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Introduction
Seaweeds are primary producers in all oceans and they release significant quantities of oxygen to the atmosphere (Mann 1973). Seaweeds are also an important food source, and they are usually used as nursery and shelter habitats by an important number of aquatic organisms (Lobban and Harrison 1994, Christie et al. 2009). As food sources, seaweeds contain a variety of nutritive and bioactive compounds (Plaza et al. 2008, 2010), such as proteins, carbohydrates, different kinds of lipids like essential fatty acids (i.e., arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid) and sterols (i.e., cholesterol), and pigments (i.e., chlorophylls and carotenoids). All of these nutritionally important biochemical components are transferred to upper levels in the marine food web (Guschina and Harwood 2006).
Seaweeds are distributed throughout different biogeographic regions according to climate. At least 2 regions with temperate and tropical conditions have been demonstrated to explain the distribution and abundance of seaweeds on the Pacific Coast of Mexico (van den Hoek 1984, Silva 1992, Paul-Chávez and Riosmena-Rodriguez 2000, Garbary 2001). Geographic distribution is usually explained by climate variations, whereby high-latitude environments (cold or temperate) have greater annual variations in climatic conditions than low-latitude environments (tropical), favoring more tolerant species with a broad geographic distribution. Thus, organisms inhabiting high-latitude environments are expected to have broader climatic tolerance compared with organisms inhabiting low-latitude environments (Stevens 1989, 1992). Differences in seaweed growth rates and biochemical composition as a response to adaptation to environmental conditions should therefore be expected. Additionally, seasonal, interannual, and species-specific variations have been associated with the biochemical composition of different seaweeds, and this association has been explained by changes in environmental parameters such as temperature, salinity, solar radiation, and nutrient availability (Xu et al. 1998, Nelson et al. 2002, Guschina and Harwood 2006, Hurtado et al. 2011, Polat and Ozogul 2013).
In Mexico, the chemoprotectant properties in seaweed biochemical composition and the nutritional value of seaweeds have been evaluated in some species from the Gulf of Mexico and the Caribbean Sea (Robledo and Freile Pelegrín 1997, Aguilera-Morales et al. 2005, Peña-Rodríguez et al. 2011). The interannual and seasonal variations in fatty acids (Nelson et al. 2002, Serviere-Zaragoza et al. 2015) and proximate composition (Serviere-Zaragoza et al. 2002) have been described for some seaweed species in temperate seawaters of the northeastern Pacific Ocean. Only 2 studies have been performed on tropical seaweed species off the Pacific coast of Mexico. In one of them, the effect of season (summer vs winter) on the proximal composition and fatty acid and amino acid contents in 5 tropical seaweeds was evaluated (Peraza-Yee 2014). More recently, the chemopreventive activities assessed in the same species evaluated in this work were demonstrated by the content of flavonoids and chlorophylls (Osuna-Ruiz et al. 2016); however, specific lipids (i.e., polyunsaturated fatty acids and phytosterols) and pigments (i.e., lutein, β-carotene, and fucoxanthin), which could also be a source of important biochemical components with high nutritional value and with chemopreventive activity, were not analyzed. This highlighted the lack of knowledge on the biochemical composition of seaweed species distributed in the tropical region off the Pacific coast of Mexico. Therefore, the aim of the present study was to assess the proximate fatty acid, sterol, and pigment composition of 7 tropical seaweed species collected from different tropical lagoons on the Pacific coast of Sinaloa, Mexico: 1 Phaephyta, Padina durvillaei Bory Saint-Vincent (Dictyotaceae); 2 Rodhophyta, Spyridia filamentosa (Wulfen) Harvey (Spyridiaceae) and Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariaceae); and 4 Chlorophyta, Ulva expansa (Setchell) Setchell & N.L. Gardner (Ulvaceae), Codium isabelae W.R. Taylor (Codiaceae), Rhizoclonium riparium (Roth) Harvey (Cladophoraceae), and Caulerpa sertularioides (S. G. Gmelin) M. A. Howe (Caulerpaceae).
Materials and methods
Sampling
Seaweeds were selected according to their seasonal appearance at each location, when abundance was highest, facilitating their sampling. All seaweed species were sampled at the same depth (3 to 5 m) in the rocky intertidal zone. Approximately 2.5 kg (about 20 to 30 specimens) of each seaweed species were hand collected in different lagoons along the coast of Sinaloa, Mexico. Rhizoclonium riparium specimens were collected in June 2013 in Urias Lagoon (23º19′0.96″N, 106º18′46.5″W); S. filamentosa and C. sertularioides were collected in October 2013 in Agiabampo-Bacorehuis Lagoon (26º20′37.2″N, 109º14′34.6″W and 26º20′26″N, 109º12′50″W, respectively); Gracilaria vermiculophylla was collected in October 2013 in Santa Maria-La Reforma Lagoon (29º50′14.7″N, 108º05′24″W); and C. isabelae, P. durvillaei, and U. expansa were collected in May 2014 in Mazatlan Bay (23º1′29.1″N, 106º25′29.7″W). All seaweed specimens were cleaned in situ with seawater (epibionts and necrotic parts were removed) and then transported to the laboratory in plastic bags covered with ice. All weighed fresh seaweed biomass was rinsed with distilled water, lyophilized (FreeZone Plus 4.5 Labconco; Kansas City, MO, USA), and grounded (0.6 mm) with a commercial grinder. Dried pooled samples were stored at -70 ºC for proximate composition, while fresh pooled subsamples were used for lipid and pigment analyses.
Proximate composition analyses
Moisture, ash content, crude protein, and ether-soluble matter (lipids) were assessed in all seaweed species according to AOAC (2006). Moisture content (%) was determined by drying 2 g of wet samples in an oven dryer (Lindberg/blue, Thermo Scientific; Waltham, MA, USA) at 105 ºC until reaching constant weight. Ash content was determined by calcination of samples at 500 ºC for 4 h in a muffle furnace (Thermolyne, Thermo Scientific; Waltham, MA, USA). Crude protein content was assessed by extracting total nitrogen using the micro-Kjeldahl technique, and protein was estimated using a conversion factor of 6.25 (Serviere-Zaragoza et al. 2002). Crude fat was determined by extraction with petroleum ether for 6 h in a Soxhlet equipment. The nitrogen-free extract (carbohydrate fraction) was obtained as the difference between measured components and total percentage of dry weights: 100 - (% moisture + %proteins + % lipids + % ash).
Fatty acid analyses
Fatty acid composition was assessed according to the method described by Serviere-Zaragoza et al. (2015). Frozen fresh pooled subsamples were thawed, and lipids were extracted with chloroform/methanol (2:1 v/v) according to Folch et al. (1957). Fatty acids were transesterified with boron-trifluoridemethanol (BF3 14% methanol, Supelco), and methyl esters were analyzed in a gas chromatograph (GC, Agilent Technologies 6890 M) equipped with a DB-23 silica column (30 m × 0.25 mm ID × 0.25 μm film thickness) and a flame ionization detector with helium as the carrier gas (0.7 mL·min−1) (temperature ramp from 110 to 220 ºC). Fatty acids were identified by comparing their retention times with those of standards (Sigma; Bellefonte, PA, USA), with the concentration of each fatty acid corrected for by correlation with the response of the corresponding standard. Data were analyzed using GC Chem Station Rev. A.10.02 (1757, Agilent Technologies, 2003).
Analysis of sterols
Total sterol composition was determined by gas chromatography according to Palacios et al. (2007) with some modifications. A fraction of total lipids previously extracted for fatty acid analysis was evaporated to dryness with N2, and sterols were transesterified with sodium methoxide at room temperature for 90 min. Released sterols were extracted in hexane. Sterols were analyzed in a GC (Agilent Technologies 6890 M) equipped with a RTx-65 fused silica column (Crossbond diphenyl dimethyl polysiloxane) (15 m × 0.25 mm ID × 0.25 μm film thickness) and a flame ionization detector with hydrogen as the carrier gas (50 psi constant pressure) (temperature ramp from 50 to 260 ºC). Sterols were identified by comparing their retention times with those of standards (Sigma; Bellefonte, PA, USA). Data were analyzed using GC Chem Station Rev. A.10.02 (1757, Agilent Technologies, 2003).
Pigment composition
Pigment composition was assessed according to the method described by Zapata et al. (2000) with the modifications described by Quintana-López et al. (2019). Briefly, fresh seaweed pooled samples were thawed, and 100-mg portions were homogenized on ice with 10 mL of acetone, avoiding light contact, and finally incubated at 4 ºC. After 24 h, the supernatant was recovered by centrifugation (3,200 × g, 10 min at 4 ºC) and stored at -70 ºC until analysis. Pigments were analyzed using a high performance liquid chromatography (HPLC) system (Agilent Technologies 1200 Infinity Series) equipped with a ZORBAX C8 column (4.6 × 100 mm, 3.5-μm particle size) and a diode-array detector (DAD) at a wavelength of 440 nm, with the temperature of the column kept at 25 ºC. The mobile phases consisted of an eluent A, which was a mixture of methanol:acetonitrile:0.25 M aqueous pyridine solution (50:25:25, V:V:V), and an eluent B, which was acetonitrile:acetone (80:20, V:V). Flow rate was fixed at 1 mL·min-1. All solvents used were HPLC grade (Tedia, OH, USA). Pigments were expressed as relative proportions and were identified by comparing their retention times with those of standards (DHI; Hoersholm, Denmark).
Statistical analyses
One-way analysis of variance (ANOVA) was performed to analyze gross chemical, fatty acid, sterol, and pigment composition data obtained from the seaweed specimens, followed by a post hoc Tukey test to assess significant differences (P < 0.05) between means. The percentage values were arcsine-transformed before analyses (Zar 1999), but results are expressed as untransformed means. Factor analysis (varimax normalized using principal components extraction) was used to extract the maximum variance from data sets of some biochemical components (fatty acids, sterols, and pigment content), with each principal component as a linear combination of most recognized bioactive compounds of seaweeds, considering factor loadings ≥0.7 and eigenvalues >1.0. The data are reported as the mean ± standard error. Analyses were run with Statistica v.13.0 (Statsoft).
Results
Proximate composition
Significant differences (P < 0.05) were observed in the crude protein and lipid compositions, nitrogen-free extract, ash, and moisture in the tissues of analyzed seaweed species (Table 1). The highest crude protein content was observed in C. sertularioides (11.96% ± 1.93 dry weight [dw]), while the lowest values were found in U. expansa and P. durvillaei (4.12% ± 0.51 and 5.87% ± 0.12 dw, respectively); intermediate values were found in S. filamentosa and G. vermiculophylla (10.4% dw, on average).
Table 1 Proximate composition (percent dry weight, % dw) in seaweed species off Sinaloa, Mexico.
| Green seaweeds | Red seaweeds | Brown seaweed | |||||||
| Ulva expansa | Caulerpa sertularioides | Rhizoclonium riparium | Codium isabelae | Spyridia filamentosa | Gracilaria vermiculophylla | Padina durvillaei | |||
| Proteins | 4.12 ± 0.51c | 11.96 ± 1.93a | 7.85 ± 0.90b | 8.90 ± 0.06ab | 10.61 ± 0.34a | 10.15 ± 0.50a | 5.87 ± 0.12bc | ||
| Lipids | 0.65 ± 0.01c | 0.92 ± 0.03b | 1.50 ± 0.01a | 0.83 ± 0.03b | 0.26 ± 0.01d | 0.36 ± 0.01d | 0.37 ± 0.03d | ||
| Nitrogen free extract1 | 59.61 ± 2.59b | 51.69 ± 1.94c | 52.2 ± 2.18c | 61.53 ± 0.10ab | 56.57 ± 0.32bc | 54.54 ± 1.09bc | 68.96 ± 0.22a | ||
| Ash | 35.66 ± 2.95ab | 35.54 ± 0.12ab | 39.08 ± 2.59a | 28.90 ± 0.30bc | 32.54 ± 0.10b | 35.02 ± 0.72ab | 24.47 ± 0.02c | ||
| Moisture2 | 84.59 ± 1.32c | 88.28 ± 0.77b | 89.83 ± 0.48b | 93.33 ± 0.46a | 93.12 ± 0.11a | 90.32 ± 0.58ab | 82.96 ± 0.28c | ||
Values are means of 3 determinations ± standard error, and they were analyzed by unifactorial analysis of variance, followed by a Tukey test to assess significant differences. Means sharing different letters within a row were significantly different (P < 0.05).
1Obtained by difference: 100% - (%moisture + %proteins + %lipid + %ash).
2Obtained from wet weight of seaweeds.
Lipid contents varied significantly (P < 0.05) among the seaweed species analyzed (Table 1). The highest lipid content was found in R. riparium (1.50% ± 0.01 dw) and the lowest in S. filamentosa, G. vermiculophylla (0.26% ± 0.01 and 0.36% ± 0.01 dw, respectively), and P. durvillaei (0.37% ± 0.03 dw).
Significant (P < 0.05) differences were observed in the ash contents of seaweed specimens (Table 1). The highest content was observed in R. riparium (39.08% ± 2.59 dw) and the lowest in P. durvillaei and C. isabelae (24.47% ± 0.02 and 28.90% ± 0.30 dw, respectively); intermediate values were observed in the remaining seaweed species.
Moisture content was significantly (P < 0.05) different among the seaweed species analyzed (Table 1). Values were within the range of 83-93%, with the highest values for C. isabelae and the lowest for P. durvillaei and U. expansa.
Fatty acids
All fatty acids significantly (P < 0.05) varied among the seaweed species analyzed (Table 2). All seaweed species had 16:0 as the major fatty acid, with the highest value in G. vermiculophylla and the lowest in R. riparium (64.51% ± 0.82 and 29.17% ± 5.15). The content of polyunsaturated fatty acids (PUFAs) was highest in R. riparium (45.19% ± 10.64), with important contributions of 18:2n-6 (23.42% ± 0.49), 18:3n-3 (13.57% ± 7.03), 20:4n-6 (2.45% ± 0.33), and 20:5n-3 (2.87% ± 1.70). The lowest PUFA content (38.99% ± 0.64) was observed in C. sertularioides, with contributions of 18:2n-6 (13.50% ± 0.67), 18:3n-3 (16.72% ± 0.71), 20:4n-6 (1.76% ± 0.07), and 20:5n-3 (3.75 ± 0.04). In P. durvillaei, PUFA content was 22.94% ± 0.38, with lower contents of 18:2n-6 (3.90% ± 0.05) and 18:3n-3 (3.34% ± 0.05), but higher content of 20:4n-6 (8.87% ± 0.09), and a value of 20:5n-3 (2.18% ± 0.12) similar to those previously reported for other seaweeds. The highest 22:6n-3 content (8.90% ± 2.19) was found in S. filamentosa, with significantly (P < 0.05) lower values for the remaining seaweed species. The content of highly unsaturated fatty acids (HUFAs) was observed within the range of 1-11%, with the highest values for P. durvillaei and S. filamentosa, followed by C. sertularioides, R. riparium, and C. isabelae, and the lowest values for G. vermiculophylla and U. expansa. The polyunsaturation index (PUI) showed higher availability of polyunsaturated fatty acids in R. riparium, C. sertularioides, and P. durvillaei (118-138) than in the remaining seaweed species (Table 2).
Table 2 Fatty acid composition (percent wet weight, % ww) in seaweed species off Sinaloa, Mexico.
| Green seaweeds | Red seaweeds | Brown seaweed | |||||||
| Ulva expansa | Caulerpa sertularioides | Rhizoclonium riparium | Codium isabelae | Spyridia filamentosa | Gracilaria vermiculophylla | Padina durvillaei | |||
| 14:0 | 2.24 ± 0.21c | 3.24 ± 0.11bc | 8.05 ± 1.53ab | 2.77 ± 0.26c | 10.57 ± 0.22a | 5.83 ± 0.13b | 2.96 ± 0.21bc | ||
| 16:0 | 48.89 ± 0.59b | 43.19 ± 0.36bc | 29.17 ± 5.15d | 33.15 ± 2.10c | 50.92 ± 1.65b | 64.51 ± 0.82a | 37.39 ± 0.33c | ||
| 18:0 | 3.76 ± 1.07ab | 1.04 ± 0.02c | 1.34 ± 0.23c | 4.57 ± 0.15a | 1.75 ± 0.10b | 5.08 ± 0.57ª | 1.46 ± 0.08c | ||
| 22:0 | 4.69 ± 0.49b | 0.54 ± 0.03c | 0.55 ± 0.07c | 9.23 ± 0.22a | 0.17 ± 0.06c | 0.10 ± 0.01c | 0.37 ± 0.44c | ||
| 16:1n-9 | 0.70 ± 0.04c | 1.65 ± 0.06b | 1.25 ± 0.16bc | 5.73 ± 0.13a | 0.54 ± 0.26d | 0.39 ± 0.01d | 0.68 ± 0.02c | ||
| 16:1n-7 | 8.45 ± 0.90ab | 5.39 ± 0.20bc | 3.40 ± 0.44c | 2.56 ± 0.92d | 6.38 ± 0.18b | 2.53 ± 0.05d | 10.20 ± 0.18ª | ||
| 18:1n-9 | 3.19 ± 0.10d | 3.78 ± 0.01c | 6.73 ± 1.51bc | 19.30 ± 0.90ª | 9.29 ± 0.20c | 15.05 ± 0.01b | 20.64 ± 0.32ª | ||
| 18:1n-7 | 18.89 ± 1.45a | 2.19 ± 0.12b | 4.32 ± 2.07b | 5.68 ± 0.70b | 6.19 ± 0.07b | 1.68 ± 0.12b | 3.35 ± 0.20b | ||
| 18:2n-6 | 2.49 ± 0.10d | 13.50 ± 0.67b | 23.42 ± 0.49a | 4.58 ± 0.74c | 0.86 ± 0.03e | 1.03 ± 0.05e | 3.90 ± 0.05cd | ||
| 18:3n-6 | 0.25 ± 0.03b | 1.44 ± 0.01a | 1.38 ± 0.50a | 0.77 ± 0.09ab | 0.06 ± 0.00b | 0.13 ± 0.02b | 0.73 ± 0.02ab | ||
| 18:3n-3 | 3.51 ± 0.03c | 16.72 ± 0.71a | 13.57 ± 7.03ab | 6.04 ± 1.15b | 2.94 ± 0.09c | 0.18 ± 0.04c | 3.34 ± 0.05c | ||
| 18:4n-3 | 1.77 ± 0.13b | 1.45 ± 0.05b | 1.11 ± 0.57bc | 0.37 ± 0.01c | 0.35 ± 0.01c | 0.08 ± 0.01c | 3.72 ± 0.10a | ||
| 20:4n-6 | 0.33 ± 0.08c | 1.76 ± 0.07bc | 2.45 ± 0.33bc | 3.02 ± 1.13b | 0.75 ± 0.15c | 3.17 ± 0.28b | 8.87 ± 0.09ª | ||
| 20:5n-3 | 0.68 ± 0.06ab | 3.75 ± 0.04ª | 2.87 ± 1.70ab | 0.57 ± 0.04b | 0.34 ± 0.06b | 0.14 ± 0.03b | 2.18 ± 0.12ab | ||
| 22:6n-3 | 0.14 ± 0.02b | 0.37 ± 0.03b | 0.39 ± 0.08b | 1.67 ± 0.13b | 8.90 ± 2.19ab | 0.11 ± 0.03b | 0.19 ± 0.12b | ||
| SAT | 59.59 ± 1.63b | 48.00 ± 0.43bc | 39.11 ± 6.49c | 49.71 ± 1.91bc | 63.41 ± 1.88b | 75.52 ± 0.28a | 42.18 ± 0.07c | ||
| MUFA | 31.23 ± 1.39ª | 13.01 ± 0.32c | 15.70 ± 4.15bc | 33.27 ± 1.33a | 22.40 ± 0.45b | 19.65 ± 0.15bc | 34.88 ± 0.44a | ||
| PUFA | 9.18 ± 0.24c | 38.99 ± 0.64ab | 45.19 ± 10.64a | 17.01 ± 1.27c | 14.19 ± 2.27c | 4.83 ± 0.28c | 22.94 ± 0.38b | ||
| HUFA | 1.16 ± 0.14c | 5.87 ± 0.12b | 5.72 ± 2.10b | 5.25 ± 1.14b | 9.99 ± 2.33a | 3.42 ± 0.26bc | 11.24 ± 0.29a | ||
| n-3/n-6 | 1.99 ± 0.11b | 1.34 ± 0.09bc | 0.63 ± 0.30bc | 1.05 ± 0.16bc | 7.46 ± 0.95a | 0.12 ± 0.02c | 0.70 ± 0.03bc | ||
| PUI | 60 ± 2c | 128 ± 1ª | 138 ± 32ª | 89 ± 4bc | 93 ± 13bc | 37 ± 1d | 118 ± 1b | ||
Values are means of 3 determinations ± standard error, and they were analyzed by unifactorial analysis of variance, followed by a Tukey test to assess significant differences. Means sharing different letters within a row were significantly different (P < 0.05). n.d., not detected; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; HUFA, highly unsaturated fatty acid; PUI, polyunsaturated index.
Sterols
Significant (P < 0.05) differences were observed in the sterol contents of the seaweeds analyzed (Table 3). The highest cholesterol+dehydrocholesterol contents (>90%) were found in S. filamentosa and G. vermiculophylla. In contrast, β-sitosterol was the major sterol (71-77%) in C. sertularioides, R. riparium, and C. isabelae. Among seaweed species, stigmasterol was highest in R. riparium (14%) and was identified as the second most important sterol in this species. In U. expansa and P. durvillaei, the major sterols were fucosterol+isofucosterol (79%), which are geometric isomers that are indistinguishable by the performed method (Jonker et al. 1985), and the signal of both compounds appeared at same retention time.
Table 3 Sterol composition (percent wet weight, % ww) in seaweed species off Sinaloa, Mexico.
| Green seaweeds | Red seaweeds | Brown seaweed | |||||||
| Ulva expansa | Caulerpa sertularioides | Rhizoclonium riparium | Codium isabelae | Spyridia filamentosa | Gracilaria vermiculophylla | Padina durvillaei | |||
| Cholesterol+dehydrocholesterol | 7.15 ± 0.42b | 0.61 ± 0.10c | 2.87 ± 0.81c | 0.80 ± 0.07c | 90.57 ± 0.17a | 91.88 ± 1.98a | 1.51 ± 0.21c | ||
| Brassicasterol | 1.68 ± 0.70b | 2.69 ± 0.29ab | 1.42 ± 0.63b | 1.21 ± 0.18b | 1.93 ± 0.24ab | 3.14 ± 0.49ab | 4.10 ± 0.53a | ||
| Campesterol | 4.53 ± 0.80b | 0.52 ± 0.1d | 3.07 ± 0.43c | 14.80 ± 0.39a | 3.52 ± 0.40bc | 0.87 ± 0.38d | 1.46 ± 0.28cd | ||
| Stigmasterol | n.d. | 1.08 ± 0.13b | 14.27 ± 0.65a | n.d. | n.d. | 1.12 ± 0.21b | 1.23 ± 0.39b | ||
| β-Sitosterol | 4.34 ± 1.10c | 76.86 ± 0.50a | 75.60 ± 0.88a | 70.92 ± 1.04b | 0.75 ± 0.07d | 0.99 ± 0.32d | 1.50 ± 0.31cd | ||
| Fucosterol+isofucosterol | 78.84 ± 4.46a | 15.00 ± 0.35b | 2.76 ± 0.17c | 0.59 ± 0.16c | 0.80 ± 0.07c | 2.00 ± 1.05c | 79.22 ± 1.34a | ||
| Unidentified sterols | 6.06 ± 2.08ab | 3.16 ± 0.34b | n.d. | 11.67 ± 1.41ª | 1.75 ± 0.43c | n.d. | 11.61 ± 2.08a | ||
Values are means of 3 determinations ± standard error, and they were analyzed by unifactorial analysis of variance, followed by a Tukey test to assess significant differences. Means sharing different letters within a row were significantly different (P < 0.05). n.d., not detected.
Pigments
All identified pigments varied significantly (P < 0.05) among the seaweed species analyzed (Table 4). The 2 major pigments found in all seaweed species were chlorophyll a and b. The former was particularly higher in G. vermiculophylla (60.27% ± 7.27), followed by S. filamentosa, P. durvillaei, U. expansa, and C. sertularioides (39-48%), and the lowest values were found in R. riparium and C. isabelae (31-33%). Chlorophyll b was higher in all green seaweeds (28-43%) and lower in S. filamentosa (<3%); it was not detected in G. vermiculophylla and P. durvillaei. Lutein content was found in considerable proportions in S. filamentosa, U. expansa, and R. riparium (33%, 18%, and 16%, respectively). Chlorophyllide a (25%) and fucoxanthin (34%) were found in considerable proportions in G. vermiculophylla and P. durvillaei, respectively. The β-carotene content values were within the range of 1.9-10.0%, with higher proportions in S. filamentosa and G. vermiculophylla, but it was not detected in C. isabelae (Table 4).
Table 4 Pigment composition (percent wet weight, % ww) in seaweed species off Sinaloa, Mexico.
| Green seaweeds | Red seaweeds | Brown seaweed | |||||||
| Ulva expansa | Caulerpa sertularioides | Rhizoclonium riparium | Codium isabelae | Spyridia filamentosa | Gracilaria vermiculophylla | Padina durvillaei | |||
| Chlorophyllide a | n.d. | 0.77 ± 0.10b | 0.92 ± 0.02b | n.d. | n.d. | 25.18 ± 7.80a | n.d. | ||
| Siphoxathin | n.d. | 3.64 ± 0.33b | n.d. | 14.46 ± 0.59a | n.d. | n.d. | n.d. | ||
| Fucoxanthin | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | 33.88 ± 0.16 | ||
| Neoxanthin | n.d. | 3.66 ± 0.76 | n.d. | n.d. | n.d. | n.d. | n.d. | ||
| Violaxanthin | 2.53 ± 0.05d | 8.05 ± 0.08a | 6.12 ± 0.11b | 1.24 ± 0.12e | n.d. | n.d. | 4.50 ± 0.10c | ||
| 19-Hex-fucoxanthin | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | 5.80 ± 0.12 | ||
| Zeaxanthin | n.d. | n.d. | n.d. | n.d. | n.d. | 7.83 ± 0.22 | n.d. | ||
| Lutein | 17.60 ± 0.46b | 2.38 ± 0.49c | 15.62 ± 0.07b | n.d. | 33.28 ± 1.16a | n.d. | n.d. | ||
| Dihydrolutein | 2.97 ± 0.15ab | n.d. | 2.50 ± 0.26b | n.d. | 3.70 ± 0.22a | n.d. | n.d. | ||
| Siphonein | n.d. | 5.80 ± 0.18b | n.d. | 9.05 ± 0.36a | n.d. | n.d. | n.d. | ||
| Astaxanthin | n.d. | 1.34 ± 0.51ab | 1.52 ± 0.12a | n.d. | 0.43 ± 0.10b | n.d. | 0.42 ± 0.03b | ||
| Chlorophyll a | 40.06 ± 0.51b | 38.88 ± 0.77b | 33.45 ± 0.38c | 31.08 ± 0.76c | 47.67 ± 1.00ab | 60.27 ± 7.27a | 40.20 ± 0.23b | ||
| Chlorophyll b | 32.59 ± 0.23c | 27.77 ± 0.34d | 36.91 ± 0.46b | 42.59 ± 0.36a | 2.99 ± 0.11e | n.d. | n.d. | ||
| Chlorophyll c2 | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | 11.05 ± 0.24 | ||
| α-Carotene | 0.83 ± 0.03c | 1.13 ± 0.26b | 1.22 ± 0.09b | 1.62 ± 0.12ab | 2.30 ± 0.44ª | n.d. | n.d. | ||
| β-Carotene | 3.41 ± 0.24cd | 5.58 ± 0.45bc | 1.86 ± 0.28d | n.d. | 9.62 ± 0.21ª | 6.73 ± 0.79b | 4.16 ± 0.12c | ||
Values are means of 3 determinations ± standard error, and they were analyzed by unifactorial analysis of variance, followed by a Tukey test to assess significant differences. Means sharing different letters within a row were significantly different (P < 0.05). n.d., not deected.
Biochemical composition obtained from principal component analysis
Fatty acids, sterols, and pigments are useful biomarkers for the identification of seaweed species within each taxonomic group. The use of some of the biochemical components not only allows discrimination among assessed variables but also helps cluster or group seaweed species according to common variability in their biochemical composition. Factor analysis showed that the variation of the selected biochemical components was described by 3 principal components (Table 5). The first component explained 39.3% of total variance, with significant contributions of chlorophyll a and β-carotene (both with 0.79), β-sitosterol (-0.90), total PUFAs (-0.72), and chlorophyll b (-0.88). The second component explained 22.3% of total variance and had significant and inverse contributions of 20:4n-6 (-0.70), 20:5n-3 (-0.70), and total HUFA (-0.87). The third component explained 18.5% of total variance and had significant contributions of 22:6n-3 (0.76) and fucosterol+isofucosterol (-0.79) (Table 5). The factor scores obtained with the selected biochemical compounds showed common variability in G. vermiculophylla and S. filamentosa, while a similar composition was obtained with C. sertularioides, R. riparium, and C. isabelae. In contrast, the compositions of U. expansa and P. durvillaei were distinctive when compared with the other seaweed species analyzed (Fig. 1).
Table 5 Factor loadings (verimax normalized) using principal component extraction.
| Variation | Factor 1 | Factor 2 | Factor 3 |
| β-sitosterol | -0.90 | 0.01 | 0.27 |
| Fucosterol + isofucosterol | 0.14 | -0.18 | -0.79 |
| 20:4n-6 | 0.12 | -0.70 | -0.54 |
| 20:5n-3 | -0.58 | -0.70 | 0.02 |
| 22:6n-3 | 0.41 | -0.10 | 0.76 |
| PUFA | -0.72 | -0.57 | 0.23 |
| HUFA | 0.19 | -0.87 | 0.28 |
| Chlorophyll a | 0.79 | 0.03 | -0.06 |
| Chlorophyll b | -0.88 | 0.40 | 0.01 |
| β-carotene | 0.79 | -0.17 | 0.38 |
| Eigenvalue | 3.90 | 2.20 | 1.80 |
| Total variance (%) | 39.30 | 22.30 | 18.50 |
| Cumulative eigenvalue | 3.90 | 6.20 | 8.00 |
| Cumulative variance (%) | 39.30 | 61.70 | 80.20 |
Bold loadings are significant (≥0.7). PUFA, polyunsaturated fatty acids; HUFA, highly unsaturated fatty acids.
Discussion
In Mexico, the biochemical compositions of some seaweed species in the Gulf of Mexico and the northeastern Pacific Ocean have been assessed, but none of the seaweed species distributed in the lower tropical region of the Gulf of California and the Pacific Ocean, particularly those from lagoons in Sinaloa, Mexico, have been evaluated. Gross chemical composition is useful for establishing not only the nutritional value of seaweeds but also that of specific components like fatty acids, sterols, and pigments, which could also be useful as biomarkers for establishing specific variations in seaweeds between biogeographic regions. The differences in the contents of biochemical components could be attributed to several intrinsic factors, such as species-specific variations, reproductive stage, and the portion of thallus analyzed, and extrinsic factors, mainly sample size, analytical technique applied, unattached epibionts or epiphytes, and the specific climatic conditions at each sampling site.
The nutritional value of the seaweeds assessed in this study showed that crude protein contents were 4-12%, 10%, and 6% for green, red, and brown seaweeds, respectively (Table 1). These values were lower than those observed in similar tropical species (U. expansa and Ulva lobata, 21-22%; Colpomenia tuberculata and P. durvillaei, 11%; and G. vermiculophylla, 23%; Peraza-Yee 2014). Compared with seaweeds inhabiting the temperate zone of the northeastern Pacific coast of Mexico, seasonal contents of crude proteins are in the range of 10-15% for Eisenia arborea, 8-14% for Macrocystis pyrifera, and 19-21% for Gelidium robustum (Serviere-Zaragoza et al. 2002). In contrast to variations in crude protein contents, the levels of total lipids, carbohydrates, and ashes were in a range (0.26-1.50%, 52-69%, and 25-39%, respectively) comparable with other tropical (0.46-1.02%, 50-73%, and 19-34%, respectively; Peraza-Yee 2014) and temperate (0.3-1.1%, 46-68%, and 7-38%, respectively; Serviere-Zaragoza et al. 2002) seaweed species. Aside from the intrinsic and extrinsic factors mentioned above that could explain the differences observed in protein contents, the conventional nitrogen-to-protein conversion factor (NPF) of 6.25 should be considered, as it usually tends to overestimate protein levels. We decided to use this NPF to compare our results with those in the studies mentioned above. Nevertheless, when the specific NPF of 5 suggested for different species of seaweeds (Lourenço et al. 2002, Angell et al. 2016) was applied to data for seaweeds analyzed in this study, protein levels were lower (1% to 3%) compared with the levels obtained with the conventional NPF (Table 1).
Although total lipid contents were similar between tropical and temperate seaweed species, some important differences were observed in fatty acid composition. Lower PUFA contents (Table 2) were obtained for the tropical red seaweeds, S. filamentosa and G. vermiculophylla (14% and 5%, respectively), compared with the seasonal contents observed for the temperate species, Gracilaria sp. (46-55%) and Gellidium robustum (38-42%) (Serviere-Zaragoza et al. 2014). In the 4 tropical green seaweeds, PUFA content values were in the range of 9-45% (Table 2), with increasing values among species as follows: U. expansa, C. isabelae, C. sertularioides, and R. riparium. These values are in the range of those reported for the tropical seaweeds U. expansa and U. lobata in the summer (15-28%) and winter (38-48%) seasons (Peraza-Yee 2014). Interestingly, PUFA content (16-48%) in the temperate seaweed Ulva lactuca was not different from the contents in the tropical species of the same genera (Serviere-Zaragoza et al. 2014). For the brown seaweed, P. durvillaei, Peraza-Yee (2014) reported the same content of saturated fatty acids (c.a. 42%) but higher contents of monounsaturated fatty acids (25%) and PUFAs (34%), compared with the contents reported in the present study. This could be due to differences in the harvest season but also to differences in environmental factors (i.e., temperature, nutrient availability, and salinity (Guschina and Harwood 2006). Further investigations are needed to establish seasonal variations and the effect of specific environmental factors for comparing the biochemical composition of these seaweeds between temperate and tropical environments.
The nutritional value of seaweeds in relation to fatty acid composition, particularly those with ≥20 carbons and the n-3 series, should also be considered. All seaweed species had 20:5n-3 (eicosapentaenoic acid) and 22:6n-3 (docosahexaenoic acid), fatty acids with known high nutritional value, which could particularly be attributed to the green seaweeds C. sertularioides and R. riparium and the brown seaweed P. durvillaei. The red seaweed S. filamentosa had the highest nutritional value because of its higher 22:6n-3 content (8.9%), while G. vermiculophylla showed the lowest since it had the lowest 20:5n-3 and 22:6n-3 contents. In general, Rhodophyta has been identified as a good source of 20:5n-3; however, the content of this PUFA is highly variable (Khotimchenko and Gusarova 2004, Kumari et al. 2010, Imbs et al. 2012, Pereira et al. 2012). The seasonal variation of 22:6n-3 in S. filamentosa off Turkey showed values of 0.69% for the spring and 3.82% for the summer (Polat and Ozogul 2013). For the tropical red seaweeds Ahnfeltia plicata, Hypnea musciformis, and Hypnea esperi, the reported 22:6n-3 contents were 0.75%, 0.96%, and 1.56%, respectively, while the 22:6n-3 contents reported for 4 brown and 9 green seaweed species were within the range of 0.05-0.78% and 0.81-5.81%, respectively (Kumari et al. 2010). The PUFA content in C. sertularioides determined in this study was similar to those reported for 3 different Caulerpa species (39% vs 29-38%, respectively), though the content of 22:6n-3 was lower (0.37% vs 2.8-3.6%, respectively), and the 20:5n-3 assessed in C. sertularioides (3.75%) was not observed in the other 3 Caulerpa species (Nagappan and Vairappan 2014).
The World Health Organization has recommended a ratio of n-3/n-6 ≥ 1 for the prevention of several cardiovascular disorders, inflammatory diseases, and cancer (Simopoulos 2009, Gómez-Candela et al. 2011). Taking into account the n-3/n-6 ratios and the PUI, which indicates the degree of unsaturation of fatty acids, reported in this study, all seaweed species investigated here could be considered appropriate for human consumption and the promotion of good health, particularly the green seaweeds C. sertularioides and R. riparium and the brown seaweed P. durvillaei. Although the n-3/n-6 ratio for the green seaweed U. expansa was >1, this value was attributed to the content of fatty acids with 18 carbons and not to the content of HUFAs (fatty acids with ≥20 carbons and ≥4 double bonds). However, this seaweed species should be still considered a good-health promoter because of its unusual fucosterol content and its considerable amount of pigments (i.e., chlorophyll a and b, lutein, and β-carotene), which are compounds with high antioxidant potential. On the other hand, the contents of phytosterols (i.e., brassicasterol, campesterol, stigmasterol, β-sitosterol, fucosterol, and isofucosterol) in green and brown seaweeds and the high contents of cholesterol+dehydrocholesterol in red seaweeds are good enough to consider these specimens as important health promoters since phytosterols have been demonstrated not only to reduce the content of total cholesterol and LDL-cholesterol in plasma, decreasing cardiovascular diseases, but also to be cytotoxic and pro-apoptotic when phytosterols oxide into oxyphytosterols, constituting alternative approaches for the prevention of carcinogenesis (García-Llatas and Rodríguez-Estrada 2011).
From another point of view, biochemical composition as a source of chemoprotectants has been largely assessed as a tool to explain different bioactivities reported in seaweed species. According to the selected biochemical components (Table 5) and results obtained with the principal component analysis, the biochemical composition was similar in red (G. vermiculophylla and S. filamentosa) and green (C. sertularioides, R. riparium, and C. isabelae) seaweeds, but it was particularly different in U. expansa and the brown seaweed (P. durvillaei) (Fig. 1). This common variability and differences in biochemical compositions explain the chemopreventive activities associated with these seaweed species. In a previous study on the same seaweed species as the ones analyzed in the present study, the antioxidant capacity was explained by the content of chlorophylls and flavonoids, but some lipophilic compounds (i.e., fatty acids and phytosterols) were not discarded as effectors of this bioactivity (Osuna-Ruiz et al. 2016). The antioxidant capacity, observed mainly in P. durvillaei and U. expansa, could also be explained by the fucosterol+isofucosterol and fucoxanthin contents, mainly in the brown seaweed. Fucoxanthin is the dominant carotenoid in brown seaweeds, and despite the susceptibility to oxidation on these seaweeds, fucoxanthin is fairly stable in the presence of common antioxidants, such as polyphenols. 1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity has been correlated with fucoxanthin content in the 2 brown seaweeds Nizamuddinia zanardinii and Cystoseira indica (Fariman et al. 2016). Fucosterol is the main phytosterol found in Phaeophyceae. It is biosynthesized through the alkylation of 24-methylenecholesterol and is known to possess anticancer and antioxidant activity (Abdul et al. 2016). However, in primitive green algae like Ulva sp., the alkylation of 24-methylenecholesterol leads to the production of a fucosterol isomer called “isofucosterol” (Lopes et al. 2013). On the other hand, fucosterol is the main sterol in brown seaweeds, but fucosterol is also characteristic of some green seaweed species, particularly U. expansa, since high proportions of this compound were determined in the present and in previous studies (78.8% in the present study, 73.8% in the study by Ilias et al. 1985); this proportions, however, constrast with the lower value (26%) (and traces of fucoesterol) found in Ulva lactuca from the Adriatic sea (Kapetanović et al. 2005). In the present research, PUFA content was found in considerable amounts in P. durvillaei but not in U. expansa (23% vs 9%). In addition to the antioxidant effect produced by pigments and sterols in P. durvillaei and U. expansa, PUFA contents could exhibit antioxidant capacity. In particular, n-3 series fatty acids showed strong radical scavenging capacity and significant inhibition of reactive oxygen species/reactive nitrogen species (ROS/RNS) production in human endothelial cells, suggesting an effect on inflammation processes that could reduce atherosclerosis and cardiovascular diseases (Richard et al. 2008).
Antioxidants inhibit oxidative reaction cascades, which decrease free-radical concentrations that produce lipid peroxidation and DNA damage or even cell death, and, consequently, induce different chronic diseases and cellular mutations that eventually cause several types of cancer (Duthie et al. 1996). Strong antimutagenic activity against aflatoxin B1 (AFB1) on Salmonella typhimurium TA98 and TA100 tester strains was detected in acetone crude extracts obtained from green (C. sertularioides and R. riparium) and red (S. filamentosa) seaweeds (Osuna-Ruiz et al. 2016). Moreover, antiproliferative activity on M12.C3.F6 cells (murine B-cell lymphoma) was also reported for extracts obtained from these seaweed species, showing a dose-response type of relationship, reaching the highest effect (25% and 29% cell proliferation inhibition) at a concentration of 100 µg·mL-1 for S. filamentosa and R. riparium, respectively; the best inhibition of cancerous cells was observed at the lowest concentrations assayed in C. sertularioides (63% and 28% for 12.5 and 25 µg·mL-1, respectively; Osuna-Ruiz et al. 2016). The highest content of PUFAs and lutein, together with chlorophylls and β-sitosterol contents, could explain the chemopreventive activity reported for R. riparium compared with that of C. sertularioides. This is also consistent with the lowest bioactivity reported for S. filamentosa, a seaweed species with the lowest values of PUFA and chlorophyll contents and no β-sitosterol experimentally assessed in the present research. High antimutagenic activity on S. typhimurium TA98, TA100, and TA102 has been reported for photosynthetic pigments (Panguestuti and Kim 2011) and for lutein, astaxanthin, and β-carotene (Bhagavathy et al. 2011). On the other hand, the highest values of β-sitosterol and fucosterol, without discarding the content of chlorophylls and carotenoids assessed in C. sertularioides, could explain the high inhibition of the proliferation of cancerous cells. Chlorophyll a, β-carotene, and lutein were identified as the main chemoprotective compounds against mutagen-induced umu C gene expression in S. typhimurium TA1535/pSK 1002 (Okai et al. 1996). Also, the possible presence of caulerpine, another abundant compound in Caulerpa spp., was not discarded. This non-toxic compound has been characterized in other species (Vidal et al. 1984) and has shown antinociceptive and anti-inflammatory properties in Caulerpa racemosa (de Souza et al. 2009). Moreover, caulerpine and PUFA (18:3n-3, 18:4n-3, and 20:5n-3) contents may explain the potent antimicrobial activity against human food pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Streptococcus sp., and Salmonella sp.), antioxidant activity (nitric oxide production and lactate dehydrogenase), and anti-inflammatory activity against the murine macrophage cell line RAW 264.7 assessed in methanolic extracts of some Caulerpa species (Nagappan and Vairappan, 2014).
In conclusion, the seaweed species studied here differ in biochemical composition and nutritional value with chemopreventive potential activity. In particular, fatty acids, sterols, and pigments in the analyzed tropical seaweeds could be useful as biomarkers to establish specific variations in seaweeds between biogeographic regions (for review see the following references: Gillan et al. 1984, Elenkov et al. 1996, Graeve et al. 2002, Galloway et al. 2012, Kai-Xion et al. 2019). According to the biochemical components that were chosen as biomarkers with known bioactivity, red (G. vermiculophylla and S. filamentosa) and green seaweeds (C. sertularioides, R. riparium, and C. isabelae) were similar in composition, but they were particularly different from the green (U. expansa) and the brown (P. durvillaei) seaweeds. The main biochemical compounds that contributed to total variability were β-sitosterol, PUFAs and HUFAs, 20:4n-6, 20:5n-3, chlorophyll a and b, and β-carotene, and to a lesser extent fucosterol+isofucosterol and 22:6n-3. Differences in the contents of these compounds could explain the chemopreventive activities previously reported for these seaweed species, suggesting neutraceutical applications in the treatment of human diseases. According to the n-3/n-6 ratio, the PUI, and the content of phytosterols, most of the seaweed species studied could be considered promoters of good health and appropriate for human consumption, particularly the green seaweeds C. sertularioides and R. riparium, and the brown seaweed P. durvillaei. Their cultivation in controlled conditions or in integrated multi-trophic culture systems is strongly recommended to evaluate the technical and economic feasibility, allowing for the preservation of the wild beds of these tropical seaweed species.
