Synthesis and characterization of mercapturic acid (N-acetyl-L-cysteine)-aflatoxin B1 adduct and its quantitation in rat urine by an enzyme immunoassay.

A simple and sensitive indirect noncompetitive enzyme immunoassay to quantitate mercapturic acid-aflatoxin B1 (AFB1) adduct in rat urine is reported. A novel procedure was developed for in vitro synthesis of an immunogen, bovine serum albumen-glutathione-aflatoxin B1 (BSA-GSH-AFB1) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Sulphydryl group's analysis confirmed the conjugation of-SH groups to AFB1. Thin-layer chromatography and spectral analysis (absorption, fluorescence, and Fourier transform infrared) of the conjugates further confirmed the formation of the adducts. Polyclonal antibodies specific to mercapturic acid-AFB1 adduct were produced against BSA-GSH-AFB1. The assay was found to be linear in the range of 100 pg-100 ng of the analyte (y = a + bx). A 50% displacement of BSA-GSH-AFB1 antibodies was achieved at an inhibitory concentration (IC50) of 11.9 ng GSH-AFB1 (r2 = 0.98) and 1.22 ng N-acetyl-L-cysteine (NAC)-AFB1 (r2 = 0.98). Spiking 5 microg/mL of reference standard to the control rat urine showed a recovery of 98 +/- 2%. The immunoassay was validated in a rodent model exposed to a single oral dose of 1 mg/kg body mass of pure AFB1. The excretion of NAC-AFB1 adduct was quantitated at the end of 24 h. The concentration of the NAC-AFB1 adduct excreted in urine as determined by the immunoassay was found to be in the range of 3.22-5.97 microg/mg creatinine. The present method may find wide application as a biochemical tool in molecular epidemiological and intervention studies with respect to human exposure to dietary aflatoxins.

Development and evaluation of thin-layer chromatography-digital image-based analysis for the quantitation of the botanical pesticide azadirachtin in agricultural matrixes and commercial formulations: comparison with ELISA.

A simple thin-layer chromatography-digital image-based analytical method has been developed for the quantitation of the botanical pesticide, azadirachtin. The method was validated by analyzing azadirachtin in the spiked food matrixes and processed commercial pesticide formulations, using acidified vanillin reagent as a postchromatographic derivatizing agent. The separated azadirachtin was clearly identified as a green spot. The Rf value was found to be 0.55, which was similar to that of a reference standard. A standard calibration plot was established using a reference standard, based on the linear regression analysis [r2 = 0.996; y = 371.43 + (634.82)x]. The sensitivity of the method was found to be 0.875 microg azadirachtin. Spiking studies conducted at the 1 ppm (microg/g) level in various agricultural matrixes, such as brinjal, tomato, coffee, and cotton seeds, revealed the recoveries of azadirachtin in the range of 67-92%. Azadirachtin content of commercial neem formulations analyzed by the method was in the range of 190-1825 ppm (microg/mL). Further, the present method was compared with an immunoanalytical method enzyme-linked immonosorbent assay developed earlier in our laboratory. Statistical comparison of the 2 methods, using Fischer's F-test, indicated no significant difference in variance, suggesting that both methods are comparable.

Antibacterial activity of biogenic silver nanoparticles synthesized with gum ghatti and gum olibanum: a comparative study.

Presently, silver nanoparticles produced by biological methods have received considerable significance owing to the natural abundance of renewable, cost-effective and biodegradable materials, thus implementing the green chemistry principles. Compared with the nanoparticles synthesized using chemical methods, most biogenic silver nanoparticles are protein capped, which imparts stability and biocompatibility, and enhanced antibacterial activity. In this study, we compared the antibacterial effect of two biogenic silver nanoparticles produced with natural plant gums: gum ghatti and gum olibanum against Gram-negative and Gram-positive bacteria. Bacterial interaction with nanoparticles was probed both in planktonic and biofilm modes of growth; employing solid agar and liquid broth assays for inhibition zone, antibiofilm activity, inhibition of growth kinetics, leakage of intracellular contents, membrane permeabilization and reactive oxygen species production. In addition, cytotoxicity of the biogenic nanoparticles was evaluated in HeLa cells, a human carcinoma cell line. Antibacterial activity and cytotoxicity of the silver nanoparticles synthesized with gum ghatti (Ag NP-GT) was greater than that produced with gum olibanum (Ag NP-OB). This could be attributed to the smaller size (5.7 nm), monodispersity and zeta potential of the Ag NP-GT. The study suggests that Ag NP-GT can be employed as a cytotoxic bactericidal agent, whereas Ag NP-OB (7.5 nm) as a biocompatible bactericidal agent.