Synthesis of novel rapanone derivatives via organocatalytic reductive C-alkylation: biological evaluation of antioxidant properties, in vivo zebrafish embryo toxicity, and docking studies.

A biologically crucial natural product rapanone 1 was isolated from Embelia ribes at the gram scale with excellent purity. Semi-synthetic analogs of 1 semi-synthesized through reductive C-alkylation could increase the therapeutic value of the compounds. Herein, a new synthetic methodology was developed as a single-step reductive C-alkylation protocol using a metal-free, room-temperature-based reaction condition that can be scaled up to gram-scale synthesis with an excellent yield of up to 93%. A straightforward purification protocol was employed for the product obtained by this method. The derivatives of 1 showed antioxidant activity, which was evaluated using DPPH and ABTS scavenging assays. Compounds 5a-5ze showed an IC50 value of 2.48-3.37 µM and 1.81-3.12 µM. Substitution by electron-donating groups on the quinone moiety seems to play an essential role in the increased antioxidant activity of compounds 5a-5i, 5v, 5w, 5zc, and 5z. Further, the in vivo embryotoxicity of 1 and its derivatives was analyzed in a zebrafish-based aquatic toxicology model. Zebrafish embryos were exposed to 1 and 5a-5ze at 20 to 160 µM concentrations. They showed reduced toxicity and a survival rate of 90-98% after 96 hpf of treatment; similarly, the compounds 5a-5i, 5v, 5w, 5zc, and 5zd did not significantly affect the hatching rates of 75.66-85.33% or developmental abnormalities of the embryos after 48 hpf of treatment. In silico molecular docking studies for the parent compound, along with its derivatives 5a-5i, 5v-5w, 5zc-5zd, and standard l-ascorbic acid (l-Aa) indicated favorable interactions with the active site of the crystal structure, coupled with the assay protein PDB:1ZB6, which was responsible for the observed biological understanding and potential.

Iodo-sulphonylation of 1,6-enynones: a metal-free strategy to synthesize N-substituted succinimides.

An iodine-mediated radical cyclization of 1,6-enynones with sulphonyl hydrazides using tert-butyl hydroperoxide (TBHP) as the oxidant has been developed for the synthesis of iodo-sulphonylated-succinimide derivatives. The notable advantages of the developed method are metal-free conditions, broad functional group tolerance, column chromatography-free purification, high stereoselectivity (E isomer), shorter reaction times, and the cascade construction of three new bonds (C-S, C-I, and C-C). The synthetic application of the iodo-functionality has been extended to the Heck coupling reaction with acrylonitrile and to the Suzuki coupling reaction with benzene boronic acid.

Cholecalciferol and metformin protect against lipopolysaccharide-induced endothelial dysfunction and senescence by modulating sirtuin-1 and protein arginine methyltransferase-1.

Endothelial cell activation through nuclear factor-kappa-B (NFkB) and mitogen-activated protein kinases leads to increased biosynthesis of pro-inflammatory mediators, cellular injury and vascular inflammation under lipopolysaccharide (LPS) exposure. Recent studies report that LPS up-regulated global methyltransferase activity. In this study, we observed that a combination treatment with metformin (MET) and cholecalciferol (VD) blocked the LPS-induced S-adenosylmethionine (SAM)-dependent methyltransferase (SDM) activity in Eahy926 cells. We found that LPS challenge (i) increased arginine methylation through up-regulated protein arginine methyltransferase-1 (PRMT1) mRNA, intracellular concentrations of asymmetric dimethylarginine (ADMA) and homocysteine (HCY); (ii) up-regulated cell senescence through mitigated sirtuin-1 (SIRT1) mRNA, nicotinamide adenine dinucleotide (NAD+) concentration, telomerase activity and total antioxidant capacity; and (iii) lead to endothelial dysfunction through compromised nitric oxide (NOx) production. However, these LPS-mediated cellular events in Eahy926 cells were restored by the synergistic effect of MET and VD. Taken together, this study identified that the dual compound effect inhibits LPS-induced protein arginine methylation, endothelial senescence and dysfunction through the components of epigenetic machinery, SIRT1 and PRMT1, which is a previously unidentified function of the test compounds. In silico results identified the presence of vitamin D response element (VDRE) sequence on PRMT1 suggesting that VDR could regulate PRMT1 gene expression. Further characterization of the cellular events associated with the dual compound challenge, using gene silencing approach or adenoviral constructs for SIRT1 and/or PRMT1 under inflammatory stress, could identify therapeutic strategies to address the endothelial consequences in vascular inflammation-mediated atherosclerosis.

Pharmacological inhibition of guanosine triphosphate cyclohydrolase1 elevates tyrosine phosphorylation of caveolin1 and cellular senescence.

The role of 2,4-diamino-6-hydroxypyrimidine (DAHP), on cellular-senescence remains unclear as differential effects of DAHP have been reported in cardiovascular and cerebrovascular systems. We investigated the effect of pharmacologically-induced guanosine-triphosphate-cyclohydrolase1 (GTPCH1)-inhibition, through DAHP, on cellular-senescence in experimentally-induced diabetic and non-diabetic Wistar rats. Cellular-senescence was evaluated through senescence-associated events, namely, cell-cycle-arrest of peripheral blood mononuclear cells (PBMNCs); myocardial DNA fragmentation, total antioxidant capacity (TAC), telomerase-activity, nicotinamide adenine dinucleotide (NAD+)-content and tyrosine14-phosphorylation of caveolin1 (pY14) in similarly-aged, pubertal Wistar rats with streptozotocin (STZ) and/or DAHP. Oxidative stress (OS) indices such as myocardial biopterin concentrations (tetrahydrobiopterin-BH4 and dihydrobiopterin-BH2) and plasma total nitrite and nitrate (NOx) were determined. DAHP, per se, exhibited distinct senescence; in addition, in STZ+DAHP (the cardiomyopathy model), there was a marked accumulation of cells in G0G1 phase, as evidenced through flow-cytometry analysis, as-well-as fragmented DNA, than the respective controls suggesting the DAHP-mediated onset of senescence in circulating cells and the myocardium, with or without STZ. Concentrations of BH4 and BH2, and NOx were impaired in STZ and/or DAHP, indicating elevated OS in the treatment groups. In the independent treatment groups or the combination treatment, typical senescence indicators including myocardial telomerase-activity, NAD+-content and TAC were significantly reduced, while there was a marked elevation in the concentrations of pY14 as compared to the respective controls, reinforcing the occurrence of senescence in PBMNCs and the myocardium. We postulate that DAHP promotes early onset of cellular-senescence, potentially through OS-mediated cellular events in diabetic or non-diabetic models.

Effect of Fadrozole Exposure on Socioreproductive Behaviors and Neurochemical Parameters in Betta splendens.

Induction of all-male population in Siamese fighting fish, Betta splendens, has potential application in ornamental fish trade. In addition, the sexually dimorphic nature of aggressive behavior exhibited by this species has made it into an emerging model for behavioral studies. Fadrozole, an aromatase inhibitor, which has been used widely in masculinization, has captivated us to use it in this study. Twenty one days postfertilization (dpf), B. splendens fry were subjected to discrete immersion treatment with various concentrations of fadrozole, and eventually, analyses of various socioreproductive behaviors and analyses of stress markers such as dopamine in brain samples, sex hormones, cortisol, and glucose in plasma samples were performed. We observed that 91% of 50 µg/L fadrozole treated fish developed as males. Interestingly, reproductive analyses of these males gave rise to two subgroups (A and B). Subsequent sociobehavioral analyses demonstrated a timid and subdued behavior in subgroup B males. Furthermore, estimation of stress markers such as dopamine levels in the brain tissue, cortisol, and glucose levels in blood plasma and sex hormone levels in blood plasma exhibited an endocrine disruption-mediated stress leading to altered behavior in these males. These findings will help in understanding the fadrozole-mediated masculinization and behavioral alterations following endocrine disruption.

Forskolin attenuates doxorubicin-induced accumulation of asymmetric dimethylarginine and s-adenosylhomocysteine via methyltransferase activity in leukemic monocytes.

Doxorubicin (DOX) is an antitumor drug, associated with cardiomyopathy. Strategies to address DOX-cardiomyopathy are scarce. Here, we identify the effect of forskolin (FSK) on DOX-induced-asymmetric-dimethylarginine (ADMA) accumulation in monocytoid cells. DOX-challenge led to i) augmented cytotoxicity, reactive-oxygen-species (ROS) production and methyltransferase-enzyme-activity identified as ADMA and s-adenosylhomocysteine (SAH) accumulation (SAH-A). However, except cytotoxicity, other DOX effects were decreased by metformin and FSK. FSK, did not alter the DOX-induced cytotoxic effect, but, decreased SAH-A by >50% and a combination of three drugs restored physiological methyltransferase-enzyme-activity. Together, protective effect of FSK against DOX-induced SAH-A is associated with mitigated methyltransferase-activity, a one-of-a-kind report.

Identification of beta-sitosterol and stigmasterol in Bambusa bambos (L.) Voss leaf extract using HPLC and its estrogenic effect in vitro.

Focus of the study is to identify a safe alternate to Hormone Replacement Therapy by identifying the presence of ß-sitosterol and stigmasterol in the hydroalcoholic extract of Bambusa bambos using HPTLC and RP-HPLC-PDA; by evaluating the estrogenic effects of extract containing ß-sitosterol and stigmasterol on the growth of MCF-7 cells in vitro. Plant material was identified by DNA sequencing analysis. Presence of ß-sitosterol and stigmasterol was confirmed by HPTLC and direct RP-HPLC-PDA. Peaks with retention time about 19.13 and 21.16min were found to be stigmasterol and ß-sitosterol in extract. Extract was not cytotoxic to MCF-7 cells in any of the dilutions. It induced cell proliferation and all the dilutions except <500µg/ml have significantly increased cell multiplication. 15.6, 31.2, 62.5 and 125µg/ml of HEBB have shown influence on the proliferation rates similar to the standard 17ß-estradiol. The results suggest that HEBB might be used as a safe alternative to estrogen replacement therapies.

Indirubin potentiates ciprofloxacin activity in the NorA efflux pump of Staphylococcus aureus.

Staphylococcus aureus is a common multidrug-resistant organism in hospital-acquired infections, and the NorA efflux pump mechanism facilitates resistance to quinone compounds. In India, Wrightia tinctoria R. Br. leaves have traditionally been used to treat skin diseases and have been explored for antibacterial and efflux pump inhibition (EPI) compounds. In this study, indirubin isolated from the chloroform extract of W. tinctoria R. Br. leaves was tested for its antibacterial activity against clinically important Gram-positive and Gram-negative bacteria and the minimum inhibitory concentration (MIC) was determined using broth microdilution. The EPI properties of indirubin were investigated using Staphylococcus aureus SA1199B, and its synergistic effects were tested with ciprofloxacin. Indirubin showed antibacterial activity against both the type strain and drug-resistant S. aureus; the MIC was determined to be 12.5 mg/l for S. aureus and 25 mg/l for Staphylococcus epidermidis. However, it synergistically (fractional inhibitory concentration index = 0.45) potentiated the activity of ciprofloxacin, probably by inhibiting the NorA efflux pump. Indirubin exhibited EPI activity nearly comparable to that of reserpine by 4-fold reduction in ciprofloxacin MIC. Our results suggest that the natural compound indirubin could be used in future therapeutic applications as a potential EPI.