Active metabolites usually exist in low concentration in plants [22, 23]. Therefore, it is necessary to select the appropriate solvent to be able to obtain extracts with high yield and best properties for the targeted biological activity. Extraction yield was used as an indicator of the effects of the extraction conditions. In this study the four solvents used to extract metabolites from H. sabdariffa calyx varied in their extraction yield with best amount obtained from the EtOH (77.2%) and 50% EtOH (58.4%) solvents respectively. This results were in agreement with finding of Grigonisa et al. [24] who reported that the highest extract yields were obtained with polar alcohol-based solvents.
The results of the present study demonstrated that the total content of polyphenol, flavonoid and Vitamin C in calyces of H. sabdariffa significantly (p ≤ 0.05) influenced by type of solvent used for extraction. EtOH and 50% EtOH extracted highest content of polyphenols while the water and acetone extracts showed the highest flavonoid and vitamin C contents respectively. Previous study by Koffi et al [25] showed that alcohol (ethanol and methanol) was better in extracting phenols than acetone. However, it would also be important to consider that the high contents of phenolics in alcoholic extracts are more likely associated with biomolecules including proteins, carbohydrates (glycosides), terpenes, chlorophyll, lipids and inorganic compounds, which could be also extracted by these solvents and consequently interfere in determination of total polyphenol by Folin-Ciocalteu reagent [26].
Total flavonoid content in the present study were best extracted by the water extract and was in contradiction to the results of Koffi et al [25] who showed that flavonoids in H. sabdariffa calyx were better extracted from the acetone extract. This difference in results could be attributed to nature of flavonoid molecules present in H. sabdariffa sample from Sudan which could be affected by many biotic and abiotic factors such as differences of cultivars, soil of cultivation, climatic conditions and storage condition [27, 28].
Furthermore, the chemical profiling of the four extracts revealed that out of the 15 phenolic standards used 12 compounds were present in H. sabdariffa with chlorogenic acid, naringenin rutin and gallic acid at noteworthy concentrations in most extracts. Generally, the highest amount of the identified compounds was obtained from the 50% EtOH extract. Previous studies [15, 16] reported the presence of most of these compounds in H. sabdariffa calyx, but to the best of our knowledge this is the first reports on comparing the effect of the four used solvents on the recovery of these compounds.
Result of the antioxidant DPPH free radical scavenging activity showed that 50% EtOH extract followed by EtOH extract exhibited the best anti-DPPH radical activity while acetone extract revealed the least activity. Previous study showed that the alcoholic (methanol) extract gave the highest DPPH value in comparison with ethyl acetate and acetone [28, 29]. As noted in the phenolic profile of these two extracts, many compounds are well known for their antiradical activity such as rutin, qurecetin [30], gallic acid [31], ellagic acid [32], chlorogenic acid, coumaric acid [33], caffeic acid [34] vanillin and catechin [35]. Although the water extract contained also considerable amount of these compounds, its antiradical activity was lower than that observed in the 50% EtOH and EtOH extracts. This might be to antagonistic effect and steric hindrances of other components which are also present in the crude extract [36]. Furthermore, the antiradical activity of the acetone extract was probably associated to its high content in vitamin C. Soobrattee et al. [37] reported that although the antiradical potential of vitamin C was found to be weaker than that of quercetin and similar to that of trolox, it can still contribute to the antiradical activity of berries and fruits.
From the Pearson’s correlation coefficient of total polyphenols, flavonoids, vitamin C with antioxidant activity, it was suggested that the antioxidant DPPH free radical scavenging activity of H. sabdariffa calyx was mainly associated to polyphenolic content (R² = 0.9836) rather than the total flavonoid content (R² = 0.0625) or vitamin C (R² = 0.3962).