# Phytosterols in Seaweeds: An Overview on Biosynthesis to Biomedical Applications
## 3 Extraction and Characterization of Phytosterols  
Extraction techniques are proficient in separating the soluble metabolites of seaweed using suitable solvents [27]. The isolation techniques to be used for the extraction of phytosterols generally depend on the type of phytosterols (free, glycosylated, and esterified) and the nature of the matrix [28]. In each technique, optimized experimental conditions and adequate parameters are required to attain the appropriate quantity and higher yields from seaweed extract. The quality of an extract is substantially influenced by various factors, such as plant/seaweed material, solvent, extraction procedure, and others [29]. There are two common extraction techniques, namely conventional and non-conventional extraction (Figure 2) [30]. Conventional techniques usually employ a large volume of organic solvents to extract adequate analytes from samples that are required for further analysis. Nonetheless, the utilization of a higher volume of organic solvents may have a negative impact on human health and the environment. Furthermore, conventional techniques are reported to possess several limitations, such as the necessity of solvents with very high purity, an extended period of time for extraction, low selectivity of extraction, requirement of solvent evaporation and thermal decomposition of heat liable compounds. These limitations demanded the discovery of new extraction techniques with relatively very low utilization of organic solvents and more advantages than conventional techniques [31]. Soxhlet extraction is one of the conventional techniques, and remains the most preferred extraction technique for phytosterols, still, today [32; 33]; it serves as a standard to many newly developed extraction techniques (Table 1) [31]. For example, [PERSON] et al. [32] extracted phytosterols from the red seaweed _Gelidium spinosum_ using the Soxhlet method and revealed the presence of stigmasterol, with a mass of 412.69 g/mol, through Fourier transform infrared (FTIR) and gas chromatography-mass spectrometry (GC-MS). In addition, maceration is another common conventional technique, which is stated to be simple and cost-effective for phytosterol extraction [34]. Figure 2: An overview of phytosterol extraction and characterization methods. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Source** & **Extraction Method** & **Methods of Analysis** & \begin{tabular}{c} **Identified** \\ **Phytosterols** \\ \end{tabular}  & **References** \\ \hline _Gelidium spinosum_ & Soxhlet method & FTIR and GC–MS & Stigmasterol & [32] \\ \hline \multirow{4}{*}{_Saccharina latissima_} & \multirow{4}{*}{saponified extract} & \multirow{4}{*}{GC–MS} & cholesterol, \\  & & & desmosterol, 24- \\  & & & methylenedcholesterol, \\  & & & fucosterol, cycloartenol, \\  & & & and unknown \(\Delta^{5}\)-sterol \\ \hline \multirow{4}{*}{_Palmaria decipients,_ _Placamini cartilagineum,_ _Iridaea cordata,_ and _Pyropia endtivifolia_} & \multirow{4}{*}{alkaline hydrolysis} & \multirow{4}{*}{GC–MS} & cholesterol, \\  & & & transiscasterol, \\  & & & transiscasterol, \\  & & & transiscasterol, \\  & & & transiscasterol, \\  & & & transiscatorol, \\  & & & transiscatorol, \\  &