# Modern Techniques for Flavonoid Extraction--To Optimize or Not to Optimize?
## 3 Sample Preparation and Hydrolysis
For example, for the determination of total flavonoid content in shoots of Brassicaceae, 30 mg of dehydrated samples per 1 mL of 80% methanol was used [55], while for fresh strawberries, using the same extraction solvent, 1 g per 10 mL of solvent was used [56]. Today, often deep eutectic solvents have been used for flavonoid extraction. For the extraction of flavonoids from wolfberries, 20:1 mg/mL [57] and for buckwheat sprouts, 100:1 mg/mL [58] of the ratio of extracted samples to solvent was used. \begin{table}
\begin{tabular}{c c c} \hline
**Sources** & **Drying Methods** & **Results** \\ \hline orange peel [47] & fresh, oven-dried and freeze-dried peel & freeze-drying preserves the concentration of the flavonoids, while oven-dried fixedenoids, while oven-dried peel presented a decrease of glycosylated flavonoids and an increase of aglycone forms \\ \hline _Helicteres hirsuta_ Lour. & hot-air drying, low-temperature-air drying, infrared drying and vacuum drying & the leaves dried under either hot-air drying at 80 \({}^{\circ}\)C, or vacuum drying at 50 \({}^{\circ}\)C yielded the highest amount of total flavonoid content \\ \hline blueberry, cherry, cranberry and strawberry [49] & hot-air drying, freeze-drying and refractance window-drying & higher levels of flavonoids were found in all the freeze-dried samples except strawberry \\ \hline _Sage_ (_Salvia officinalis_ L) [50] & air-drying at shade and ambient 22 \({}^{\circ}\)C, drying in a hot air oven with natural convection at 45 \({}^{\circ}\)C and 65 \({}^{\circ}\)C, drying in a microwave oven at 600 W/30 g and 800 W/30 g of fresh plant; drying in an infrared moisture analyzer at 45 \({}^{\circ}\)C and 65 \({}^{\circ}\)C. \\ \hline _Cosmos caudatus_ [51] & air-, oven- and freeze-drying & the freeze-drying and air drying were the best methods for flavonoids \\ \hline _Allium cpa_ red cv. and _A. sativum_ [52] & microwave, air-drying, or freeze-drying & microwave and freeze-dried samples show similar profile \\ \hline _Stevia rebuudiana_ leaves [53] & hot air-drying, freeze-drying and shade-drying & the majority of the compounds analyzed reached their maximum values with the freeze-drying method \\ \hline \end{tabular}
\end{table} 
Table 3: _Cont._Flavonoids are present in complex forms in matrices, and hydrolysis has often been used to remove sugar units from glycosides. Hydrolysis can be acidic, basic, or enzymatic [59]. For example, [PERSON] et al. [60] obtained the best results by refluxing at 80 \({}^{\circ}\)C for 2 h with 1.2 M HCl in 50% aqueous methanol with the addition of 2 mg ascorbic acid as an antioxidant. [PERSON] et al. [61] compared basic (1 g defatted flour was suspended in 25 mL MeOH:H\({}_{2}\)O 7:3 \(v\)/\(v\) (0.1 M NaOH); the solution was sonicated at 60 \({}^{\circ}\)C for 1 h, then CH\({}_{3}\)COOH was added until the pH was 6.5-7.0) and acid hydrolysis (1 g defatted flour was extracted with 25 mL MeOH:H2 SO4 9:1 \(v\)/\(v\); the solution was sonicated at 60\({}^{\circ}\)C for 2 h) were used for the extraction of total flavonoids from millet and reported that acid hydrolysis was able to extract the highest amount of total phenolic compounds, while basic hydrolysis underestimated the phenolic concentration. Sometimes enzymatic processes and fermentation are also used [41] before extraction to improve the extraction efficiency. Enzymatic hydrolysis prior to extraction can boost extraction efficiency by improving solvent dispersion, mass transfer, particle size reduction, and contact area [62]. [PERSON] et al. [63] and [PERSON], et al. [41] studied the potential of enzyme-assisted supercritical extraction of phenolic from _Medicago sativa_ leaves. The degradation of the plant material was enhanced using commercially available multi enzyme preparation containing five different enzymes: xylanase, beta-glucanase, cellulase, amylase and protease. The results showed that the extraction yield was about twice as high when enzymatic hydrolysis was performed before extraction.