Statin therapy influences endothelial cell morphology and F-actin cytoskeleton structure when exposed to static and laminar shear stress conditions

AimsTo determine how statin drugs (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) affect endothelial cell (EC) shape and F-actin cytoskeleton arrangement in the presence of physiologically relevant wall shear stress (WSS) of 12.5 dyn/cm².Main methodsHuman abdominal aortic endothelial cells (HAAECs) were cultured to a confluent monolayer within three dimensional tissue culture models and presheared for 6 h at 12.5 dyn/cm² within a continuous flow loop. Statins were added to the perfusion media and the perfusion was continued for a further 24 h. ECs were then analyzed for morphology and F-actin cytoskeleton arrangement using light microscopy and laser scanning confocal microscopy.Key findingsECs became rounded with a significantly higher shape index with the addition of 10 μM simvastatin under both static and flow conditions. F-actin cytoskeleton structure was disorganized and fragmented with statin treatment under static and flow conditions. Neither of these findings were observed with the addition of both simvastatin and 200 μM mevalonate, confirming regulation through the cholesterol biosynthesis pathway.SignificanceEC morphology and F-actin cytoskeleton arrangement are regulated through the cholesterol biosynthesis pathway and are therefore impacted by statin treatment. ECs treated with statins became rounded, which is usually associated with unhealthy cells in regions of the vasculature prone to developing atherosclerotic plaques.     

Homocysteine and inflammation as main determinants of oxidative stress in the elderly

Oxidative stress is commonly observed in the elderly and could be involved in age-related diseases. However, the determinants of superoxide anion overproduction are not clearly understood. Superoxide anion production was evaluated using a lucigenin-based chemiluminescence method in 478 elderly subjects (304 women, 174 men; 79.5 ± 7.1 years). Homocysteine (HCy) metabolism (homocysteinemia, vitamin B12, plasma, and erythrocyte folates), inflammation (CRP, fibrinogen, α-1 acid glycoprotein), lipid parameters (total cholesterol, triglycerides, HDL and LDL cholesterol), and nutritional parameters (albumin, transthyretin) were determined. The results show that HCy levels (p < 0.001) and superoxide anion production (p = 0.04) increase with aging, but CRP does not. Highest HCy (> 20 μM) (OR 1.83 (1.09–3.07), p = 0.02) and CRP over 5 mg/L (adjusted OR 2.01 (1.15–3.51), p = 0.01) are the main determinants in superoxide anion production in the elderly. These clinical data are confirmed in an in vitro study using THP-1 monocyte-like cells. Incubation with HCy thiolactone (HTL) (0–200 μM) and LPS (0–20 ng/ml) dramatically enhances NADPH oxidase expression and activation. Moreover, a synergic action was evidenced for low concentrations of HTL (20 μM) and LPS (5 ng). Taken together, the clinical data and in vitro experiments support the hypothesis that moderate homocysteinemia and low-grade inflammation synergically enhance NADPH oxidase activity in the elderly.     

Statins directly suppress cytokine production in murine intraepithelial lymphocytes

Statins, inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are known not only as cholesterol-lowering agents but also as anti-inflammatory mediators. However, their regulatory effect on intestinal mucosal immunity remains unclear. The present study examined the possible direct effects of statin on intestinal intraepithelial lymphocytes (IELs), the front line cells of the intestinal mucosal immune system. Murine IELs were isolated from the small intestines of C57BL/6 mice. IELs activated with anti-CD3/CD28 monoclonal antibodies produced interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and IL-4 in significant numbers; however, they did not produce IL-5. Both simvastatin and lovastatin suppressed IEL production of IFN-γ, TNF-α, IL-2, and IL-4 in a dose-dependent manner, whereas 48-h treatment with high concentrations (5 × 10^?5 M) of simvastatin and lovastatin did not affect the number of IELs. The suppressive effect of the simvastatin was significantly restored by the addition of mevalonate, farnesyl pyrophosphate ammonium salt, and geranylgeranyl pyrophosphate ammonium salt, which are downstream metabolites of HMG-CoA. These findings suggest that statins have direct suppressive effects on the production of T helper 1-cytokines and IL-4 in IELs; these effects are associated with inhibition of the mevalonate pathway to some extent.     

Synergistic antifungal activity of statin–azole associations as witnessed by Saccharomyces cerevisiae- and Candida utilis-bioassays and ergosterol quantification

BackgroundFrequent opportunist fungal infections and the resistance to available antifungal drugs promoted the development of new alternatives for treatment, like antifungal drug combinations.AimsThis work aimed to detect the antifungal synergism between statins and azoles by means of an agar-well diffusion bioassay with Saccharomyces cerevisiae ATCC 32051 and Candida utilis Pr_1–2 as test strains.MethodsSynergistic antifungal effects were tested by simultaneously adding a sub inhibitory concentration (SIC) of statin (atorvastatin, lovastatin, pravastatin, rosuvastatin or simvastatin) plus a minimal inhibitory concentration (MIC) of azole (clotrimazole, fluconazole, itraconazole, ketoconazole or miconazole) to yeast-embedded YNB agar plates, and a positive result corresponded to a yeast growth inhibition halo higher than that produced by the MIC of the azole alone. Yeast cell ergosterol quantification by RP-HPLC was used to confirm statin–azole synergism, and ergosterol rescue bioassays were performed for evaluating statin-induced ergosterol synthesis blockage.ResultsGrowth inhibition was significantly increased when clotrimazole, fluconazole, itraconazole, ketoconazole and miconazole were combined with atorvastatin, lovastatin, rosuvastatin and simvastatin. Highest growth inhibition increments were observed on S. cerevisiae (77.5%) and C. utilis (43.2%) with a SIC of simvastatin plus a MIC of miconazole, i.e. 4 + 2.4 μg/ml or 20 + 4.8 μg/ml, respectively. Pravastatin showed almost no significant effects (0–7.6% inhibition increase). Highest interaction ratios between antifungal agents corresponded to simvastatin–miconazole combinations and were indicative of synergism. Synergism was also confirmed by the increased reduction in cellular ergosterol levels (S. cerevisiae, 40% and C. utilis, 22%). Statin-induced ergosterol synthesis blockage was corroborated by means of ergosterol rescue bioassays, pravastatin being the most easily abolished inhibition whilst rosuvastatin being the most ergosterol-refractory.ConclusionsSelected statin–azole combinations might be viable alternatives for the therapeutic management of mycosis at lower administration doses or with a higher efficiency.     

Simvastatin inhibits the core promoter of the TXNRD1 gene and lowers cellular TrxR activity in HepG2 cells

Thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing redox-active enzyme that is thought to be important during carcinogenesis. We have recently shown that treatment with statins, HMGCoA reductase inhibitors, reduces the levels of TrxR1 in liver of both rat and human. The reduced TrxR1 levels were correlated with inhibited hepatocarcinogenesis in a rat model. The aim of the present study was to investigate if statins affect the activity of the human TXNRD1 core promoter, which guides expression of TrxR1, and if the effects by statins on TrxR1 expression in liver could be reproduced in a cellular model system. We found that simvastatin and fluvastatin decreased cellular TrxR activity in cultured human liver-derived HepG2 cells with approximately 40% (p < 0.05). Simvastatin, but not fluvastatin or atorvastatin, also reduced the TXNRD1 promoter activity in HepG2 cells by 20% (p < 0.01). In line with this result, TrxR1 mRNA levels decreased with about 25% in non-transfected HepG2 cells upon treatment with simvastatin (p < 0.01). Concomitant treatment with mevalonate could not reverse these effects of simvastatin, indicating that other mechanisms than HMGCoA reductase inhibition was involved. Also, simvastatin did not inhibit sulforaphane-derived stimulation of the TXNRD1 core promoter activity, suggesting that the inhibition by simvastatin was specific for basal and not Nrf2-activated TrxR1 expression. In contrast to simvastatin, the two other statins tested, atorvastatin or fluvastatin, did not influence the TrxR1 mRNA levels. Thus, our results reveal a simvastatin-specific reduction of cellular TrxR1 levels that at least in part involves direct inhibitory effects on the basal activity of the core promoter guiding TrxR1 expression.     

Protection of oxidative stress induced apoptosis in osteosarcoma cells by dihydromyricetin through down-regulation of caspase activation and up-regulation of BcL-2

Current study was aimed to investigate the effect of dihydromyricetin on hydrogen peroxide induced oxidative stress in the osteosarcoma cells. MTT assay showed that hydrogen peroxide treatment at a concentration of 100 μM caused a significant (p < 0.005) reduction in the viability of MG63 cells. However, reduction in cell viability caused by 100 μM concentration of hydrogen peroxide was completely prevented on incubation with 30 μM dose of dihydromyricetin. Treatment with 100 μM concentration of hydrogen peroxide for 24 h led to condensation of chromatin material, rounding of cell shape and detachment of cells. The results from flow cytometry using annexin V-FITC and PI double staining showed apoptosis induction in 47.84 ± 5.21% cells on treatment with 100 μM concentration of hydrogen peroxide compared to 2.32 ± 0.54% in controlcells. The apoptotic alterations in MG63 cell morphology were prevented significantly on pre-treatment with 30 μM doses of dihydromyricetin for 48 h. Annexin V-FITC and PI staining showed reduction of hydrogen peroxide induced apoptotic cell percentage to 3.07 ± 0.86% on pre-treatment of MG63 cells with 30 μM dose of dihydromyricetin. Western blot analysis showed a significant increase in the activation of caspase-3 and -9 on treatment of MG63 cells for 24 h with 100 μM concentration of hydrogen peroxide. The expression level of Bcl-2 was decreased significantly by 100 μM concentration of hydrogen peroxide in MG63 cells. However, pre-treatment of MG63 cells with 30 μM dose of dihydromyricetin for 48 h significantly prevented hydrogen peroxide induced increase in caspase-3 and -9 levels and reduction in Bcl-2 level. Thus dihydromyricetin prevents hydrogen peroxide induced reduction in viability and induction of apoptosis in MG63 cells through down-regulation of caspase activation and up-regulation of Bcl-2 levels.     

Effects of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on rat CYP1A2 expression and metabolism of model CYP1A2 probe substrates

This study explored the effects of Danshen on metabolism/pharmacokinetics of model CYP1A2 substrates and hepatic CYP1A2 expression in rats. The effects of Danshen and tanshinones on CYP1A2 activity was determined by metabolism of model substrates in vitro (phenacetin) and in vivo (caffeine). HPLC was used to determine model substrates/metabolites. The effect of Danshen on CYP1A2 expression was determined by Western blot. Tanshinones (1.25–50 μM) competitively inhibited phenacetin O-deethylation in vitro. Inhibition kinetics studies showed the K_i values were in the order: dihydrotanshinone (3.64 μM), cryptotanshinone (4.07 μM), tanshinone I (22.6 μM) and tanshinone IIA (23.8 μM), furafylline (35.8 μM), a CYP1A2 inhibitor. The Ki of Danshen extract (mainly tanshinones) was 72 μg/ml. Acute Danshen extract treatment (50–200 mg/kg, i.p.) decreased metabolism of caffeine to paraxanthine, with overall decrease in caffeine clearance (14–22%); increase in AUC (11–25%) and plasma T_1/2 (12–16%). Danshen treatment with (100 mg/kg/day, i.p. or 200 mg/kg/day, p.o.) for three or fourteen days showed similar pharmacokinetic changes of the CYP1A2 probe substrate without affecting CYP1A2 expression. This study demonstrated that major tanshinones competitively inhibited the metabolism of model CYP1A2 probe substrates but had no effect on rat CYP1A2 expression.     

Casearin X exhibits cytotoxic effects in leukemia cells triggered by apoptosis

Clerodane diterpenes have demonstrated cytotoxic, antiplasmodial and anti-ulcer properties. In the present work, we determined the cytotoxic effect of casearin L (Cas L), O (Cas O) and X (Cas X) and (?)-hardwickiic acid isolated from Casearia sylvestris leaves, and investigated the underlying mechanisms involved in in vitro cell death induced by Cas X in HL-60 leukemia cells (0.7, 1.5 and 3.0 μM). Cytotoxicity tests demonstrated that Cas X was the most active compound studied, showing greater cytotoxic effects against CEM and HL-60 lines (IC₅₀ of 0.4 μM) and human peripheral blood mononuclear cells (PBMC, IC₅₀ of 1.2 μM). After 24 h exposure, Cas X caused a decrease in 5-bromo-20-deoxyuridine (BrdU) incorporation (36.6 and 24.5% labeling at 0.7 and 1.5 μM, respectively), reduction in viability, and increase in apoptotic and necrotic leukemia cells in a dose-dependent manner evidenced by the trypan blue and AO/EB (acridine orange/ethidium bromide) assays. Moreover, Cas X-treated cells exhibited nuclear fragmentation and cytoplasmic vacuolization depending on the concentration tested. These characteristics of apoptosis or secondary necrosis were confirmed by flow cytometry which revealed DNA fragmentation, phosphatidylserine externalization, activation of the effector caspases 3/7 and mitochondrial depolarization. We then found evidence that Cas X causes cell death via apoptotic pathways, corroborating the potential of casearins as compounds with promising antitumor-related properties.     

Tyrosine phosphorylation of cytochrome c as a signaling event in frozen thawed buffalo spermatozoa at the cross-roads of capacitation and apoptosis

Considering the importance of cytochrome c in both life and death, it was of significant interest to investigate the expression of cytochrome c, its tyrosine phosphorylation status and immunolocalization patterns in a frozen-thawed buffalo sperm cell in comparison to in vitro capacitated [heparin (10 μg/ml) induced, for 4 h] and stress [apoptotic (10 μM staurosporine), oxidative (25 μM H₂O₂) and osmotic (180 mM NaCl) for 4 h] induced conditions. Proteins were subjected to immunoblotting and probed by using monoclonal anti-phosphotyrosine antibodies. A significant (p < 0.05) increase in expression of tyrosine phosphorylated cytochrome c was observed in capacitated buffalo sperm in comparison to frozen-thawed samples. cAMP protein kinase-A dependent and extracellular signal-regulated kinase independent tyrosine phosphorylation of cytochrome c was found during in vitro capacitation of buffalo spermatozoa. Localized increase in cytochrome c and tyrosine phosphorylated proteins were observed in frozen thawed and capacitated sperm. The information generated in this study can be used to understand the molecular mechanism of regulation of an apoptotic protein (cytochrome c) by tyrosine phosphorylation (a capacitation marker) in a frozen thawed sperm cell which could be a good target to combat apoptosis.     

Phenolic compounds composition and physiological attributes of Matricaria chamomilla grown in copper excess

Four-week old plants of chamomile (Matricaria chamomilla) cultivated in nutrient solution were exposed to copper (3, 60 and 120 μM) for 10 days. At 120 μM, Cu decreased dry mass production, water, chlorophyll and nitrogen content in both the leaf rosettes and roots. Five phenolic acids were detected in methanol extracts of the leaf rosettes (protocatechuic, p-hydroxybenzoic, vanillic, chlorogenic and salicylic acid) and six additional compounds (gentisic, syringic, caffeic, sinapic and o-/p-coumaric acid) were released after acid hydrolysis. Most of the 11 phenolic acids detected increased in 60 μM Cu but in the 120 μM treatment their contents were lower or not significantly different from the control. Among coumarin-related compounds, (Z)- and (E)-2-ß-d-glucopyranosyloxy-4-methoxycinnamic acids increased in 60 and 120 μM Cu while herniarin rose in the 3 and 60 μM Cu by the end of the experiment. The amounts of umbelliferone were not affected by any of the doses tested. These facts in relation to antioxidative properties of phenolic metabolites are also discussed. The malondialdehyde content of the leaf rosettes was not affected by exposure of plants to 120 μM Cu in a time-course experiment but in the roots a sharp increase was observed after 24 and 48 h of treatment. At 120 μM, Cu stimulated a 9-fold higher K⁺ loss than the 60 μM treatment while at the lowest concentration it stimulated potassium uptake. Cu accumulation in the roots was 3-, 49- and 71-fold higher than that in the leaf rosettes in the 3, 60, and 120 μM Cu treatments, respectively. Results suggest that 120 μM Cu dose is limiting for chamomile growth under the conditions of present research.     

Cytotoxic activity of naphthoquinones with special emphasis on juglone and its 5-O-methyl derivative

The cytotoxicity of nine naphthoquinones (NQ) was assayed against HL-60 (leukaemia), MDA-MB-435 (melanoma), SF-295 (brain) and HCT-8 (colon), all human cancer cell lines, and peripheral blood mononuclear cells (PBMC), as representatives of normal cells, after 72 h of incubation. 5-Methoxy-1,4-naphthoquinone was the most active compound, showing IC₅₀ values in the range of 0.31 (1.7 μM) in HL-60 to 0.88 μg/mL (4.7 μM) in SF-295 and IC₅₀ of 0.69 μg/mL (3.7 μM) against PBMC. With the introduction of a bromo-substituent in position 2 or 3 of juglone, the IC₅₀ significantly decreased, regardless of the position on the NQ moiety. However, compared with juglone methyl ether, the halogen substitution decreased the activity. To further understand the mechanism underlying the cytotoxicity of 5-methoxy-1,4-naphthoquinone, studies involving DNA fragmentation, cell cycle analysis, phosphatidyl serine externalization, mitochondrial depolarization and activation of caspases 8 and 3/7 were performed in HL-60 cell line, using doxorubicin as a positive control. The results indicate that the cytotoxic 5-methoxy-1,4-naphthoquinone activates caspases 8 and 3/7 and thus induces apoptosis independent of mitochondria.     

Source of isopentenyl diphosphate for taxol and baccatin III biosynthesis in cell cultures of Taxus baccata

To achieve a better understanding of the metabolism and accumulation of taxol and baccatin III in cell cultures of Taxus, three cell lines (I, II and III) of T. baccata were treated (on day 7) with several concentrations of fosmidomycin (100, 200 and 300 μM), an inhibitor of the non-mevalonate branch of the terpenoid pathway, or mevinolin (1, 3 and 5 μM), an inhibitor of the mevalonate branch, in both cases in presence and absence of 100 μM methyl jasmonate (MeJ). They were compared with lines treated only with the elicitor MeJ as well as an untreated control with respect to growth, viability and production of taxol and baccatin III. The results show that the cell line type was an important variable, mainly for taxane accumulation. The blocking effect of fosmidomycin on taxane production was significantly greater than that of mevinolin in all the cell lines, clearly suggesting that the isopentenyl diphosphate (IPP) used for the taxane ring formation was mainly formed via the non-mevalonate pathway. However, the significant reduction in the content of taxol (on average 3.8-fold) and baccatin III (on average 4.3-fold) in line I when treated with the elicitor together with mevinolin concentrations of 5 and 1 μM, respectively, also suggests that both non-mevalonate and mevalonate pathways are involved in the biosynthesis of the two taxanes as a result of cytosolic IPP and/or other prenyl diphosphate transport to the plastids. The observation that the inhibitory effect of fosmidomycin or mevilonin on taxol and baccatinn III yield does not interfere with methyl jasmonate elicitation is discussed.     

Copper toxicity and sulfur metabolism in Chinese cabbage are affected by UV radiation

Biomass production, dry matter content, specific leaf area and pigment content of Chinese cabbage were all quite similar, when plants were grown in the absence or presence of UV-A + B (2.2 mW cm⁻²). Elevated Cu²⁺ concentrations (2–10 μM) in the root environment and UV radiation had negative synergistic effects for Chinese cabbage and resulted in a more rapid and stronger decrease in plant biomass production and pigment content. The quantum yield of photosystem II photochemistry (F_v/F_m) was only decreased at ≥5 μM Cu²⁺ in the presence of UV radiation, when leaf tissue started to become necrotic. The enhanced Cu toxicity in the presence of UV was largely due to a UV-induced enhanced accumulation of Cu in both roots and shoots. An enhanced Cu content strongly affected the uptake and assimilation of sulfur in plants. The total sulfur content of the root increased at ≥2 μM Cu²⁺ in presence of UV and at 10 μM Cu²⁺ in absence of UV and that of the shoot increased at ≥2 μM Cu²⁺ in presence of UV and at ≥5 μM Cu²⁺ in absence of UV. In the shoot it could be attributed mainly to an increase in sulfate content. Moreover, there was a strong increase in the water-soluble non-protein thiol content upon Cu²⁺ exposure in the root and, to a lesser extent in the shoot, both in the presence and absence of UV. The regulation of the uptake of sulfate responded to the occurrence of Cu toxicity directly, since it was more rapidly affected in the presence than in the absence of UV radiation. For instance, the expression and activity of the high affinity sulfate transporter, Sultr1;2, were enhanced at ≥2 μM in the presence of UV, and at ≥5 μM Cu²⁺ in the absence of UV. In the shoot, the expression of the vacuolar sulfate transporter, Sultr4;1, was upregulated at ≥5 μM Cu²⁺ in the presence and absence of UV whilst the expression of a second vacuolar sulfate transporter, Sultr4;2, was upregulated at 10 μM Cu²⁺ in the presence of UV. It is suggested that high Cu tissue levels may interfere/react with the signal compounds involved in the regulation of expression and activity of sulfate transporters. The expression of adenosine 5′-phosphosulfate reductase in the root was hardly affected and was slightly down-regulated at 2 μM in the presence of UV and at 10 μM in the absence of UV. The expression and activity of sulfate transporters were enhanced upon exposure at elevated Cu²⁺ concentrations; this may not be simply due to a greater sulfur demand at higher Cu levels, but more likely is the consequence of Cu toxicity, since it occurred more rapidly in the presence compared to the absence of UV.     

Effect of mercury and gold on growth,nutrient uptake,and anatomical changes in Chilopsis linearis

Plants of Chilopsis linearis were grown with 0, 50, 100, and 200 μM Hg [as Hg(CH₃COO)₂] and 0 and 50 μM Au (as KAuCl₄) in hydroponics. The results showed that seedling grown with 50 μM Au + 50 μM Hg and 50 μM Au + 100 μM Hg had roots 25 and 55% shorter than control roots, respectively. The element uptake determination using ICP/OES demonstrated that Hg at 50 and 100 μM (with and without Au) significantly increased (p < 0.05) the S concentration in leaves. On the other hand, the concentration of Fe significantly increased in roots of plants treated with Au–Hg. In addition, the stems of plants treated with Hg at 100 μM, with and without Au, had 239 and 876 mg Hg/kg dry biomass (d wt), respectively. Also, at 50 μM Hg, with and without Au, stems accumulated 375 and 475 mg Hg/kg d wt. The Hg concentration in leaves (287 mg Hg/kg d wt) was higher (p < 0.05) for the treatment containing 50 μM Au + 100 μM Hg. Without Au, the Hg concentration in leaves decreased to 75 mg Hg/kg d wt. Toxicity symptoms induced by Hg in cortex cells and the vascular system were lower in plants exposed to 50 μM Au + 50 μM Hg compared to plants exposed to 50 μM Hg only. Further, the SEM micrographs revealed deposition of Au–Hg particles inside the root. Although the concentrations of Hg used in this study showed different degree of toxicity, the plants displayed good agronomic value.     

Design,synthesis, antiviral and cytostatic activity of ω-(1H-1,2,3-triazol-1-yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues

The efficient synthesis of a new series of polyhydroxylated dibenzyl ω-(1H-1,2,3-triazol-1-yl)alkylphosphonates as acyclic nucleotide analogues is described starting from dibenzyl ω-azido(polyhydroxy)alkylphosphonates and selected alkynes under microwave irradiation. Selected O,O-dibenzylphosphonate acyclonucleotides were transformed into the respective phosphonic acids. All compounds were evaluated in vitro for activity against a broad variety of DNA and RNA viruses and for cytostatic activity against murine leukemia L1210, human T-lymphocyte CEM and human cervix carcinoma HeLa cells. Compound (1S,2S)-16b exhibited antiviral activity against Influenza A H3N2 subtype (EC₅₀ = 20 μM—visual CPE score; EC₅₀ = 18 μM—MTS method; MCC >100 μM, CC₅₀ >100 μM) in Madin Darby canine kidney cell cultures (MDCK), and (1S,2S)-16k was active against vesicular stomatitis virus and respiratory syncytial virus in HeLa cells (EC₅₀ = 9 and 12 μM, respectively). Moreover, compound (1R,2S)-16l showed activity against both herpes simplex viruses (HSV-1, HSV-2) in HEL cell cultures (EC₅₀ = 2.9 and 4 μM, respectively) and feline herpes virus in CRFK cells (EC₅₀ = 4 μM) but at the same time it exhibited cytotoxicity toward uninfected cell (MCC ⩾ 4 μM). Several other compounds have been found to inhibit proliferation of L1210, CEM as well as HeLa cells with IC₅₀ in the 4–50 μM range. Among them compounds (1S,2S)- and (1R,2S)-16l were the most active (IC₅₀ in the 4–7 μM range).     

Discovery of small molecules that inhibit melanogenesis via regulation of tyrosinase expression

5,6,7,8-Tetrahydro-4H-cyclohepta[d]isoxazole derivatives were synthesized and evaluated as a novel class of inhibitors for α-melanocyte-stimulating hormone (α-MSH) induced melanogenesis in a mouse melanoma B16F10 cell line. Compound 8e (IC₅₀ = 0.67 μM), 8h (IC₅₀ = 1.01 μM) and 9b (IC₅₀ = 0.99 μM) exhibited a potent inhibitory activity approximately 85- to 126-fold greater than kojic acid, a well-known potent inhibitor. A biochemical study indicates that the activity of this series should be displayed via down-regulation of the expression of tyrosinase.     

Synthesis and biological evaluation of indole derivatives as α-amylase inhibitor

A series of twenty indole hydrazone analogs (1–21) were synthesized, characterized by different spectroscopic techniques such as ¹H NMR and EI-MS, and screened for α-amylase inhibitory activity. All analogs showed a variable degree of α-amylase inhibition with IC₅₀ values ranging between 1.66 and 2.65 μM. Nine compounds that are 1 (2.23 ± 0.01 μM), 8 (2.44 ± 0.12 μM), 10 (1.92 ± 0.12 μM), 12 (2.49 ± 0.17 μM), 13 (1.66 ± 0.09 μM), 17 (2.25 ± 0.1 μM), 18 (1.87 ± 0.25 μM), 20 (1.83 ± 0.63 μM), and 19 (1.97 ± 0.02 μM) showed potent α-amylase inhibition when compared with the standard acarbose (1.05 ± 0.29 μM). Other analogs showed good to moderate α-amylase inhibition. The structure activity relationship is mainly focusing on difference of substituents on phenyl part. Molecular docking studies were carried out to understand the binding interaction of the most active compounds.     

Cajanol,a novel anticancer agent from Pigeonpea [Cajanus cajan (L.) Millsp.] roots,induces apoptosis in human breast cancer cells through a ROS-mediated mitochondrial pathway

Cajanol (5-hydroxy-3-(4-hydroxy-2-methoxyphenyl)-7-methoxychroman-4-one) is an isoflavanone from Pigeonpea [Cajanus cajan (L.) Millsp.] roots. As the most effective phytoalexin in pigeonpea, the cytotoxic activity of cajanol towards cancer cells has not been report as yet. In the present study, the anticancer activity of cajanol towards MCF-7 human breast cancer cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of cajanol, cell cycle distribution, DNA fragmentation assay and morphological assessment of nuclear change, ROS generation, mitochondrial membrane potential (ΔΨm) disruption, and expression of caspase-3 and caspase-9, Bax, Bcl-2, PARP and cytochrome c were measured in MCF-7 cells. Cajanol inhibited the growth of MCF-7 cells in a time and dose-dependent manner. The IC₅₀ value was 54.05 μM after 72 h treatment, 58.32 μM after 48 h; and 83.42 μM after 24 h. Cajanol arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of cajanol towards cancer cells in vitro.     

Morroniside cinnamic acid conjugate as an anti-inflammatory agent

A morroniside cinnamic acid conjugate was prepared and evaluated on E-selectin mediated cell–cell adhesion as an important role in inflammatory processes. 7-O-Cinnamoylmorroniside exhibited excellent anti-inflammatory activity (IC₅₀ = 49.3 μM) by inhibiting the expression of E-selectin; further, it was more active than another cinnamic-acid-conjugated iridoid glycoside (harpagoside; IC₅₀ = 88.2 μM), 7-O-methylmorroniside, and morroniside itself. As a result, 7-O-cinnamoylmorroniside was observed to be a potent inhibitor of TNF-α-induced E-selectin expression.     

Rapid in vitro micropropagation of Agapanthus praecox

In vitro micropropagation and acclimatization for the ornamental Agapanthus praecox, are reported. The influence of different growth regulators on shoot multiplication from shoot-tip explants of A. praecox was investigated. Prolific shoot multiplication (47.3 ± 1.96 shoots per explant) was achieved on Murashige and Skoog (MS) medium supplemented with 22.2 μM benzyladenine (BA), 2.9 μM indole-3-acetic acid (IAA), and 4.5 μM thidiazuron (TDZ). Shoots were rooted on half-strength MS basal medium supplemented with 5.7 μM IAA and 2.5 μM 2-isopentenyladenine (2iP) with 11.3 ± 0.78 roots per shoot. The in vitro-raised plants were established successfully in a 1:1 (v/v) vermiculite:sand mixture when maintained in a greenhouse with 100% survival. The elongated shoots (more than 5 cm in length) were treated for rooting and acclimatization in a moistened (5.7 μM IAA and 2.5 μM 2iP) vermiculite:sand (1:1 v/v) mixture, first in the misthouse and then in the greenhouse. Rooting and acclimatization was achieved simultaneously (100%) in the misthouse which was followed by greenhouse cultivation. This system can be used for rapid mass clonal propagation of A. praecox, for conservation strategies, commercial production, gene transformation studies and to produce phytomedicines.