4.2.2. Qualitative and Quantitative Analysis for Flavonoids and Phenolics

Qualitative analysis: The presence of flavonoids (alkaline reagent test) and phenolic (FeCl<sup>3</sup> test) compounds in the aqueous garlic extract was checked using a protocol published by Alsahli et al. with slight modifications [38].

Quantitative analysis of total phenol and flavonoids content: Total phenol content was estimated using Folin– Ciocalteau reagent as described previously with slight modifications [26]. Gallic acid (0–100 µg/mL) served as a reference point. The total phenolic content in garlic extract was determined from the calibration plot and expressed as mg gallic acid equivalents (GAE). All assays were performed in triplicate. Results are expressed as mg gallic acid equivalent per g dry extract.

Total phenolic content (TPC) = C × V/M

where C is the concentration of gallic acid in mg/mL that was obtained from gallic acid calibration curve, V is the volume of plant extract in mL, and M is the weight of pure plant extract in grams (g).

The aluminum chloride (AlCl3) colorimetric method was used to determine total flavonoid content in garlic extract as described previously [12,39]. The calibration curve was plotted using quercetin (0–250 µg/mL). Garlic extract (50 µg/mL) or standard quercetin solution was added to 2% AlCl<sup>3</sup> (500 µL). The resultant solution was incubated for 1 h with occasional stirring. The absorbance of reaction mixture was estimated using a spectrophotometer at 430 nm using ethanol as blank, as 2% AlCl<sup>3</sup> solution was prepared in ethanol. Total flavonoid content was determined to be quercetin equivalent (mg/g) (mg QUE/g).

Total flavonoid content (TFC) = Z × V/m

where Z is concentration of quercetin (mg/mL); V is volume (mL) of sample used in the extraction; m is weight of pure dried sample used (g).
