Antioxidant Activity and α-Glucosidase Inhibitory Activities of the Polycondensate of Catechin with Glyoxylic Acid

In order to investigate polymeric flavonoids, the polycondensate of catechin with glyoxylic acid (PCG) was prepared and its chemically antioxidant, cellular antioxidant (CAA) and α-glucosidase inhibitory activities were evaluated. The DPPH and ABTS radical scavenging activities and antiproliferative effect of PCG were lower than those of catechin, while PCG had higher CAA activity than catechin. In addition, PCG had very high α-glucosidase inhibitory activities (IC₅₀ value, 2.59 μg/mL) in comparison to catechin (IC₅₀ value, 239.27 μg/mL). Inhibition kinetics suggested that both PCG and catechin demonstrated a mixture of noncompetitive and anticompetitive inhibition. The enhanced CAA and α-glucosidase inhibitor activities of PCG could be due to catechin polymerization enhancing the binding capacity to the cellular membrane and enzymes.     

Antioxidant and anticholinesterase investigations of Rumex hastatus D. Don: potential effectiveness in oxidative stress and neurological disorders

Background

Rumex species are traditionally used for the treatment of neurological disorders including headache, migraine, depression, paralysis etc. Several species have been scientifically validated for antioxidant and anticholinestrase potentials. This study aims to investigate Rumex hastatus D. Don crude methanolic extract, subsequent fractions, saponins and flavonoids for acetylcholinestrase, butyrylcholinestrase inhibition and diverse antioxidant activities to validate its folkloric uses in neurological disorders. Rumex hastatus crude methanolic extract (Rh. Cr), subsequent fractions; n-hexane (Rh. Hex), chloroform (Rh. Chf), ethyl acetate (Rh. EtAc), aqueous fraction (Rh. Aq), crude saponins (Rh. Sp) and flavonoids (Rh. Fl) were investigated against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at various concentrations (125, 250, 500, 1000 μg/mL) using Ellman’s spectrophotometric analysis. Antioxidant potentials of Rh. Sp and Rh. Fl were evaluated using DPPH, H₂O₂ and ABTS free radical scavenging assays at 62.5, 125, 250, 500, 1000 μg/mL.

Results

All the test samples showed concentration dependent cholinesterase inhibition and radicals scavenging activity. The AChE inhibition potential of Rh. Sp and Rh. Fl were most prominent i.e., 81.67 ± 0.88 and 91.62 ± 1.67 at highest concentration with IC₅₀ 135 and 20 μg/mL respectively. All the subsequent fractions exhibited moderate to high AChE inhibition i.e., Rh. Cr, Rh. Hex, Rh. Chf, Rh. EtAc and Rh. Aq showed IC₅₀ 218, 1420, 75, 115 and 1210 μg/mL respectively. Similarly, against BChE various plant extracts i.e., Rh. Sp, Rh. Fl, Rh. Cr, Rh. Hex, Rh. Chf, Rh. EtAc and Rh. Aq resulted IC₅₀ 165, 175, 265, 890, 92, 115 and 220 μg/mL respectively. In DPPH free radical scavenging assay, Rh. Sp and Rh. Fl showed comparable results with the positive control i.e., 63.34 ± 0.98 and 76.93 ± 1.13% scavenging at 1 mg/mL concentration (IC₅₀ 312 and 104 μg/mL) respectively. The percent ABTS radical scavenging potential exhibited by Rh. Sp and Rh. Fl (1000 μg/mL) were 82.58 ± 0.52 and 88.25 ± 0.67 with IC₅₀ 18 and 9 μg/mL respectively. Similarly in H₂O₂ scavenging assay, the Rh. Sp and Rh. Fl exhibited IC₅₀ 175 and 275 μg/mL respectively.

Conclusion

The strong anticholinesterase and antioxidant activities of Rh. Sp, Rh. Fl and various fractions of R. hastatus support the purported ethnomedicinal uses and recommend R. hastatus as a possible remedy for the treatment of AD and neurodegenerative disorders.     

Antischistosomal,antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents

BackgroundGhana is endemic for some neglected tropical diseases (NTDs) including schistosomiasis, onchocerciasis and lymphatic filariasis. The major intervention for these diseases is mass drug administration of a few repeatedly recycled drugs which is a cause for major concern due to reduced efficacy of the drugs and the emergence of drug resistance. Evidently, new treatments are needed urgently. Medicinal plants, on the other hand, have a reputable history as important sources of potent therapeutic agents in the treatment of various diseases among African populations, Ghana inclusively, and provide very useful starting points for the discovery of much-needed new or alternative drugs.Methodology/Principal findingsIn this study, extracts of fifteen traditional medicines used for treating various NTDs in local communities were screened in vitro for efficacy against schistosomiasis, onchocerciasis and African trypanosomiasis. Two extracts, NTD-B4-DCM and NTD-B7-DCM, prepared from traditional medicines used to treat schistosomiasis, displayed the highest activity (IC₅₀ = 30.5 μg/mL and 30.8 μg/mL, respectively) against Schistosoma mansoni adult worms. NTD-B2-DCM, also obtained from an antischistosomal remedy, was the most active against female and male adult Onchocera ochengi worms (IC₅₀ = 76.2 μg/mL and 76.7 μg/mL, respectively). Antitrypanosomal assay of the extracts against Trypanosoma brucei brucei gave the most promising results (IC₅₀ = 5.63 μg/mL to 18.71 μg/mL). Incidentally, NTD-B4-DCM and NTD-B2-DCM, also exhibited the greatest antitrypanosomal activities (IC₅₀ = 5.63 μg/mL and 7.12 μg/mL, respectively). Following the favourable outcome of the antitrypanosomal screening, this assay was selected for bioactivity-guided fractionation. NTD-B4-DCM, the most active extract, was fractionated and subsequent isolation of bioactive constituents led to an eupatoriochromene-rich oil (42.6%) which was 1.3-fold (IC₅₀ <0.0977 μg/mL) more active than the standard antitrypanosomal drug, diminazene aceturate (IC₅₀ = 0.13 μg/mL).Conclusion/SignificanceThese findings justify the use of traditional medicines and demonstrate their prospects towards NTDs drug discovery.     

Anticholinesterse and antioxidant investigations of crude extracts,subsequent fractions,saponins and flavonoids of atriplex laciniata L.: potential effectiveness in Alzheimer’s and other neurological disorders

Background

Atriplex laciniata L. was investigated for phenolic, flavonoid contents, antioxidant, anticholinesterase activities, in an attempt to explore its effectiveness in Alzheimer’s and other neurological disorders. Plant crude methanolic extract (Al.MeF), subsequent fractions; n-hexane (Al.HxF), chloroform (Al.CfF), ethyl acetate (Al.EaF), aqueous (Al.WtF), Saponins (Al.SPF) and Flavonoids (Al.FLVF) were investigated for DPPH, ABTS and H₂O₂ free radical scavenging activities. Further these extracts were subjected to acetylcholinesterase (AChE) & butyrylcholinesterase (BChE) inhibitory activities using Ellman’s assay. Phenolic and Flavonoid contents were determined and expressed in mg Gallic acid GAE/g and Rutin RTE/g of samples respectively.

Results

In DPPH free radicals scavenging assay, Al.FLVF, Al.SPF and Al.MeF showed highest activity causing 89.41 ± 0.55, 83.37 ± 0.34 and 83.37 ± 0.34% inhibition of free radicals respectively at 1 mg/mL concentration. IC₅₀ for these fractions were 33, 83 and 82 μg/mL respectively. Similarly, plant extracts showed high ABTS scavenging potential, i.e. Al.FLVF (90.34 ± 0.55), Al.CfF (83.42 ± 0.57), Al.MeF (81.49 ± 0.60) with IC₅₀ of 30, 190 and 70 μg/ml respectively. further, H₂O₂ percent scavenging was highly appraised in Al.FLVF (91.29 ± 0.53, IC₅₀ 75), Al.SPF (85.35 ± 0.61, IC₅₀ 70) and Al.EaF (83.48 ± 0.67, IC₅₀ 270 μg/mL). All fractions exhibited concentration dependent AChE inhibitory activity as; Al.FLVF, 88.31 ± 0.57 (IC₅₀ 70 μg/mL), Al.SPF, 84.36 ± 0.64 (IC₅₀ 90 μg/mL), Al.MeF, 78.65 ± 0.70 (IC₅₀ 280 μg/mL), Al.EaF, 77.45 ± 0.46 (IC₅₀ 270 μg/mL) and Al.WtF 72.44 ± 0.58 (IC₅₀ 263 μg/mL) at 1 mg/mL. Likewise the percent BChE inhibitory activity was most obvious in Al.FLVF 85.46 ± 0.62 (IC₅₀ 100 μg/mL), Al.CfF 83.49 ± 0.46 (IC₅₀ 160 μg/mL), Al.MeF 82.68 ± 0.60 (IC₅₀ 220 μg/mL) and Al.SPF 80.37 ± 0.54 (IC₅₀ 120 μg/mL).

Conclusions

These results stipulate that A. laciniata is enriched with phenolic and flavonoid contents that possess significant antioxidant and anticholinestrase effects. This provide pharmacological basis for the presence of compounds that may be effective in Alzheimer’s and other neurological disorders.     

New Anti-Inflammatory Metabolites by Microbial Transformation of Medrysone

Microbial transformation of the anti-inflammatory steroid medrysone (1) was carried out for the first time with the filamentous fungi Cunninghamella blakesleeana (ATCC 8688a), Neurospora crassa (ATCC 18419), and Rhizopus stolonifer (TSY 0471). The objective was to evaluate the anti-inflammatory potential of the substrate (1) and its metabolites. This yielded seven new metabolites, 14α-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (2), 6β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (3), 15β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (4), 6β,17α-dihydroxy-6α-methylpregn-4-ene-3,11,20-trione (5), 6β,20S-dihydroxy-6α-methylpregn-4-ene-3,11-dione (6), 11β,16β-dihydroxy-6α-methylpregn-4-ene-3,11-dione (7), and 15β,20R-dihydroxy-6α-methylpregn-4-ene-3,11-dione (8). Single-crystal X-ray diffraction technique unambiguously established the structures of the metabolites 2, 4, 6, and 8. Fungal transformation of 1 yielded oxidation at the C-6β, -11β, -14α, -15β, -16β positions. Various cellular anti-inflammatory assays, including inhibition of phagocyte oxidative burst, T-cell proliferation, and cytokine were performed. Among all the tested compounds, metabolite 6 (IC₅₀ = 30.3 μg/mL) moderately inhibited the reactive oxygen species (ROS) produced from zymosan-induced human whole blood cells. Compounds 1, 4, 5, 7, and 8 strongly inhibited the proliferation of T-cells with IC₅₀ values between <0.2–10.4 μg/mL. Compound 7 was found to be the most potent inhibitor (IC₅₀ < 0.2 μg/mL), whereas compounds 2, 3, and 6 showed moderate levels of inhibition (IC₅₀ = 14.6–20.0 μg/mL). Compounds 1, and 7 also inhibited the production of pro-inflammatory cytokine TNF-α. All these compounds were found to be non-toxic to 3T3 cells (mouse fibroblast), and also showed no activity when tested against HeLa (human epithelial carcinoma), or against PC3 (prostate cancer) cancer cell lines.     

Inhibition of fungal ABC transporters by unnarmicin A and unnarmicin C, novel cyclic peptides from marine bacterium

Novel inhibitors of fungal ATP-binding cassette transporters were obtained by screening compounds and crude extracts from marine-derived fungi and bacteria using disk diffusion assays of Saccharomyces cerevisiae strains overexpressing a variety of fungal multi-drug efflux pumps. The cyclodepsipeptides unnarmicin A and unnarmicin C were able to sensitize cells overexpressing azole drug pumps ScPdr5p, CaCdr1p, CgCdr1p, and CgPdh1p to sub-MIC concentrations of fluconazole without affecting the growth of CaCdr2p and CaMdr1p overexpressing cells. Unnarmicin A and unnarmicin C were potent inhibitors of rhodamine 6G efflux of CaCdr1p expressing cells with IC₅₀ values of 3.61 and 5.65 μM, respectively. They inhibited the in vitro CaCdr1p ATPase activity at IC₅₀ values of 0.495 and 0.688 μM, respectively. And most importantly, they were able to sensitize azole-resistant Candida albicans clinical isolates to fluconazole. Unnarmicin A and unnarmicin C are candidate efflux pump inhibitors with the potential to be used as adjuvants for antifungal chemotherapy.     

Investigations of anticholinestrase and antioxidant potentials of methanolic extract,subsequent fractions,crude saponins and flavonoids isolated from Isodon rugosus

Background

Based on the ethnomedicinal uses and the effective outcomes of natural products in various diseases, this study was designed to evaluate Isodon rugosus as possible remedy in oxidative stress, alzheimer’s and other neurodegenerative diseases. Acetylecholinestrase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of crude methanolic extract (Ir.Cr), resultant fractions (n-hexane (Ir.Hex), chloroform (Ir.Cf), ethyl acetate (Ir.EtAc), aqueous (Ir.Aq)), flavonoids (Ir.Flv) and crude saponins (Ir.Sp) of I. rugosus were investigated using Ellman’s spectrophotometric method. Antioxidant potential of I. rugosus was determined using DPPH, H₂O₂ and ABTS free radicals scavenging assays. Total phenolic and flavonoids contents of plant extracts were determined and expressed in mg GAE/g dry weight and mg RTE/g of dry sample respectively.

Results

Among different fractions Ir.Flv and Ir.Cf exhibited highest inhibitory activity against AChE (87.44 ± 0.51, 83.73 ± 0.64%) and BChE (82.53 ± 0.71, 88.55 ± 0.77%) enzymes at 1 mg/ml with IC₅₀ values of 45, 50 for AChE and 40, 70 μg/ml for BChE respectively. Activity of these fractions were comparable to galanthamine causing 96.00 ± 0.30 and 88.61 ± 0.43% inhibition of AChE and BChE at 1 mg/ml concentration with IC₅₀ values of 20 and 47 μg/ml respectively. In antioxidant assays, Ir.Flv, Ir.Cf, and Ir.EtAc demonstrated highest radicals scavenging activities in DPPH and H₂O₂ assays which were comparable to ascorbic acid. Ir.Flv was found most potent with IC₅₀ of 19 and 24 μg/ml against DPPH and H₂O₂ radicals respectively. Whereas antioxidant activates of plant samples against ABTS free radicals was moderate. Ir.Cf, Ir.EtAc and Ir.Cr showed high phenolic and flavonoid contents and concentrations of these compounds in different fractions correlated well to their antioxidant and anticholinestrase activities.

Conclusion

It may be inferred from the current investigations that the Ir.Sp, Ir.Flv and various fractions of I. rugosus are good sources of anticholinesterase and antioxidant compounds. Different fractions can be subjected to activity guided isolation of bioactive compounds effective in neurological disorders.     

In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC₅₀: 7.54 ± 1.10 μM), 5e (IC₅₀: 9.00 ± 0.97 μM), and 5 h (IC₅₀: 9.57 ± 0.62 μM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC₅₀: 32.18 ± 1.66 µM), 5 h (IC₅₀: 31.47 ± 1.42 µM), and 5 s (IC₅₀: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.

Highlights

1. A series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.
2. Compound 5g exhibited promising activity (IC₅₀ = 7.54 ± 1.10 μM) against α-glucosidase.
3. Compound 5s exhibited promising activity (IC₅₀ = 30.91 ± 0.86 μM) against α-amylase.
4. In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.

     

Antioxidant,inhibition of α-glucosidase and suppression of nitric oxide production in LPS-induced murine macrophages by different fractions of Actinidia arguta stem

In traditional systems of medicine, fruits, leaves, and stems of Actinidia arguta (Sieb. et Zucc.) Planch. ex Miq. have been used to treat various inflammatory diseases. The present study determined the proximate composition, antioxidant, anti-inflammatory, and hypoglycemic potential of A. arguta stem. Phenolic composition of hot water extract and its sub-fractions was determined by Folin–Ciocalteu’s reagent method. In vitro antioxidant activities of the samples were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging assays. Anti-inflammatory activity of different fractions was investigated through the inhibition of nitric oxide (NO) production in lipopolysaccharide (1 μg/ml) stimulated RAW 264.7 cells. In addition, inhibition of α-glucosidase activity of hot water extract was determined using p-nitrophenyl-α-d-glucopyranoside (pNPG) as a substrate. Ethyl acetate (557.23 mg GAE/g) fraction contains higher level of total phenolic content. The antioxidant activity evaluated by DPPH radical scavenging assay showed a strong activity for ethyl acetate (IC₅₀ of 14.28 μg/ml) and n-butanol fractions (IC₅₀ of 48.27 μg/ml). Further, ethyl acetate fraction effectively inhibited NO production in RAW 264.7 cells induced by lipopolysaccharide (LPS) than other fractions (nitrite level to 32.14 μM at 200 μg/ml). In addition, hot water extract of A. arguta stem exhibited appreciable inhibitory activity against α-glucosidase enzyme with IC₅₀ of 1.71 mg/ml. The obtained results have important consequence of using A. arguta stem toward the development of effective anti-inflammatory drugs.     

Establishment of Hairy Root Cultures by Agrobacterium Rhizogenes Mediated Transformation of Isatis Tinctoria L. for the Efficient Production of Flavonoids and Evaluation of Antioxidant Activities

In this work, Isatis tinctoria hairy root cultures (ITHRCs) were established as an alternative source for flavonoids (FL) production. I. tinctoria hairy root line V was found to be the most efficient line and was further confirmed by the PCR amplification of rolB, rolC and aux1 genes. Culture parameters of ITHRCs were optimized by Box-Behnken design (BBD), and eight bioactive FL constituents (rutin, neohesperidin, buddleoside, liquiritigenin, quercetin, isorhamnetin, kaempferol and isoliquiritigenin) were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total FL accumulation of ITHRCs (24 day-old) achieved was 438.10 μg/g dry weight (DW), which exhibited significant superiority as against that of 2 year-old field grown roots (341.73 μg/g DW). Additionally, in vitro antioxidant assays demonstrated that ITHRCs extracts exhibited better antioxidant activities with lower IC₅₀ values (0.41 and 0.39, mg/mL) as compared to those of field grown roots (0.56 and 0.48, mg/mL). To the best of our knowledge, this is the first report describing FL production and antioxidant activities from ITHRCs.     

Anti-inflammatory and antioxidant activities of Sargassum horneri extract in RAW264.7 macrophages

[Purpose]In this study, we investigated whether a 70% ethanolic (EtOH) extract of Sargassum horneri had antioxidant and anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated macrophage-like RAW 264.7 cells.[Methods]The proximate composition, fatty acids, amino acids, and dietary fiber of S. horneri, various biologically active compounds, and antioxidant activity were analyzed.[Results]The DPPH and ABTS free radical scavenging activities, as well as the reduction power, of the S. horneri extract used here were significantly increased in a concentration-dependent manner. This indicates that S. horneri contains bioactive compounds, such as phenols and flavonoids, that have excellent antioxidant activity. The cellular viability and metabolic activity results confirmed that the extract had no discernible toxicity at concentrations up to 100 μg/mL. The levels of nitrites and cytokines (PGE₂, TNF-α and IL-6), which mediate pro-inflammatory effect, were significantly inhibited by treatment with either 50 or 100 μg/mL S. horneri extract, whereas that of IL-1β was significantly inhibited by treatment with 100 μg/mL of the extract. Similarly, the expression of iNOS and COX-2 proteins also decreased according to 50 or 100 μg/mL extract concentrations. NF-κB binding to DNA was also significantly inhibited by treatment with 100 μg/mL of extract.[Conclusion]These results suggest that 70% EtOH extracts of S. horneri can relieve inflammation caused by disease or high intensity exercise.     

Selamectin Is the Avermectin with the Best Potential for Buruli Ulcer Treatment

A comprehensive analysis was done to evaluate the potential use of anti-parasitic macrocyclic lactones (including avermectins and milbemycins) for Buruli ulcer (BU) therapy. A panel containing nearly all macrocyclic lactones used in human or in veterinary medicine was analyzed for activity in vitro against clinical isolates of Mycobacterium ulcerans. Milbemycin oxime and selamectin were the most active drugs against M. ulcerans with MIC values from 2 to 8 μg/mL and 2 to 4 μg/mL, respectively. In contrast, ivermectin and moxidectin, which are both in clinical use, showed no significant activity (MIC> 32 μg/mL). Time-kill kinetic assays showed bactericidal activity of selamectin and in vitro pharmacodynamic studies demonstrated exposure-dependent activity. These data together with analyses of published pharmacokinetic information strongly suggest that selamectin is the most promising macrocyclic lactone for BU treatment.     

Discovery of orally active chalcones as histone lysine specific demethylase 1 inhibitors for the treatment of leukaemia

Histone lysine specific demethylase 1 (LSD1) has emerged as an attractive molecule target for the discovery of potently anticancer drugs to treat leukaemia. In this study, a series of novel chalcone derivatives were designed, synthesised and evaluated for their inhibitory activities against LSD1 in vitro. Among all these compounds, D6 displayed the best LSD1 inhibitory activity with an IC₅₀ value of 0.14 μM. In the cellular level, compound D6 can induce the accumulation of H3K9me1/2 and inhibit cell proliferation by inactivating LSD1. It exhibited the potent antiproliferative activity with IC₅₀ values of 1.10 μM, 3.64 μM, 3.85 μM, 1.87 μM, 0.87 μM and 2.73 μM against HAL-01, KE-37, P30-OHK, SUP-B15, MOLT-4 and LC4-1 cells, respectively. Importantly, compound D6 significantly suppressed MOLT-4 xenograft tumour growth in vivo, indicating its great potential as an orally bioavailable candidate for leukaemia therapy.     

Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant,Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T₁) and reached a maximum after the completion of CaDPA release (T_release) and spore cortex lysis (T_lysis).     

Flavonoid constituents,cytotoxic and antioxidant activities of Gleditsia triacanthos L. leaves

Gleditsia triacanthos L. is a deciduous tree belonging to the family Fabaceae. It possesses important biological activities as anti-mutagenic, anticancer, cytotoxic and treating rheumatoid arthritis. The total ethanol extract (EtOHE) and successive extracts (petroleum ether, chloroform, ethyl acetate, and aqueous ethanol) were prepared from the leaves. Eight flavone glycosides and two flavone aglycones named vicenin-I (1), vitexin (2), isovitexin (3), orientin (4), isoorientin (5), luteolin-7-O-ß-glucopyranoside (6), luteolin-7-O-ß-galactopyranoside (7), apigenin-7-O-ß-glucopyranoside (8), luteolin (9) and apigenin (10) were isolated from the aqueous ethanol extract of G. triacanthos L. leaves. Potent cytotoxic activity of the EtOHE extract was observed against the liver (IC₅₀ = 1.68 μg), breast (IC₅₀ = 0.74 μg), cervix (IC₅₀ = 1.28 μg), larynx (IC₅₀ = 0.67 μg) and colon (IC₅₀ = 2.50 μg) cancer cell lines. Cytotoxic activity of compounds 2, 4, 6 and 8 against, the liver, breast and colon cancer cell lines was also proved. Evaluation of the in-vivo antioxidant activity of the EtOHE and successive extracts revealed that the highest activity was exhibited by 100 mg of EtOHE (97.89% potency) as compared with vitamin E (100% potency). Compound 6 showed 91.8% free radical scavenging activity.     

Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (K_s, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (K_d, ≤3 nM) and prothioconazole-desthio the lowest (K_d, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC₅₀s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC₁₀₀, >256 μg ml⁻¹). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC₁₀₀ [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml⁻¹) with the exception of clotrimazole, which was as potent as malachite green (MIC₁₀₀, ∼1 μg ml⁻¹). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC₅₀, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC₁₀₀, ∼1 to 2 μg ml⁻¹) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.     

Screening of ethnic medicinal plants of South India against influenza (H1N1) and their antioxidant activity

Antiviral activity against H1N1 influenza was studied using ethnic medicinal plants of South India. Results revealed that Wrightia tinctoria (2.25 μg/ml) was one of the best antidotes against H1N1 virus in terms of inhibitory concentration of 50% (IC₅₀) whereas the control drug Oseltamivir showed 6.44 μg/ml. Strychnos minor, Diotacanthus albiflorus and Cayratia pedata showed low cytotoxicity (>100) to the MDCK (Malin darby canine kidney) cells by cytotoxicity concentration of 50% (CC₅₀) and possessed antiviral activity suggesting that these plants can be used as herbal capsules for H1N1 virus. W. tinctoria and S. minor showed high therapeutic indexes (TI) such as 12.67 and 21.97 suggesting that those plants can be used for anti-viral drug development. The CC₅₀ values of Eugenia singampattiana (0.3 μg/ml), Vitex altissima (42 μg/ml), Salacia oblonga (7.32 μg/ml) and Salacia reticulata (7.36 μg/ml) resulted in cytotoxicity of the MDCK cells, due to their high phenolic content. Findings from this study state that the plant W. tinctoria can be a potent source for third generation anti-viral drug development against H1N1.     

Anti-inflammatory components of the Vietnamese starfish Protoreaster nodosus

Background

In the present study, we examined the inhibitory effects of a methanolic extract, dichloromethane fraction, water layer, and polyhydroxylated sterols (1–4) isolated from the Vietnamese starfish Protoreaster nodosus on pro-inflammatory cytokine (IL-12 p40, IL-6, and TNF-α) production in LPS-stimulated bone marrow-derived dendritic cells (BMDCs) using enzyme-linked immunosorbent assays (ELISA).

Results

The methanolic extract and dichloromethane fraction exerted potent inhibitory effects on the production of all three pro-inflammatory cytokines, with IC₅₀ values ranging from 0.60 ± 0.01 to 26.19 ± 0.64 μg/mL. Four highly pure steroid derivatives (1–4) were isolated from the dichloromethane fraction and water layer of P. nodosus. Potent inhibitory activities were also observed for (25S) 5α-cholestane-3β,4β,6α,7α,8β,15α,16β,26-octol (3) on the production of IL-12 p40 and IL-6 (IC_50s = 3.11 ± 0.08 and 1.35 ± 0.03 μM), and for (25S) 5α-cholestane-3β,6α,8β,15α,16β,26-hexol (1) and (25S) 5α-cholestane-3β,6α,7α,8β,15α,16β,26-heptol (2) on the production of IL-12 p40 (IC_50s = 0.01 ± 0.00 and 1.02 ± 0.01 μM). Moreover, nodososide (4) exhibited moderate inhibitory effects on IL-12 p40 and IL-6 production.

Conclusion

This is the first report of the anti-inflammatory activity from the starfish P. nodosus. The main finding of this study is the identification oxygenated steroid derivatives from P. nodosus with potent anti-inflammatory activities that may be developed as therapeutic agents for inflammatory diseases.     

Rapid Identification of α-Glucosidase Inhibitors from Phlomis tuberosa by Sepbox Chromatography and Thin-Layer Chromatography Bioautography

Alpha-glucosidase inhibitors currently form an important basis for developing novel drugs for diabetes treatment. In our preliminary tests, the ethyl acetate fraction of Phlomis tuberosa extracts showed significant α-glucosidase inhibitory activity (IC₅₀ = 100 μg/mL). In the present study, a combined method using Sepbox chromatography and thin-layer chromatography (TLC) bioautography was developed to probe α-glucosidase inhibitors further. The ethyl acetate fraction of P. tuberosa extracts was separated into 150 individual subfractions within 20 h using Sepbox chromatography. Then, under the guidance of TLC bioautography, 20 compounds were successfully isolated from these fractions, including four new diterpenoids [14-hydroxyabieta-8,11,13-triene-11-carbaldehyde-18-oic-12-carboxy-13-(1-hydroxy-1-methylethyl)-lactone (1), 14-hydroxyabieta-8,11,13-triene-17-oic-12-carboxy-13-(1-hydroxy-1-methylethyl)-lactone (2), 14,16-dihydroxyabieta-8,11,13-triene-15,17-dioic acid (3), and phlomisol (15,16-eposy-8,13(16),14-labdatrien-19-ol) (4)], and 16 known compounds. Activity estimation indicated that 15 compounds showed more potent α-glucosidase inhibitory effects (with IC₅₀ values in the range 0.067–1.203 mM) than the positive control, acarbose (IC₅₀ = 3.72 ± 0.113 mM). This is the first report of separation of α-glucosidase inhibitors from P. tuberosa.     

Streptomycin Induced Stress Response in Salmonella enterica Serovar Typhimurium Shows Distinct Colony Scatter Signature

We investigated the streptomycin-induced stress response in Salmonella enterica serovars with a laser optical sensor, BARDOT (bacterial rapid detection using optical scattering technology). Initially, the top 20 S. enterica serovars were screened for their response to streptomycin at 100 μg/mL. All, but four S. enterica serovars were resistant to streptomycin. The MIC of streptomycin-sensitive serovars (Enteritidis, Muenchen, Mississippi, and Schwarzengrund) varied from 12.5 to 50 μg/mL, while streptomycin-resistant serovar (Typhimurium) from 125–250 μg/mL. Two streptomycin-sensitive serovars (Enteritidis and Mississippi) were grown on brain heart infusion (BHI) agar plates containing sub-inhibitory concentration of streptomycin (1.25–5 μg/mL) and a streptomycin-resistant serovar (Typhimurium) was grown on BHI containing 25–50 μg/mL of streptomycin and the colonies (1.2 ± 0.1 mm diameter) were scanned using BARDOT. Data show substantial qualitative and quantitative differences in the colony scatter patterns of Salmonella grown in the presence of streptomycin than the colonies grown in absence of antibiotic. Mass-spectrometry identified overexpression of chaperonin GroEL, which possibly contributed to the observed differences in the colony scatter patterns. Quantitative RT-PCR and immunoassay confirmed streptomycin-induced GroEL expression while, aminoglycoside adenylyltransferase (aadA), aminoglycoside efflux pump (aep), multidrug resistance subunit acrA, and ribosomal protein S12 (rpsL), involved in streptomycin resistance, were unaltered. The study highlights suitability of the BARDOT as a non-invasive, label-free tool for investigating stress response in Salmonella in conjunction with the molecular and immunoassay methods.