Here, we investigate the effect of the enzymatic hydrolysis and anion exchange chromatographic purification on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant and antitumor activities of the collected mother fraction (S1), the enzymatically-hydrolyzed fraction (V45), and the eight column effluent fractions (F1–F8). Chemical analysis and evaluation of the biological activities of these fractions will, thus, be employed to study this effect.
2.1. Chemical Analysis
The total protein, carbohydrate, and sulfate contents in each collected fraction are listed in
Table 1, along with the degree of polymerization (DP).
Among the investigated fractions, S1 has the highest % carbohydrates (
Table 1). With the enzymatic hydrolysis of S1 to produce V45, the carbohydrate and sulfate contents decreased at the expense of the protein content. This is probably because Viscozyme broke down the saccharide linkages and, as a result of centrifugation and decantation, some of the broken soluble sugars were removed. Among the eight column fractions, F4–F8 have the highest sulfate contents, with a corresponding degree of polymerization of 3–5. In addition, F4 has the highest protein content while F7 and F8 contain traces of proteins. It can also be deduced from the table that the algal extract (S1) contains comparable contents of carbohydrates and sulfates. The same holds true for each of fractions F4, F5, and F8.
Following the degree of polymerization trend, it can be deduced that fractions with lower DP were eluted first from the column and these represent monosaccharides and disaccharides. Later, the longer chain sugars, oligosaccharides, were eluted. This is because they were more strongly bound to the column by virtue of their charge and therefore required an eluent with higher ionic strength.
2.2. FTIR Characterization
All employed fractions were analyzed using FTIR spectroscopy. The main functional groups present in the analyzed fractions are summarized in
Table 2.
As clear from
Table 2, S1 and V45 along with all column fractions show bands in the range 3500–3200 cm
−1 corresponding to the stretching vibration of the hydroxyl group. They also exhibit bands in the range 1450–1350 cm
−1, which can be ascribed to the stretching vibration of sulfate group (S=O bond) [
22,
23,
24], in addition to the amide peaks at the wavelength range of 1670–1600 cm
−1 [
25].
Aromatic ester bands appeared at 1250–1310 cm
−1 in F6, F7, F8, and S1 [
26]. In addition, sulfoxide bands appeared in S1, V45, F1, F2, and F8 at 1055, 1058, 1059, 1075, and 1025 cm
−1, respectively, and these could be attributed to the stretching vibration of S=O [
27]. Only F8 and S1 showed bands corresponding to ester sulfate at 833 and 848 cm
−1, respectively [
28,
29,
30]. The ester sulfate bond is confirmed by the presence of both aromatic ester and sulfoxide bands in F8 and S1. As a result of hydrolysis, some of the ester sulfate bonds in S1 could have been broken and, thus, they appeared only in the highest molecular weight fraction (F8). Although the ester sulfate and aromatic ester bands did not appear in V45, these groups were probably present but in low concentrations that could not be detected by FTIR.
For fractions F3, F4, F5, F6, and F7, peaks appeared at wavenumbers of 1116, 1115, 1113, and 1111 cm
−1, respectively. These correspond to the C=S stretching vibration of thiocarbonyl [
23]. The absence of thiocarbonyl bands in S1 and V45 does not indicate the absence of thiocarbonyl groups since their concentrations are expected to be higher in the fractions than in S1 or V45 and, hence, were detectable in the fractions but not in S1 or V45.
Furthermore, the band pertaining to the O–H bending vibration of phenolic groups appeared at 1365 cm
−1 in F3 only, while that corresponding to the thiol stretching vibration (S–H) appeared only in F8 at 2583 cm
−1 [
31,
32]. In conclusion, all fractions along with S1 and V45 contain hydroxyl, sulfate, and amide groups. All fractions together with S1 and V45 and apart from (F3–F7) contain sulfoxide groups. F3–F7, however, possess thiocarbonyl groups instead.
2.3. Antioxidant Activity
The antioxidant activities of the column effluent fractions at different concentrations are depicted in
Figure 2A, along with those of S1 and V45. The relevant IC50 values together with that of ascorbic acid (AA) are presented in
Figure 2B.
As can be seen from the figure, the antioxidant activity is concentration dependent for S1 and all column fractions. It is also clear from the figure that at all concentrations, the activity of the mother fraction (S1) decreased with enzymatic hydrolysis. Furthermore, the anion exchange purification of the enzymatically hydrolyzed fraction did not improve the activity. The same finding was reported in a study on
Ganoderma atrum mushrooms [
19], and in another study performed on polysaccharides extracted from the brown seaweed
Saragasum pallidum. In the latter, purification of the crude extracts onto DEAE column reduced their antioxidant activities [
18].
Amongst the eight column effluent fractions (
Figure 2), F2 and F4 are the only two fractions that showed their highest activities above 60% and these were achieved at 1000 μg/mL. These activities are slightly lower than that of V45 (63%) and are comparable to that of fraction F3 at the same concentration. The fractions F5 and F8 also showed comparable antioxidant activities but at lower concentrations.
The mechanism of DPPH scavenging activity is that of hydrogen transfer from the antioxidant to the DPP hydrazyl (radical) to convert it into DPP hydrazine. This is to avoid the presence of the active free radicals, which have the capability of degenerating the proteins, lipids, and DNA in human bodies or foods on the shelf, and, hence, lead to degenerative diseases [
33]. The hydrogen transfer is suggested to take place through a possible reaction between the radical and amine or amide groups present in the antioxidant [
34]. As deduced from the FTIR analysis, the amide group is shown in all fractions and, therefore, all of them have antioxidant activity. The highest in protein content are F2 and F4 and they are the only two fractions that showed antioxidant activity above the 60% so it could be attributed to the higher amount of protein, which indicates higher content of amide groups. However, there is no direct correlation between the antioxidant activity of the tested fractions and their protein content and this is because there are other functional groups that could be involved in the antioxidation reaction such as OH groups.
It can also be noted that the fractions with relatively higher antioxidant activities (F2–F5 and F8) all possess 1 < DP ≤ 3 except for fraction F8. The relatively high activity of F8 despite its high DP could be due to its possession of various functional groups probably at high concentrations. In addition, each of fractions F4, F5, and F8 along with S1 possess comparable carbohydrate and sulfate contents, as previously mentioned. This might suggest that the carbohydrate-to-sulfate ratio influences the antioxidant activity. Fractions F6 and F7, on the other hand, exhibited lower activities compared to the aforementioned fractions although they have a DP of 3. This could be owed to their possession of the highest sulfate contents in combination with the lowest protein contents amongst the investigated fractions. The negative effect of sulfates on the antioxidant activity was demonstrated in a study conducted on crude polysaccharides extracted from five different algal species. Therein, it was suggested that polysaccharides of the
Laminaria japonica had the least antioxidant activity and the highest sulfate content due to the consumption of hydroxyl groups, a major player in the antioxidation process, with the sulfate groups [
35]. Amongst all the column fractions, F1 with unity DP showed the lowest activity probably because it constitutes only monosaccharides.
These findings suggest that various functional groups could be involved in the antioxidation process and these include hydroxyl, sulfate, and amide groups. Furthermore, DP plays an important role in the antioxidation capacity.
2.4. Antitumor Activity
As a preliminary investigation, the lethality percentage for each algal fraction on each of four cancer cell lines was determined to test its antitumor activity. Fractions with lethality of above 75% were considered promising. As can be seen in
Table 3, four fractions S1, F4, F5, and F8 showed % lethality above 75. Fractions S1, F4, and F8 were lethal to HCT116, HePG2, and MCF7 cancer cells, respectively; whereas F5 was almost 100% lethal to both HePG2 and MCF7, and 80% lethal to A549 cancer cells.
Further investigations were performed on the fractions that showed 75% lethality or more. Dose–response curves (
Figure 3) were generated for these fractions (S1, F4, F5, and F8) and used to determine LC50, LC0, and Hill coefficient values. Results obtained for both LC50 and LC90 are compiled in
Table 4. LC50 and LC90 represent the concentration of the extract or fractions that led to death of the cancer cells by up to 50% and 90%, respectively.
To examine the dependence of lethality on concentration, lethalities of the promising fractions at various concentrations were determined. The effect of concentration on the %lethality of S1 fraction on HCT116 cells is depicted in
Figure 4A, where it can be observed that lethality is concentration dependent. Clearly, no lethality is detected for concentrations below or equal to 1.56 μg/mL; however, lethality increases with increasing concentration and its behavior follows a polynomial function trend with a correlation factor (R
2) of 0.998. As for 1.56 ≤ concentrations ≤ 25 μg/mL, the lethality increases linearly with concentration (R
2 = 0.998). Furthermore, the lethality-concentration profile for fraction F8 on breast cancer cells MCF7 is similar to that of S1 on HCT116 colon cancer cells (
Figure 4B), and the correlation between lethality and concentration is linear for 1.56 ≤ concentrations ≤ 25 μg/mL (R
2 = 0.981). F4, on the other hand, shows strong lethal effect on HePG2 human hepatocellular carcinoma cells as can be seen in
Figure 4C, where no lethality is exhibited for concentrations below 0.38 μg/mL, then it increases linearly with concentration in the range (0.38–6.25) μg/mL (R
2 = 0.996) and finally reaches a plateau at 25 μg/mL. This indicates that F4 has a strong antitumor activity with regard to HePG2 cells, even at relatively low concentrations.
Among the tested fractions, F5 showed a distinct behavior (
Figure 4D), where it exhibited the best antitumor activity, with an almost 100% lethality on MCF7 and HePG2 at 100 and 60 μg/mL, respectively. This is in addition to 80% lethality on A549 at 100 μg/mL and the lethality-concentration relation was linear (R
2 = 0.999) in this case.
From the above, it can be concluded that S1, F4, F5, and F8 have potent antioxidant and antitumor activities probably since they are the only fractions that contain comparable amounts of carbohydrates and sulfates, i.e., carbohydrate-to-sulfate ratio of 1:1. Also notable is that F4 and F5 are both active on HePG2 cells and they are the only two among the tested fractions that share the same functional groups of hydroxyl, amide, sulfate, and thiocarbonyl, as well as the same approximate DP of 3. Fractions S1 and F8, on the other hand, showed antitumor activities on HCT116 and MCF7 cells, respectively; despite their high DP (high molecular weight). This could possibly be due to their carbohydrate-to-sulfate ratio of 1 and to their possession of a variety of functional groups. They both share hydroxyl, amide, sulfate, aromatic ester, sulfoxide, and ester sulfate groups, while F8 possesses additional thiol and phenolic groups.
Interestingly, F6 and F7 showed no antitumor activity and relatively low antioxidant activity relative to F4 and F5, although they share the same DP of 3 with these two fractions. This could be attributed to their low protein content and hence their possession of less functional amide groups as compared to fractions 4 and 5. This is in addition to their highest sulfate contents amongst other fractions, which could have affected their antitumor activity as was the case with their antioxidant activity. Moreover, F6 and F7 share the same functional groups of hydroxyl, amide, sulfate, thiocarbonyl, and aromatic ester.
Another fraction that showed no antitumor activity, even though it comprises the same main functional groups present in fractions F4 and F5 (hydroxyl, amide, sulfate, and thiocarbonyl), is F3. This could be ascribed to its possession of unequal ratio of sulfate to carbohydrate content and probably due to its lower DP as compared to F4 and F5. However, F3 possesses an additional phenolic group that could have possibly played a role in imparting an antioxidant activity for this fraction that is comparable to those of F4 and F5. Antioxidant activity has been reported in the literature to be directly related to the phenolic content [
21,
36].
2.6. Sugar Analysis by HPLC
The sugar analysis (
Table 6) was performed only on the mother algal extract together with F4, F5, and F8 since they exhibited the best antitumor activities among other fractions. They also showed potent antioxidant activities.
As clear from the table, the most antitumor active fraction F5 is the highest in glucuronic acid while the second highest fraction in glucuronic acid is F4. Both of these fractions, as have been previously discussed, showed lethality for HePG2. In addition to their antitumor activity and as mentioned earlier, both F4 and F5 showed potent antioxidant activities that are, however, lower than that of S1. In a study conducted on the antioxidant activity of polysaccharides extracted from three different algal species, it was concluded that both the sulfate and glucuronic acid contents improve the activity. It was also reported that the sulfate groups are involved in the antitumor process by binding to the cationic protein on the cell surface avoiding its proliferation [
13]. In the present work, F4 and F5 have higher glucuronic acid contents than S1, but lower sulfate contents. Hence, it can be inferred that the higher sulfate content of S1 could have led to its higher antioxidant activity, while the higher contents of glucuronic acid in F4 and F5 could be responsible for their higher antitumor activities. In addition to their highest glucuronic acid contents, F4 and F5 both have the lowest molar percentages of rhamnose. This is in contrary with S1 and F8 where rhamnose is the most abundant sugar unit. A distinguishable feature that is only pertinent to F5 is the possession of comparable contents of rhamnose and glucuronic acid on the one hand, and the same for glucose and arabinose on the other hand.
In addition to sugar composition, the complexity of the polysaccharide in terms of the variety of sugar units constituting its chain is another important influencing factor. Previous studies reported that better lung cancer A549 antitumor activity was exhibited with the more complex polysaccharides that contained different kinds of monosaccharides. The more complex ones comprised eight kinds of monosaccharides as opposed to other samples that contained three and five types only [
38]. Sugar analysis in the present study revealed that F5 contains the five tested monomers while F4 and F8 comprise only four monomers and this could explain the high antitumor activity exhibited by F5 on three types of cells rather than only one type as is the case with other fractions. However, the algal extract S1 contains five monomers but is active on one cell line only, which suggests that the molecular weight along with the complexity of the molecule should be taken into consideration. F5 showed better antitumor performance than S1 probably due to its lower DP, as previous studies showed that antitumor activity is enhanced with lower molecular weights [
18,
39]. In addition, F5 comprises a thiocarbonyl group while S1 possesses an ester sulfate group instead [
38]. Most importantly, the superior activity of F5 relative to other fractions could be ascribed to a feature that is characteristic only of this fraction. This feature is having comparable contents of proteins, sulfates, and carbohydrates, in addition to having comparable contents of rhamnose and glucuronic acid, and the same for glucose and arabinose.
To sum up, S1 along with F4, F5, and F8 showed potent antioxidant as well as antitumor activities due to a number of factors. The ratio of carbohydrate to sulfate content could be majorly responsible for this activity since they all possess a carbohydrate to sulfate ratio of about 1:1. Furthermore, other factors could affect the biological activity of a molecule as was shown earlier; namely, DP, type of characteristic functional groups like sulfate, hydroxyl, and amide, as well as polymer complexity and monomer percentage. Additional reported factors include, but are not limited to, the arrangement of functional groups and monomer distribution and branching [
18,
40]. It remains to be determined whether the SPs of the crude extract S1 and the isolated fractions F4, F5, and F8 of the green algae effect on cell proliferation was attributed to genetic or epigenetic alterations in the cellular milieu. Jose and colleagues [
41] reported that SPs isolated from the brown marine algae
Padina tetrastromatica reduced the expression levels of genes known to promote cell growth, angiogenesis, and metastasis. Moreover, anti-cancer therapeutic agents such as oxaliplatin were proven to be associated with epigenetic modulations as depicted by the miRNA expression profile in HCT116 in response to the treatment [
42]. It is also possible that the SPs are contributing to the modification of the aberrant glycosylation on the surface of cancer cells tumor-associated carbohydrates [
43]. As stated and referenced earlier, the extracts properties and biological activities, to a great extent, depend on the number and the location of their sulfate groups [
44].