A UPLC-DAD-Based Bio-Screening Assay for the Evaluation of the Angiotensin Converting Enzyme Inhibitory Potential of Plant Extracts and Compounds: Pyrroquinazoline Alkaloids from Adhatoda vasica as a Case Study.

Angiotensin converting enzyme (ACE) plays a crucial role in regulating blood pressure in the human body. Identification of potential ACE inhibitors from medicinal plants supported the idea of repurposing these medicinal plants against hypertension. A method based on ultra-performance liquid chromatography (UPLC) coupled with a diode array detector (DAD) was used for the rapid screening of plant extracts and purified compounds to determine their ACE inhibitory activity. Hippuryl-histidiyl-leucine (HHL) was used as a substrate, which is converted into hippuric acid (HA) by the action of ACE. A calibration curve of the substrate HHL was developed with the linear regression 0.999. The limits of detection and quantification of this method were found to be 0.134 and 0.4061 mM, respectively. Different parameters of ACE inhibitory assay were optimized, including concentration, incubation time and temperature. The ACE inhibition potential of Adhatoda vasica (methanolic-aqueous extract) and its isolated pyrroquinazoline alkaloids, vasicinol (1), vasicine (2) and vasicinone (3) was evaluated. Compounds 1-3 were characterized by various spectroscopic techniques. The IC50 values of vasicinol (1), vasicine (2) and vasicinone (3) were found to be 6.45, 2.60 and 13.49 mM, respectively. Molecular docking studies of compounds 1-3 were also performed. Among these compounds, vasicinol (1) binds as effectively as captopril, a standard drug of ACE inhibition.

Flow Injection-High Resolution-Electrospray Ionization-Mass Spectrometry (FI-HR-ESI-MS) Method for the Screening of Antimicrobial Pharmaceutical Drugs and Compounds against Klebsiella pneumoniae.

The development of drug resistant microorganism is a global threat. Therefore, screening of more compounds for antimicrobial potentials is needed. Hence, a rapid method was developed for the screening of antimicrobial drugs and compounds against Klebsiella pneumoniae using Flow Injection Analysis-High Resolution-Mass Spectrometry. The method was optimized for bacterial culture time and concentration of drugs. IC50 values for the drugs were calculated from the percent intensity of 704.5207 m/z of K. pneumoniae at 5 hrs incubation. This mass was proposed as diacylglycerophosphoethanolamine and observed as a potential biomarker of K. pneumoniae for the evaluation of inhibition potential of antimicrobial drugs and compounds. The calculated values for half maximal inhibitory concentration of cefixime, gentamicin and enrofloxacin were 0.052, 0.028 and 0.042 µg/mL, respectively. Ten compounds were also screened against the developed method, among them one compound (RSE-6) was found to be active with IC50 value of 45.08 µg/mL. The obtained results were further compared with MIC values, obtained from micro dilution and Alamar blue assay after 24 hrs incubation. In comparison to these methods, developed method is sensitive, reproducible, rapid and robust for the determination of IC50 value or inhibition potential of the drugs and compounds even at early incubation period of 5 hours.

Screening of inhibitors of angiotensin-converting enzyme (ACE) employing high performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (HPLC-ESI-QqQ-MS).

Angiotensin-converting enzyme (ACE) plays a key role in regulating blood pressure in the body by converting the angiotensin I (AI) into angiotensin II (AII). Angiotensin II is a potent vaso-active peptide that causes arterioles to constrict, resulting in increased blood pressure. A rapid and sensitive method for the identification of inhibitors of ACE was developed, and optimized employing HPLC-ESI-QqQ-MS. In this assay, angiotensin I substrate was converted into the product angiotensin II with the catalytic action of ACE. A calibration curve for depleting concentration of angiotensin I was developed and linearity of R2=0.999 with a remarkably low concentration of substrate range 20-200nM. The limit of detection and quantification of angiotensin I was found to be 1.93 and 5.84nM, respectively. The enzymatic reaction was optimized for incubation time, concentration, and volume of enzyme and substrate. All reactions were performed at 37°C at pH7.5 with standard incubation time of 20min. Two standard inhibitors, Captopril and Lisinopril, were checked through the newly developed method for their inhibitory potential, and their IC50 values were found to be 3.969 and 0.852µM, respectively. Reproducibility and precision analysis of different experiments showed <9.9% RSD. The developed method can be used for the identification of new ACE inhibitors.

Photodegradation of moxifloxacin in aqueous and organic solvents: a kinetic study.

The kinetics of photodegradation of moxifloxacin (MF) in aqueous solution (pH 2.0-12.0), and organic solvents has been studied. MF photodegradation is a specific acid-base catalyzed reaction and follows first-order kinetics. The apparent first-order rate constants (kobs) for the photodegradation of MF range from 0.69 × 10(-4) (pH 7.5) to 19.50 × 10(-4) min(-1) (pH 12.0), and in organic solvents from 1.24 × 10(-4) (1-butanol) to 2.04 × 10(-4) min(-1) (acetonitrile). The second-order rate constant (k2) for the [H(+)]-catalyzed and [OH(-)]-catalyzed reactions are 6.61 × 10(-2) and 19.20 × 10(-2) M(-1) min(-1), respectively. This indicates that the specific base-catalyzed reaction is about three-fold faster than that of the specific acid-catalyzed reaction probably as a result of the rapid cleavage of diazabicyclononane side chain in the molecule. The kobs-pH profile for the degradation reactions is a V-shaped curve indicating specific acid-base catalysis. The minimum rate of photodegradation at pH 7-8 is due to the presence of zwitterionic species. There is a linear relation between kobs and the dielectric constant and an inverse relation between kobs and the viscosity of the solvent. Some photodegraded products of MF have been identified and pathways proposed for their formation in acid and alkaline solutions.