# Phytosterols in Seaweeds: An Overview on Biosynthesis to Biomedical Applications
## 3 Extraction and Characterization of Phytosterols
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Source** & **Extraction Method** & **Methods of Analysis** & **Identified Phytosterols** & **References** \\ \hline  & solvent extraction and & NMR (\({}^{1}\)H and \({}^{13}\)C), IR & \(\beta\)-sitosterol, fucosterol & [44] \\ _Sargassum elegans_ & column & and mass spectral data & \(\beta\)-sitosterol, fucosterol & [44] \\  & chromatography & & \\ \hline Phaeophyta (_Cystophora_ & & & \\ _pectinata_, _Pollospora_ & & & \\ _comosa_, _Scytothalia_ & & & \\ _dorycarpa_, _Carrogglosum_ & & & \\ _confluens_, _E. radiata_, & & & \\ _Sargassum lacerifolium_, & & & \\ _Perthalia caudate_, & & & \\ _Codium harreeyi_, & & & \\ _Scytothalia dorycarpa_, & & & \\ _Hypnea valida_, & & & \\ _Cystophora monilifera_, & & & \\ _Hormosia banksia_, & & & \\ _Myridoansan_ & & & \\ _integrifolium_, & & & \\ _integri_, & & & \\ _algae sp., _Cystophora_ & & & \\ _subfracnata_), & & & \\ _Rhodophyta_ & & & \\ _(Austrophyllis harreeyana_, & & & \\ _Rhodophyllis_ & & & \\ _membaneca_), and & & & \\ chloroformyta (_Codium_ & & & \\ _fragile_) & & & \\ \hline _Asscea minabilis_, _A._ & & & cholesterol, \\ _atricularis_, _Desmarestia_ & & & desmosterol, \\ _anceps_, and _Phaeurus_ & & & \\ _antarcticus_ & & & \\ \hline _Ecklonia stolonifera_ & silica gel column & \({}^{1}\)H and \({}^{13}\)C NMR & fucosterol & [47] \\  & chromatography & & & \\ \hline Rhodophyta (_Gracilaria_ & & & \\ _vermectinolophila_, & & & \\ _Pterocladiella tentaus_, & & & \\ _Palisada intermedia_, & & & \\ _Chrysymenia wrighttii_, & & & \\ _Gracilaria elegans_, & & & \\ _Gracletopia asiatic_, & & & \\ _Laurencia ackumarne_) and & & & \\ Phaeophyta (_Ecklionopsis_ & & & \\ _radicosa_, _Sargassum thunbergai_, _Ecklonia_ & & & \\ _kurome_, _Eisenia arbora_, & & & \\ _Sargassum pillilferum_, _S._ & & & \\ _fusiforme_, _U. pinnatifida_, & & & \\ _Ecklonia cava_) & & & \\ \hline _S. horneri_ & total lipid extraction & RP-HPLC & fucosterol & [49] \\  & using methanol & & \\ \hline _Hizikia fusiformein_ & ethanol extraction and & LC/ electrospray & \\ _chromatographic_ & & ionization (ESI)-MS & fucosterol & [50] \\  & separation & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._Apart from these conventional techniques, phytosterol extraction has also been shown to be accomplished using many other non-conventional extraction techniques, such as microwave-assisted extraction (MAE), ultrasonic-assisted extraction (UAE), enzyme-assisted extraction (EAE), pulsed electric field-assisted extraction (PEFAE), pressurized liquid extraction (PLE), and supercritical fluid extraction (SFE) (Figure 2) [31]. For instance, [PERSON] et al. [52] have employed MAE with high-speed countercurrent chromatography (liquid-liquid partition chromatography) and a UV detector to extract, separate, and purify phytosterols from the edible brown seaweeds _S. fusiforme_ and _U. pinnatifida_. By employing these extraction and chromatographic techniques, the authors were able to obtain 1.5 mg of 24-methylenediolesterol and 13 mg of fucosterol from 15 g of _U. pinnatifida_ and 0.3 mg of 24-methylenediolesterol and 4.6 mg of fucosterol from 15 g of _S. fusiforme_. Remarkably, [PERSON] et al. [53] have performed a comparative analysis of various phytosterol extraction techniques on cocoa butter such as Soxhlet, ultrasonic, supercritical carbon dioxide, and supercritical carbon dioxide with co-solvents. The authors concluded that the highest phytosterol content was obtained when using supercritical carbon dioxide with a cosolvent. The separation process usually follows the extraction process. In this context, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) techniques are extensively used separation techniques (Figure 2). After the separation process, phytosterols in the analytes are confirmed via various analytical methods. Preliminary screening of phytosterols can be performed through Salkowski test [54], in which the formation of brown ring confirms the presence of phytosterols.