R. Srinivas

Garlic Attenuates Cardiac Oxidative Stress via Activation of PI3K/AKT/Nrf2-Keap1 Pathway in Fructose-Fed Diabetic Rat

Background Cardiovascular complication due to diabetes has remained a major cause of death. There is an urgent need to intervene the cardiac complications in diabetes by nutritional or pharmacological agents. Thus the present study was designed to find out the effectiveness of garlic on cardiac complications in insulin-resistant diabetic rats. Methods and Results SD rats were fed high fructose (65%) diet alone or along with raw garlic homogenate (250 mg/kg/day) or nutrient-matched (65% corn starch) control diet for 8 weeks. Fructose-fed diabetic rats showed cardiac hypertrophy, increased NFkB activity and increased oxidative stress. Administration of garlic significantly decreased (p<0.05) cardiac hypertrophy, NFkB activity and oxidative stress. Although we did not observe any changes in myocardial catalase, GSH and GPx in diabetic heart, garlic administration showed significant (p<0.05) increase in all three antioxidant/enzymes levels. Increased endogenous antioxidant enzymes and gene expression in garlic treated diabetic heart are associated with higher protein expression of Nrf2. Increased myocardial H2S levels, activation of PI3K/Akt pathway and decreased Keap levels in fructose-fed heart after garlic administration might be responsible for higher Nrf2 levels. Conclusion Our study demonstrates that raw garlic homogenate is effective in reducing cardiac hypertrophy and fructose-induced myocardial oxidative stress through PI3K/AKT/Nrf2-Keap1 dependent pathway.

Naringin ameliorates gentamicin-induced nephrotoxicity and associated mitochondrial dysfunction, apoptosis and inflammation in rats: Possible mechanism of nephroprotection

Gentamicin-induced nephrotoxicity has been well documented, although its underlying mechanisms and preventive strategies remain to be investigated. The present study was designed to investigate the protective effect of naringin, a bioflavonoid, on gentamicin-induced nephrotoxicity and to elucidate the potential mechanism. Serum specific renal function parameters (blood urea nitrogen and creatinine) and histopathology of kidney tissues were evaluated to assess the gentamicin-induced nephrotoxicity. Renal oxidative stress (lipid peroxidation, protein carbonylation, enzymatic and non-enzymatic antioxidants), inflammatory (NF-kB [p65], TNF-α, IL-6 and MPO) and apoptotic (caspase 3, caspase 9, Bax, Bcl-2, p53 and DNA fragmentation) markers were also evaluated. Significant decrease in mitochondrial NADH dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and mitochondrial redox activity indicated the gentamicin-induced mitochondrial dysfunction. Naringin (100mg/kg) treatment along with gentamicin restored the mitochondrial function and increased the renal endogenous antioxidant status. Gentamicin induced increased renal inflammatory cytokines (TNF-α and IL-6), nuclear protein expression of NF-κB (p65) and NF-κB-DNA binding activity and myeloperoxidase (MPO) activity were significantly decreased upon naringin treatment. In addition, naringin treatment significantly decreased the amount of cleaved caspase 3, Bax, and p53 protein expression and increased the Bcl-2 protein expression. Naringin treatment also ameliorated the extent of histologic injury and reduced inflammatory infiltration in renal tubules. U-HPLS-MS data revealed that naringin co-administration along with gentamicin did not alter the renal uptake and/or accumulation of gentamicin in kidney tissues. These findings suggest that naringin treatment attenuates renal dysfunction and structural damage through the reduction of oxidative stress, mitochondrial dysfunction, inflammation and apoptosis in the kidney.

Identification and characterization of stressed degradation products of metoprolol using LC/Q‐TOF‐ESI‐MS/MS and MSn experiments

A rapid, specific and reliable isocratic high-performance liquid chromatography combined with quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed and validated for the identification and characterization of stressed degradation products of metoprolol. Metoprolol, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified conditions. The drug showed extensive degradation under oxidative and hydrolysis (acid and base) stress conditions. However, it was stable to thermal, neutral and photolysis stress conditions. A total of 14 degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on a C(18) column (4.6 × 250 mm, 5 µm). To characterize degradation products, initially the mass spectral fragmentation pathway of the drug was established with the help of MS/MS, MS(n) and accurate mass measurements. Similarly, fragmentation pattern and accurate masses of the degradation products were established by subjecting them to LC-MS/QTOF analysis. Structure elucidation of degradation products was achieved by comparing their fragmentation pattern with that of the drug. The degradation products DP(2) (m/z 153) and DP(14) (m/z 236) were matched with impurity B, listed in European Pharmacopoeia and British Pharmacopoeia, and impurity I, respectively. The LC-MS method was validated with respect to specificity, linearity, accuracy and precision.

Baicalein alleviates doxorubicin-induced cardiotoxicity via suppression of myocardial oxidative stress and apoptosis in mice.

AIMS Doxorubicin is a widely used anthracycline derivative anticancer drug. Unfortunately, the clinical use of doxorubicin has the serious drawback of cardiotoxicity. In this study, we investigated whether baicalein, a bioflavonoid, can prevent doxorubicin-induced cardiotoxicity in vivo and we delineated the possible underlying mechanisms. MAIN METHODS Male BALB/c mice were treated with either intraperitoneal doxorubicin (15 mg/kg divided into three equal doses for 15 days) and/or oral baicalein (25 and 50 mg/kg for 15 days). Serum markers of cardiac injury, histology of heart, parameters related to myocardial oxidative stress, apoptosis and inflammation were investigated. KEY FINDINGS Treatment with baicalein reduced doxorubicin-induced elevation of serum creatine kinase-MB isoenzyme (CK-MB), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and ameliorated the histopathological damage. Baicalein restored the doxorubicin-induced decrease in both enzymatic and non-enzymatic myocardial antioxidants and increased the myocardial expression of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Further studies showed that baicalein could inverse the Bax/Bcl-2 ratio, suppress doxorubicin-induced p53, cleaved caspase-3 and PARP expression and prevented doxorubicin-induced DNA damage. Baicalein treatment also interferes with doxorubicin-induced myocardial NF-κB signaling through inhibition of IκBα phosphorylation and nuclear translocation of p65 subunit. Doxorubicin elevated iNOS and nitrites levels were also significantly decreased in baicalein treated mice. However, we did not find any significant change (p>0.05) in the myocardial TNF-α and IL-6 levels in control and treated animals. SIGNIFICANCE Our finding suggests that baicalein might be a promising molecule for the prevention of doxorubicin-induced cardiotoxicity.

In vivo metabolic investigation of moxifloxacin using liquid chromatography/electrospray ionization tandem mass spectrometry in combination with online hydrogen/deuterium exchange experiments.

RATIONALE Tuberculosis is a leading cause of death from an infectious disease and moxifloxacin is an effective drug as compared to other fluoroquinolones. To date only two metabolites of the drug are known. Therefore, the present study on characterization of hitherto unknown in vivo metabolites of moxifloxacin using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is undertaken. METHODS In vivo metabolites of moxifloxacin have been identified and characterized by using LC/ESI-MS/MS in combination with an online hydrogen/deuterium (H/D) exchange technique. To identify in vivo metabolites, blood, urine and faeces samples were collected after oral administration of moxifloxacin to Sprague-Dawley rats. The samples were prepared using an optimized sample preparation approach involving protein precipitation, liquid-liquid extraction followed by solid-phase extraction and LC/MS/MS analysis. RESULTS A total of nine phase I and ten phase II metabolites of moxifloxacin have been identified in urine samples including N-sulphated, glucuronide and hydroxylated metabolites which are also observed in plasma samples. In faeces samples, only the N-sulphated metabolite is observed. The structures of metabolites have been elucidated based on fragmentation patterns, accurate mass measurements and online H/D exchange LC/MS/MS experiments. Online H/D exchange experiments are used to support the identification and structural characterization of drug metabolites. CONCLUSIONS A total of 19 in vivo metabolites of moxifloxacin have been characterized using LC/ESI-MS/MS in combination with accurate mass measurements and online H/D exchange experiments. The main phase I metabolites of moxifloxacin are hydroxylated, decarbonylated, desmethylated and desmethylhydroxylated metabolites which undergo subsequent phase II glucuronidation pathways.

Diisopropylaminoisocyanide and DMAD in multiple component reactions (MCRs): novel synthesis of substituted 1-amino-3-pyrrolin-2-ones by reaction with aldehydes and dicarbonyl compounds

The zwitterion generated from diisopropylaminoisocyanide and dimethyl acetylenedicarboxylate (DMAD) reacts with a variety of carbonyl and dicarbonyl compounds affording substituted 1-aminopyrrolin-2-ones.

Identification of hydrolytic and isomeric N-oxide degradants of vilazodone by on line LC–ESI–MS/MS and APCI–MS

The present study reports the degradation behavior of a new antidepressant drug, vilazodone, under various stress conditions as per International Conference on Harmonization guidelines (ICH, Q1A(R2). The investigation involved monitoring decomposition of the drug under hydrolytic (acidic, basic and neutral), oxidative, photolytic and thermal stress conditions and identifying degradation products. A rapid, precise, accurate and robust ultra high performance liquid chromatography (UPLC) method has been developed on a Waters CSH Phenyl-Hexyl column (100 mm × 2.1 mm, 1.7 μm) using gradient elution of 10mM ammonium acetate buffer (pH 5.0) and acetonitrile as mobile phase. The drug was found to be degraded in hydrolytic (acidic and basic) and oxidative conditions, whereas it was stable under neutral hydrolytic, photolytic and thermal stress conditions. The method was extended to quadrupole time-of-flight mass spectrometry (QTOF-MS) for the structural characterization of degradation products. It has been observed that isomeric N-oxide degradation products were formed under oxidative stress condition. The exact location of N-oxidation in the drug was investigated using atmospheric pressure chemical ionization (APCI) due to the formation of characteristic fragment ions. These fragment ions resulted from Meisenheimer rearrangement owing to thermal energy activation at the vaporizer of APCI source. All degradation products were comprehensively characterized by UPLC-ESI-MS/MS and UPLC-APCI-MS experiments. The most probable mechanisms for the formation of degradation products have also been proposed. The method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per ICH guidelines.

Differentiation of Positional Isomers of Hybrid Peptides Containing Repeats of β-Nucleoside Derived Amino Acid (β-Nda-) and L-Amino Acids by Positive and Negative Ion Electrospray Ionization Tandem Mass Spectrometry (ESI-MSn)

A new class of positional isomeric pairs of -Boc protected oligopeptides comprised of alternating nucleoside derived β-amino acid (β-Nda-) and L-amino acid residues (alanine, valine, and phenylalanine) have been differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-MSn). The protonated dipeptide positional isomers with β-Nda- at the N-terminus lose CH3OH, NH3, and C2H4O2, whereas these processes are absent for the peptides with L-amino acids at the N-terminus. Instead, the presence of L-amino acids at the N-terminus results in characteristic retro-Mannich reaction involving elimination of imine. A good correlation has been observed between the conformational structure of the peptides and the abundance of yn+ and bn+ ions in MSn spectra. In the case of tetrapeptide isomers that are reported to form helical structures in solution phase, no yn+ and bn+ ions are observed when the corresponding amide -NH- participates in the helical structures. In contrast, significant yn+ and bn+ ions are formed when the amide -NH- is not involved in the H-bonding. In the case of tetra- and hexapeptides, it is observed that abundant bn+ ions are formed, presumably with stable oxazolone structures when the C-terminus of the bn+ ions possessed L-amino acid and the β-Nda- at the C-terminus appears to prevent the cyclization process leading to the absence of corresponding bn+ ions.

Differentiation of three pairs of Boc-β,γ- and γ,β-hybrid peptides by electrospray ionization tandem mass spectrometry

A new series of Boc-N-beta(3), gamma(4)-/gamma(4), beta(3)-isomeric hybrid peptides (containing repeats of beta(3)-Caa and gamma(4)-Caas, Caa = C-linked carbo beta(3)-/gamma(4)-amino acids derived from D-xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion-trap and high resolution quadrupole time-of-flight/tandem mass spectrometry (Q-TOF MS/MS). MS(n) of protonated isomeric peptides and [M+H-Boc+H](+) produce characteristic fragmentation involving the peptide backbone, the Boc-group, and the side chain. The positional isomers are differentiated from one another by the presence of y(n)(+), b(n)(+), and other fragment ions of different m/z values. It is observed that the peptides with beta-Caa at the N-terminus produce extensive fragmentation, whereas gamma-Caa gave rise to much less fragmentation. Peptides with gamma-Caa at the N-terminus lose NH(3), whereas this process is absent for the carbopeptides with beta-Caa at the N-terminus. Two pairs of dipeptide diastereomers are clearly differentiated by the collision-induced dissociation (CID) of their protonated molecules. The loss of 2-methylprop-1-ene is more pronounced for Boc-NH-(R)-beta-Caa-(R)-gamma-Caa-OCH(3) (6) and Boc-NH-(R)-gamma-Caa-(R)-beta-Caa-OCH(3) (12), whereas it is insignificant or totally absent for its protonated diastereomeric pair Boc-NH-(S)-beta-Caa-(S)-gamma-Caa-OCH(3) (1) and Boc-NH-(S)-gamma-Caa-(S)-beta-Caa-OCH(3) (7). Further, ESI negative ion tandem mass spectrometry has also been found to be useful for differentiating these isomeric peptide acids.

Identification of forced degradation products of tamsulosin using liquid chromatography/electrospray ionization tandem mass spectrometry.

A rapid and gradient high-performance liquid chromatography combined with quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed for the identification and structural characterization of stressed degradation products of tamsulosin. Tamsulosin, a selective α1-adrenoceptor antagonist, was subjected to forced degradation studies under hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress conditions as per ICH guidelines Q1A (R2). The drug degraded significantly under hydrolytic (base and neutral), thermal, oxidative and photolytic conditions, while it was stable to acid hydrolytic stress conditions. A total of twelve degradation products were formed and the chromatographic separation of the drug and its degradation products were achieved on a GRACE C-18 column (250mm×4.6mm, 5μm). All the degradants have been identified and characterized by LC/ESI-MS/MS and accurate mass measurements. To elucidate the structures of degradation products, fragmentation of the [M+H](+) ions of tamsulosin and its degradation products was studied by using LC-MS/MS experiments combined with accurate mass measurements. The product ions of all the protonated degradation products were compared with the product ions of protonated tamsulosin to assign most probable structures for the observed degradation products.