Comparative application of biological and ninhydrin- derivatized spectrophotometric assays in the evaluation and validation of amikacin sulfate injection

Abstract Instrumental techniques are preferred over bioassay methods for antibiotic quantification mainly due to speed and ability to quantify metabolites in biological samples; however, the potency and biological activity of these drugs cannot be assessed. Two methods - agar well diffusion (bio-assay) and spectrophotometric methods were used to evaluate amikacin sulfate injection. Agar plates were inoculated with S. aureus inoculum; zones of inhibition from its susceptibility to amikacin were obtained, while spectrophotometric absorption at 650 nm of ninhydrin- derivatized amikacin in phosphate buffer (pH 8) was measured. Methods performance showed linearity from 1 - 16 µgmL-1 (bioassay, r = 0.9994) and 10-50 µgmL-1 (spectrophotometric, r = 0.9998). Molar absorptivity was 2.595 x 104 Lmol-1cm-1. Limits of detection and quantification were 1.07 and 3.24 µgmL-1 respectively for bioassay method, while corresponding values for spectrophotometric method were 0.98 and 2.97 µg mL-1. Relative standard deviations were ≤ 2.0% for both methods, with recoveries from 95.93 - 100.25%. Amikacin in brands ranged from 97.53 ± 2.68 to 100.84 ± 1.82%, student's t-test was ≤ 2.78 (n = 4) with respect to label claim for both methods. Experimental paired t-test (t = 2.07; n = 4) and F-test (F = 3.94; n = 4) values indicated no significant difference between both methods, hence comparable and can jointly be used in quality control assessment of antibiotics

Some bioactive potentials of two biflavanols isolated from Garcinia kola on cadmium-induced alterations of raw U937 cells and U937-derived macrophages.

OBJECTIVE: To investigate the abilities of two flavonoids - Garcinia biflavanol-1 (GB-1) and Garcinia biflavanol-2 (GB-2) from Garcinia kola (G. kola) in reducing cadmium-induced effects on raw U937 cells and U937-derived macrophages. METHODS: Macrophage U937 cells were incubated with cadmium followed by treatment with the flavonoids and cell viability assessed via trypan blue staining. In the other experiment, the U937 cells were transformed to the macrophage form and treated with cadmium in order to activate them. The cells were later incubated with the flavonoids and finally the supernatant of each cell culture was analysed for the secretion of nitric oxide, catalyse activity, and the release of tumour necrosis factor-alpha, interleukin-1 and interleukin-2 as indices of macrophage activation. Quercetin (a flavonol) was used as the reference flavonoid in all experiments. RESULTS: It revealed that the flavonoids significantly increased the viability of the cells and also reduced the cadmium-induced activation of the macrophage cells in a concentration-dependent manner. The flavanols GB-1 and GB-2 possessed higher activities than quercetin in all cases (P<0.05). Garcinia biflavanol-2 possessed a higher bioactivity than GB-1 significantly (P<0.05). CONCLUSIONS: In addition to corroborating the several reported importance of G. kola as a potential neutraceutical and pharmacological condiment, the study also clearly indicates the role hydroxylation especially at the 3'- position of polyphenols could play in enhancing bioactivities of flavonoids.

Hibiscus sabdariffa extractivities on cadmium-mediated alterations of human U937 cell viability and activation.

OBJECTIVE: To investigate the effect of the anthocyanin-rich extract of Hibiscus sabdariffa (H. sabdariffa) calyx on the viability of cadmium-treated U937 cells and cadmium-mediated activation of U937-derived macrophages. METHODS: The macrophage cell line U937 was treated with cadmium (0.1 µ mol/L) and later incubated with the anthocyanin-rich extract and cell viability was assessed via trypan blue staining. In the other experiment, the U937 cells were transformed to the macrophage form by treatment with phorbol 12, myristate 13, and acetate and incubated with cadmium (10 µ mol/L). The anthocyanin-rich extract was added to the cells later and subsequently, the supernatant of each cell culture was analysed for the production of tumour necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), nitric oxide, and catalase activity as indices for the activation of macrophages. RESULTS: It revealed that the anthocynanin-rich extract significantly (P < 0.05) increased the viability of the cells which was suppressed by cadmium when compared to quercetin dihydrate. The extract also reduced the cadmium-mediated production of the markers of macrophage-activation when compared to quercetin dihydrate. In both experiments, the activity of the extract was concentration-dependent (P < 0.05). CONCLUSIONS: The findings show that H. sabdariffa possesses significant immunoprotective effect. These corroborate the immense reported antioxidant and medicinal potential of the calyces of the plant which could be exploited for pharmacological and neutraceutical advantages.