## Results and Discussion
### Relative Quantification  
To further validate the results, the amount of metabolites detected from the \({}^{1}\)H NMR spectra were relatively quantified based on the variable importance in the projection (VIP) values of its partial least squares discriminant analysis (PLSDA) model (Figure S3B). In this regard, metabolites with VIP value greater than 0.5 (considered as top-ranked metabolites) were selected to determine if they might influence the clustering of the X-variable (identified metabolites) in the PCA loading plot [32]. Figure 3A,B show the graph with the metabolites and their relative quantities in the different extracts. The hexane extract was characterized by significantly higher amounts of arachidonic acid (binned at 1.26 ppm), \(\alpha\)-linolenic acid (binned at 1.30 ppm), astaxanthin (binned at 1.34 ppm), lutein (binned at 0.86 ppm), fucoxanthin (binned at 1.22 ppm), and Chl \(c_{1}\) (binned at 9.82 ppm) in comparison to the other extracts. In addition, the chloroform extract contained higher levels of canthaxanthin (binned at 1.22 ppm), palmitic acid (binned at 1.66 ppm), stearic acid (binned at 1.78 ppm), isoleucine (binned at 0.94 ppm), violaxanthin (binned at 0.98 ppm), zeaxanthin (binned at 1.10 ppm), cholesterol (binned 0.70 ppm) and leucine (binned at 1.70 ppm) in comparison to the acetone extract. However, lower numbers of metabolites were observed in the 70% ethanol extract. These results indicated that medium and non-polar solvents such as chloroform, acetone and hexane were the best solvents to use for extraction of the microalgae, allowing a good recovery of the hydrophobic compounds such as fatty acids, lipids and pigments. Furthermore, according to the Tukey honest significant difference (HSD) pairwise comparison (Table S1), the carotenoid contents of the chloroform, acetone and hexane extracts, represented by spectral signals of fucoxanthin, astaxanthin, violaxanthin and lutein, were highly significant (\(P\leq 0.001\)) compared to those in the methanol and 70% ethanol extracts. The amounts of fatty acids, such as arachidonic acid and \(\alpha\)-linolenic acids, were significantly higher (\(P\leq 0.001\)) in the hexane extract when compared to the other extracts. However, the higher amount palmitic and stearic acids were highly significant (\(P\leq 0.001\)) in the acetone and chloroform extracts. The amount of the amino acid such as isoleucine was significantly higher (\(P\leq 0.001\)) in all the extracts other than the 70% ethanol extract. Figure 2: Score plot of principal component analysis (PC1 versus PC2) in (**A**) and loading plot (**B**) obtained from five different solvent extractions of _Chaotecros calcitrans_. The plot elipse represents 95% hotelling T2 confidence. Effect of Different Solvent Extractions on Total Phenolic Content (TPC), 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging and Nitric Oxide Inhibitory Activities  
As shown in Figure 4, the five different solvent systems contributed variation to the total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and nitric oxide inhibitory activities of _C. calcitrans_. Acetone and chloroform extracts exhibited comparably high percentages of DPPH free radical scavenging activity (Figure 4A). The percentage inhibition for acetone extract was 43.01% whereas for chloroform extracts with 35.03% of inhibition. There were significant differences (_P_ < 0.05) in all solvent systems with the exception that ethanol and methanol extracts, 70% ethanol and methanol extracts of _C. calcitrans_ were statistically similar with only 18.61 and 18.51% of ability to scavenge the free radicals. On the other hand, hexane extract showed the lowest DPPH scavenging activity with only 13.71%. The results in the present study are contrary to the results reported from previous study by [PERSON] et al.