# Extraction of Bioactive Compounds from _Ulva lactuca_
## 2 Materials and Methods
### Methods
#### 2.3.2 Solvent Extractions  
Pure ethanol, water, and ethyl acetate, as well as a mixture of ethanol/water 70:30 (_v/v_) were investigated for their efficiency in the extraction of carotenoids and phenolics from _U. lactuca_ biomass. For the solvent screening, preliminary extractions were conducted. Specifically, 100 mL of each solvent was added to 10 g biomass and the extractions were carried out under continuous magnetic stirring in double-walled glass vessels for 12 h. A thermostat was employed to control the extraction temperature which was set at 25 \({}^{\circ}\)C.  
Using the same experimental setup, the effect of some of the most influential extraction parameters, namely time, temperature, and the ratio of biomass to solvent, was investigated using the ethanol/water mixture as a solvent. The extraction conditions are presented in detail in Table 1. After the completion of each extraction, the extract was collected via centrifugation at 4000 rpm for 15 min. Subsequently, total phenolics, total carotenoids, antioxidant activity of the extracts, and the extraction yield were measured. Determination of Total Phenolics Content (TPC)  
Total phenolics content in _U. lactuca_ extracts was estimated spectrophotometrically using Folin-Ciocalteu reagent as described by [PERSON] et al., 1965 [27]. Specifically, 7.9 mL of distilled water and 0.1 mL of extract were transferred in glass vials and the mixture was homogenized by vortexing. Afterwards, the addition of 0.5 mL of Folin-Ciocalteu reagent and homogenization were realized, followed by the addition of 1.5 mL (20% _w/v_) Na\({}_{2}\)CO\({}_{3}\) solution. The final mixture was incubated for 30 min in a water bath at 40 \({}^{\circ}\)C and its absorbance was subsequently measured at 765 nm using a SHIMADZU UV-1900, UV-VIS spectrophotometer and compared to a gallic acid calibration curve. The measurement was conducted in triplicate. Determination of Total Carotenoids Content (TCC)  
The estimation of total carotenoids content was carried out spectrophotometrically according to Association of Official Analytical Collaboration (AOAC) methods [28]. After each extraction, the absorbance of 3 mL of extract was measured at 450 nm and total \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Extract Abbreviation** & \
\begin{tabular}{c} **Extraction Duration** \\ **(Hours)** \\ \end{tabular} & **Temperature (\({}^{\circ}\)C)** &
\begin{tabular}{c} **Biomass to Solvent** \\ **Ratio (_v/v_)** \\ \end{tabular} \\ \hline E1 & 6 & 25 & 1:10 \\ E2 & 10 & 25 & 1:10 \\ E3 & 12 & 25 & 1:10 \\ E4 & 12 & 25 & 1:20 \\ E5 & 12 & 25 & 1:40 \\ E6 & 16 & 25 & 1:10 \\ E7 & 16 & 25 & 1:20 \\ E8 & 16 & 25 & 1:40 \\ E9 & 24 & 25 & 1:10 \\ E10 & 12 & 40 & 1:10 \\ E11 & 12 & 40 & 1:40 \\ E12 & 3 & 60 & 1:10 \\ E13 & 6 & 60 & 1:10 \\ E14 & 12 & 60 & 1:10 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Extraction conditions using ethanol/water (70:30 _v/v_) as solvent. carotenoids content was calculated according to Equation (1) acquired by the standard \(\beta\)-carotene calibration curve:  
\[\text{TCC}=(6.9691\text{Abs}\;450\;\text{nm}-0.1286) \tag{1}\]  
Determination of Extraction Yield  
After each extraction, the collected extract was employed in order to determine the extraction yield achieved. Particularly, the extracts were weighed, placed in a round bottom flask and the ethanol/water 70:30 _v/v_ solvent was evaporated using a rotary evaporator at 45 \({}^{\circ}\)C and 100 mbar. Consequently, the remaining residue was dried at 100 \({}^{\circ}\)C for 1 h and the obtained dry extract was weighed. The extraction yield was gravimetrically determined as wt% of the biomass. Determination of Antioxidant Activity (IC50)  
Antioxidant activity was assessed using the 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) assay [29], which is a simple, well-established method, and among the most frequently used in the literature. Briefly, 100 \(\upmu\)L of _U. lactuca_ extracts were added to a 3 mL methanolic solution of DPPH (0.03% _v/v_).