Bioassays were developed as relative potency assays in which the activity of test samples was determined by comparison to an in-house reference standard.
An aliquot of the extract (final assay concentration of tested samples corresponded to the extracts obtained from 0.01, 0.03, 0.1 and 3 g dry weight of C. aquatica per mL) was evaporated to dryness and biological activity of the extracts was determined by a cress bioassay as described latter.
An aliquot of the extract (final assay concentration of tested samples corresponded to the extracts obtained from 0.3, 1, 3, 10, 30, 100 and 300 mg dry weight of B. brizantha shoots per mL) was evaporated to dryness, dissolved in a 0.2 mL of methanol and added to a sheet of filter paper (No. 2) in a 3-cm Petri dish.
The cytotoxicity effect of test samples was performed by 5-diphenyl-2H-tetrazolium bromide (MTT) assay [25].
For APTT assay, 25 μL of tested samples were mixed with 50 μL of citrated normal rabbit serum, and then APTT assay reagent was added.
Principle of this assay is to detect ability of test sample to reduce DPPH free radicals.
The assay was performed with 1.5 μl of test sample, after being zeroed with TRIS-EDTA (TE) buffer (pH 7.4).
Briefly for collagenase inhibition assay, 20 μl of tested sample was diluted with 50 μl buffer-solution (50 mM HEPES, 10 mM CaCl2, 0.05% Brij-35 and 1 mM DTNB in DMSO).
For elastase inhibition assay, 20 μl of tested sample was diluted with 65 μl buffer-solution containing 100 mM HEPES, 500 mM NaCl and 0.05% Tween20 in DMSO in a 96-well plate.
Validation of the 96-plex assay using independent test samples of known origin was successful; a subsequent survey of market samples revealed a high level of breed label conformity.
