# Phytosterols in Seaweeds: An Overview on Biosynthesis to Biomedical Applications
## 3 Extraction and Characterization of Phytosterols
Besides the conventional Salkowski method, FTIR and nuclear magnetic resonance are widely utilized to analyze phytosterols. However, precise determination of phytosterols could be achieved through employing mass spectrometry analysis [32; 33]. For example, GC-MS analysis of the ethanolic extracts of 18 seaweeds revealed the presence of 14 compounds including 3 sterols, cholesterol, \(\beta\)-sitosterol, and fucosterol [55]. In another study, the presence of a significant amount of fucosterol in brown seaweed _C. barbata_ and the highest level of \(\beta\)-sitosterol in _H. tuna_ and _C. bursa_ were revealed through GC-MS analysis [43]. Similarly, [PERSON] et al. [33] have isolated sterols from four red seaweeds namely _P. decipients_, _P. endivifioulia_, _I. cordata_, and _P. carilagineum_ using alkaline hydrolysis extraction method and showed the presence of sterols such as fucosterol, \(\beta\)-sitosterol, stigmasterrol, brassic asteroid, cholesterol and camesterol through GC-MS analysis. Correspondingly, [PERSON] et al. [56] have also employed GC-MS analysis to reveal the presence of phytosterols such as fucosterol, isofucosterol, brassicasterol, chondillasterol and cholest-4-en-3-one in the chloroform/methanol extracts of _U. armorica_ and _S. chordalis_. In another study, [PERSON] et al. [57] used \({}^{1}\)H nuclear magnetic resonance (NMR) to analyze fractions of the marine brown seaweed _C. foeniculacea_ that were extracted using a chloroform methanol water mixture and isolated using reversed-phase HPLC (RP-HPLC). Through \({}^{1}\)H NMR analysis, the authors revealed the presence of fucosterol and an epimeric combination of saringosterol in the seaweed fractions. Recently, a one-step preparative method for separation of phytosterols was put forward by [PERSON] et al. [41] wherein the authors \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Source** & **Extraction Method** & **Methods of Analysis** &
\begin{tabular}{c} **Identified** \\ **Phystosterols** \\ \end{tabular} & **References** \\ \hline _A. utricularis, Ascoseira_ & & & ergosterol, & \\ _mirabilis, Cystosphaera_ & & brassicasterol, & \\ _jacquinti, D. anccs,_ & ultrasound irradiation & LC-MS/MS & fucosterol, \(\beta\)-sitosterol, & [6] \\ _Durvillana antractica,_ & & campesterol, & \\ and _Himanutthallus_ & & cholesterol, and & \\ _grandifolins_ & & stigmasterrol) & \\ \hline \multirow{3}{*}{_Porphyna dentata_} & methanol extraction & HPLC- evaporative & cholesterol, \(\beta\) & \\ \cline{1-1}  & and silica gel column & light scattering detector & -sitosterol, and & [51] \\ \cline{1-1}  & chromatography & (ELSD) & campesterol & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: \(Cont.\)successfully utilized the high-speed countercurrent chromatographic method to extract fucosterol (23.7 mg), and saringosterol (3.1 mg) from the crude extract of _S. horneri_ using two-phase solvent system. Furthermore, the authors have characterized the phytosterols through \({}^{1}\)H and \({}^{13}\)C NMR structural analysis. In addition, high-performance thin-layer chromatography (HPTLC, a technique for target-directed identification of active leads in a group of compounds) combined with biochemical and microchemical derivativizations revealed the presence of phytosterols and phenolic lipids in the ethyl acetate extract of 19 marine algae samples in addition to displaying bioactivity such as antioxidant, \(\alpha\)-amylase and acetylcholinesterase inhibitors activities [41]. In a study by [PERSON] et al. [47], methanolic extract of _E. stolonifera_ was fractionated using various solvents and the fraction with strong bioactivity was further purified to yield fucosterol (99% purity, determined by HPLC) and identified by \({}^{1}\)H and \({}^{13}\)C NMR methods to study the neuroprotective effects of fucosterols. Through various techniques, the sterol profiling of diverse seaweeds from different regions has already been done by many research groups, which has subsequently helped to gratify the demand for phytosterols in the global market.