito, Garden pea, Desi chickpea and Kabuli chickpea could be useful as natural antithrombotic anti-inflammatory materials.

Redox and non-redox mechanism of in vitro cyclooxygenase inhibition by natural quinones.

In this study, ten anthra-, nine naphtho-, and five benzoquinone compounds of natural origin and five synthetic naphthoquinones were assessed, using an enzymatic in vitro assay, for their potential to inhibit cyclooxygenase-1 and -2 (COX-1 and COX-2), the key enzymes of the arachidonic acid cascade. IC50 values comparable with COX reference inhibitor indomethacin were recorded for several quinones (primin, alkannin, diospyrin, juglone, 7-methyljuglone, and shikonin). For some of the compounds, we suggest the redox potential of quinones as the mechanism responsible for in vitro COX inhibition because of the quantitative correlation with their pro-oxidant effect. Structure-relationship activity studies revealed that the substitutions at positions 2 and 5 play the key roles in the COX inhibitory and pro-oxidant actions of naphthoquinones. In contrast, the redox mechanism alone could not explain the activity of primin, embelin, alkannin, and diospyrin. For these four quinones, molecular modeling suggested similar binding modes as for conventional nonsteroidal anti-inflammatory drugs (NSAIDs).

Determination of oxygen radical absorbance capacity of black cumin (Nigella sativa) seed quinone compounds.

In this study, the antioxidant capacities of main quinone constituents of Nigella sativa seeds, namely dithymoquinone (1), thymohydroquinone (2) and thymoquinone (3), were compared using DPPH and ORAC methods. The best scavenging activity was produced by 2, which showed a remarkable activity of 2.60 Trolox equivalents (TE) in a concentration range between 1.6 and 6.4 microg/mL and IC50 value of 2.4 microg/mL in ORAC and DPPH assays, respectively. Contrastingly, 3 possessed only weak DPPH scavenging efficacy (IC50 = 170 microg/mL) but significant antioxidative action of 1.91 TE in ORAC assay. No effect has been observed for 1. Additionally, modified protocol for synthesis of 2 has been developed with aim to enhance its availability for further studies as well as for its future potential use. Based on the results of this study, we conclude that 2 could be considered as a compound with prospective antioxidative properties.

Evaluation of antimicrobial and anti-inflammatory activities of seed extracts from six Nigella species.

Seed extracts from six species of the genus Nigella (Family Ranunculaceae)-Nigella arvensis, Nigella damascena, Nigella hispanica, Nigella nigellastrum, Nigella orientalis, and Nigella sativa-obtained by successive extraction with n-hexane, chloroform, and methanol, were tested for their antimicrobial activity against 10 strains of pathogenic bacteria and yeast using the microdilution method as well as for anti-inflammatory properties by in vitro cyclooxygenase (COX)-1 and COX-2 assay. Chemical characterization of active extracts was carried out including free and fixed fatty acid analysis. Comparison of antimicrobial activity showed that N. arvensis chloroform extract was the most potent among all species tested, inhibiting Gram-positive bacterial and yeast strains with minimum inhibitory concentration (MIC) values ranging from 0.25 to 1 mg/mL. With the exception of selective inhibitory action of n-hexane extract of N. orientalis on growth of Bacteroides fragilis (MIC = 0.5 mg/mL), we observed no antimicrobial activity for other Nigella species. Anti-inflammatory screening revealed that N. sativa, N. orientalis, N. hispanica, N. arvensis n-hexane, and N. hispanica chloroform extracts had strong inhibitory activity (more than 80%) on COX-1 and N. orientalis, N. arvensis, and N. hispanica n-hexane extracts were most effective against COX-2, when the concentration of extracts was 100 microg/mL in both COX assays. In conclusion, N. arvensis, N. orientalis, and N. hispanica seeds, for the first time examined for antimicrobial and anti-inflammatory effects, revealed their significant activity in one or both assays.

In vitro inhibitory effects of thymol and quinones of Nigella sativa seeds on cyclooxygenase-1- and -2-catalyzed prostaglandin E2 biosyntheses.

Dithymoquinone, thymohydroquinone, thymol and thymoquinone, compounds derived from N. sativa seeds, were investigated for their in vitro anti-inflammatory activities using cyclooxygenase-1 (COX-1) and -2 (COX-2) assays. Our results show that all substances tested possess significant inhibitory activity against at least one COX form at concentrations comparable to the active one of indomethacin. Thymol was the most active against COX-1 with an IC (50) value of 0.2 microM while thymohydroquinone and thymoquinone exhibited the strongest inhibitory effect on COX-2 with IC (50) values of 0.1 and 0.3 microM, respectively. Moreover, dithymoquinone and thymoquinone showed a limited COX-2-specific inhibition. We conclude that dithymoquinone, thymohydroquinone, thymol and thymoquinone can participate in the general anti-inflammatory activity of N. sativa and suggest that these agents should be further studied for possible use as non-steroidal anti-inflammatory drugs.