2.1. Cytotoxicity Assay
The growing interest in plant-derived compounds for commercial human applications requires the assessment of their possible toxicity. Cytotoxicity studies in cell lines can be considered as a first step in the development of pharmaceutical, cosmetic or food products; toxicity levels (minimal or no toxicity) should be verified [16
]. In this test, HT-29 cells, the human colon adenocarcinoma cell line expressing the characteristics of mature intestinal cells, such as enterocytes or mucus-producing cells [17
] were used. As seen in Figure 1
, the incubation with extracts at low concentrations (1 and 10 μg/mL) did not reveal significant changes in the MTT conversion rates associated with cell viability. Nothogenia
sp. and M. laminarioides
extracts negatively affected the cellular metabolism of HT-29 cells at 24 and 48 h, respectively, at the maximum concentration evaluated (1000 μg/mL); however, no extract decreased the cell viability in the same way as the positive control (DMSO) that reduced viability to 17.1 ± 1.6% and 18.4 ± 1.4% at 24 and 48 h of exposure, respectively. In general, no statistically significant differences were observed between the type of extract (ethanol and acetone) or between the incubation times (24 and 48 h). According to Galindo et al. [18
], a cytotoxic effect can be considered when the viability is less than 75%. None of the seaweed extracts reduced the cellular viability to that level, so they may be considered to not be cytotoxic.
The diversity of compounds derived from algae can generate different actions; these can stimulate growth or apoptosis. Nothogenia
sp. and Pyropia
sp. extracts slightly decreased cell viability at higher concentrations, therefore, they could be analyzed from a chemotherapeutic perspective. Seaweed extracts could also be used as antitumoral agents. Using different cell types can show different responses towards a specific compound or plant extract. Some authors indicate that polyphenols are toxic to certain types of cancer cells that proliferate rapidly but are nontoxic to others [19
2.2. Total Polyphenol Content and Antioxidant Capacities
Both the species and extraction method significantly affect (p
< 0.05) the total polyphenol content (TP) of the extracts (Table 1
). The seaweed with the highest TP was D. antarctica
(cochayuyo), whose acetone and ethanol extracts contained 7.4 ± 0.2 and 6.7 ± 0.7 mg gallic acid equivalents (GAE)/g dry seaweed, respectively, followed by Pyropia
sp., whose acetone and ethanol extracts contained 6.2 ± 0.2 and 4.8 ± 0.3 mg GAE/g, respectively. In most cases, conventional extraction with acetone yielded higher values of TP (10%–20%) than HPLE with ethanol (p
< 0.05). Acetone, being a dipolar compound, has an intermediate polarity and solubilizes solutes with a similar relative polarity. Wang et al. [21
] found that acetone (70%) was the most suitable solvent to recover algae polyphenols. Nonetheless, acetone is a toxic solvent not permitted for human consumption. However, HPLE with ethanol (50%) allowed for seaweed extracts that are of food grade, which is mandatory for future applications such as nutraceuticals and functional food ingredients.
Two complementary methods were applied to determine the antioxidant activity of the extracts: the oxygen radical absorbance capacity (ORAC) test, which corresponds to an electron transfer model, and the DPPH assay that determines the transfer of hydrogen atoms.
Statistically significant differences were observed among the species studied (p
< 0.05), with brown seaweed extracts being the ones with the highest antioxidant activities in both extraction methods. The high antioxidant activity of brown species could be due mainly to their content of phlorotannins, whose phenolic ring can eliminate the reactive species of oxygen [23
Ethanol D. antarctica
extracts presented the highest antioxidant activity, both in the ORAC (680.1 ± 11.0 μmol ET/g dry seaweed) and DPPH (48.5 ± 4.2 μmol ET/g dry seaweed) assays (Table 1
). Thus, HPLE with ethanol is a feasible food grade alternative to conventional extraction since it allows obtaining polyphenol extracts with higher antioxidant activity (p
< 0.05). It is worth mentioning that the TP extraction yields differed only between 10–20%. Contrarily, for the antioxidant activity, the HPLE extracts presented two and 10 times higher DPPH and ORAC values, respectively, as compared to the extracts obtained using acetone. The advantage of using HPLE with ethanol is clear; the antioxidant capacity is associated with the potential benefic effect of polyphenols on human health, and above all because ethanol is a food grade solvent.
The major compounds contributing to the overall antioxidant activity in seaweeds are frequently phenolic compounds and polysaccharides; they may be found alone or associated with other components such as polyphenols, amino acid, protein, lipids, and sometimes polysaccharide conjugates [27
] (Jacobsen et al., 2019). Since some seaweed polyphenol compounds may be conjugated with different types of sugars, the chemical structure of these conjugated compounds makes them more soluble in alcoholic solvents compared to acetone. This may explain the higher antioxidant activity of our extracts. The higher solubility of sugars can be associated with the dielectric constant of ethanol (24.55 at 25 °C), which is closer to water (78.54 at 25 °C) than acetone (20.7 at 25 °C) [28
2.3. Anti-Enzymatic Activities
One of the strategies to manage type-2 diabetes is through the inhibition of enzymes such as α-amylase and α-glucosidase. The selective inhibition of these enzymes reduces the hydrolysis of carbohydrates and the rate of glucose uptake into the bloodstream [29
]. The ability of the seaweed extracts to inhibit the activity of α-amylase and α-glucosidase at different concentrations was determined.
The α-amylase activity did not decrease significantly with any of the ethanol seaweed extracts within the concentration range studied (Figure 2
). Acetone extracts of D. antarctica
sp. inhibited the activity of this enzyme, although to a lesser extent than acarbose. The acetone extract of D. antarctica
(2000 µg of dry extract/mL) decreased the α-amylase activity to 56.6 ± 2.0% and Gelidium
sp. to 77.9 ± 2.1%. In contrast, acarbose (concentration ≥ 1000 μg/mL) decreased the enzyme activity to 37.5 ± 0.4%.
Like α-amylase inhibitors, α-glucosidase inhibitors can slow down the breakdown and absorption of dietary carbohydrates [30
]. The acetone and ethanol extracts of D. antarctica
and the acetone extract of L. spicata
were the most effective inhibitors of α-glucosidase (Figure 3
). The D. antarctica
acetone extract almost completely inhibited the enzymatic activity (to 0.7 ± 0.3%), followed by the acetone extract of L. spicata
(to 1.2 ± 0.3%) and by the ethanol extract of D. antarctica
(to 3.1 ± 0.4%); all of them to a concentration of 1000 μg dry extract/mL. These extracts were much more effective than the commercial enzymatic inhibitor acarbose (1000 μg/mL), which reduced the enzymatic activity only to 40.4 ± 1.1%. The ethanol and acetone extracts of Gelidium
sp., M. laminarioides
sp. did not show any significant inhibitory effect against α-glucosidase in the concentration range assessed (1, 10, 100, 1000 μg dry extract/mL). In general, the differences in the inhibition capacity among the extracts would be derived from their compositions, which in turn is the result of the seaweed species and extraction methods used.
values for the α-glucosidase (Table 2
) of acetone extracts of D. antarctica
and L. spicata
(the only two seaweed species that generated inhibition higher than 50%) were 466 and 479.21 μg/mL, respectively, and both were lower than the IC50
of acarbose (797.9 μg/mL). Hence, D. antarctica
and L. spicata
have an effective inhibitory effect on the activity of α-glucosidase. Brown algae were better inhibitors of α-glucosidase than red algae such as Gelidium
sp., M. laminarioides
sp. Likewise, other authors found that the water extract of Laminaria digitata
, a member of the Laminariaceae family and a brown seaweed, was effective in inhibiting α-glucosidase [11
The diversity and abundance of some compounds may hamper the interactions with the enzyme at a molecular level; however, it is possible that the purification of extracts may enhance the enzymatic inhibitory activity. Therefore, our crude algae extracts were not as effective in inhibiting these enzymes as the pure polyphenols of vegetable origin (IC50
~ 5 μg/mL) [14
] or purified polyphenol extracts from pomegranate extracts (IC50
of 278 μg/mL) or black tea (IC50
of 64 μg/mL) [31
Several reports have been published on the established enzyme inhibitors, such as acarbose, miglitol, voglibose and nojirimycin, and their favorable effects on blood glucose levels after food intake [32
]. This fact is attributed to their capacity to diminish the assimilation of sugars, both monosaccharides and more complex carbohydrates. It seems appropriate to ingest the potent inhibitors of α-glucosidase and α-amylase to control the release of glucose from saccharides in the intestine. However, the complete inhibition of both enzymes could result in the poor absorption of nutrients. Inhibitors should be administered in doses that allow all carbohydrates to be digested. Otherwise, undigested carbohydrates will enter the colon and, as a result of bacterial fermentation, will lead to side effects such as flatulence. The most adequate inhibitors should delay the digestion and absorption of carbohydrates without overloading the colon with them [34
]. It has been proposed that the administration of products presenting the moderate inhibition of α-amylase and strong inhibition of α-glucosidase is an effective therapeutic strategy that could decrease the availability of monosaccharides for absorption in the intestine [35
]. Hence, the consumption of some of our seaweed extracts may be a preferred alternative for modulating the glycemic index of some food products.
The enzymatic inhibition capacity (α-amylase and α-glucosidase) of seaweed extracts followed the same trend as their antioxidant activities. The ethanol and acetone extracts of D. antarctica, the species with the highest average antioxidant activity (ORAC and DPPH), presented the highest α-glucosidase inhibition activity. Consequently, antioxidant activity indices can be used to optimize the design of the extraction process.