# Pigments Content (Chlorophylls, Fucoxanthin and Phycobiliproteins) of Different Commercial Dried Algae

^{*}

^{†}

## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Reagents

_{3}) was from Honeywell Riedel-de-Haën (Seelze, Germany). Disodium hydrogen phosphate (Na

_{2}HPO

_{4}) and sodium tetraborate decahydrate (Na

_{2}B

_{4}O

_{7}·10H

_{2}O) were from Merck (Darmstadt, Germany). Potassium carbonate was from Panreac (Barcelona, Spain). Acid-free methanol was prepared by adding magnesium carbonate to methanol (kept at 4 °C after preparation). Phosphate buffer (pH 6.8) was prepared with 10 mM Na

_{2}HPO

_{4}, 10 mM Na

_{2}B

_{4}O

_{7}·10H

_{2}O and 5 mM NaN

_{3}. Ultrapure water, used to prepare DMSO–water (4:1, v/v), was treated in a Milli-Q water purification system (Millipore, Bedford, MA, USA).

#### 2.2. Samples

#### 2.3. Pigments Extraction

#### 2.3.1. 100%. Methanol

- $\mathrm{Chl}a\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=-2.0780\times \left({\mathrm{A}}_{632}{-\mathrm{A}}_{750}\right)-6.5079\times \left({\mathrm{A}}_{652}{-\mathrm{A}}_{750}\right)+16.2127\times \left({\mathrm{A}}_{665}{-\mathrm{A}}_{750}\right)-2.1372\times \left({\mathrm{A}}_{696}{-\mathrm{A}}_{750}\right)(\pm 0.0070)$
- $\mathrm{Chl}b\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=-2.9450\times \left({\mathrm{A}}_{632}{-\mathrm{A}}_{750}\right)-32.1228\times \left({\mathrm{A}}_{652}{-\mathrm{A}}_{750}\right)+13.8255\times \left({\mathrm{A}}_{665}{-\mathrm{A}}_{750}\right)-3.0097\times \left({\mathrm{A}}_{696}{-\mathrm{A}}_{750}\right)\left(\pm 0.0212\right)$
- $\mathrm{Chl}c\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=34.0115\times \left({\mathrm{A}}_{632}{-\mathrm{A}}_{750}\right)-12.7873\times \left({\mathrm{A}}_{652}{-\mathrm{A}}_{750}\right)+1.4489\times \left({\mathrm{A}}_{665}{-\mathrm{A}}_{750}\right)-2.5812\times \left({\mathrm{A}}_{696}{-\mathrm{A}}_{750}\right)(\pm 0.0120)$
- $\mathrm{Chl}d\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=-0.3411\times \left({\mathrm{A}}_{632}{-\mathrm{A}}_{750}\right)+0.1129-\left({\mathrm{A}}_{652}{-\mathrm{A}}_{750}\right)-0.2538\times \left({\mathrm{A}}_{665}{-\mathrm{A}}_{750}\right)+12.9508\times \left({\mathrm{A}}_{696}{-\mathrm{A}}_{750}\right)(\pm 0.0031)$
- $\mathrm{Total}\mathrm{Chl}\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=\mathrm{Chl}a+\mathrm{Chl}b+\mathrm{Chl}c+\mathrm{Chl}d$
- $\mathrm{Carotenoids}\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=4\times \left({\mathrm{A}}_{480}{-\mathrm{A}}_{750}\right)$

#### 2.3.2. 100%. Ethanol

- $\mathrm{Chl}a\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=0.0604\times \left({\mathrm{A}}_{632}{-\mathrm{A}}_{750}\right)-4.5224\times \left({\mathrm{A}}_{649}{-\mathrm{A}}_{750}\right)+13.2969\times \left({\mathrm{A}}_{665}{-\mathrm{A}}_{750}\right)-1.7453\times \left({\mathrm{A}}_{696}{-\mathrm{A}}_{750}\right)(\pm 0.0053)$
- $\mathrm{Chl}b\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=-4.1982\times \left({\mathrm{A}}_{632}{-\mathrm{A}}_{750}\right)+25.7205\times \left({\mathrm{A}}_{649}{-\mathrm{A}}_{750}\right)-7.4096\times \left({\mathrm{A}}_{665}{-\mathrm{A}}_{750}\right)-2.7418\times \left({\mathrm{A}}_{696}{-\mathrm{A}}_{750}\right)(\pm 0.0142)$
- $\mathrm{Chl}c\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=28.4593\times \left({\mathrm{A}}_{632}{-\mathrm{A}}_{750}\right)-9.9944\times \left({\mathrm{A}}_{649}{-\mathrm{A}}_{750}\right)-1.9344\times \left({\mathrm{A}}_{665}{-\mathrm{A}}_{750}\right)-1.8093\times \left({\mathrm{A}}_{696}{-\mathrm{A}}_{750}\right)(\pm 0.0084)$
- $\mathrm{Chl}d\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=-0.2007\times \left({\mathrm{A}}_{632}{-\mathrm{A}}_{750}\right)+0.0848\times \left({\mathrm{A}}_{649}{-\mathrm{A}}_{750}\right)-0.1909\times \left({\mathrm{A}}_{665}{-\mathrm{A}}_{750}\right)+12.1302\times \left({\mathrm{A}}_{696}{-\mathrm{A}}_{750}\right)(\pm 0.0023)$
- $\mathrm{Total}\mathrm{Chl}\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=\mathrm{Chl}a+\mathrm{Chl}b+\mathrm{Chl}c+\mathrm{Chl}d$

#### 2.3.3. 90%. Acetone

- $\mathrm{Chl}a\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=-0.3319\times \left({\mathrm{A}}_{630}{-\mathrm{A}}_{750}\right)-1.7485\times \left({\mathrm{A}}_{647}{-\mathrm{A}}_{750}\right)+11.9442\times \left({\mathrm{A}}_{664}{-\mathrm{A}}_{750}\right)-1.4306\times \left({\mathrm{A}}_{691}{-\mathrm{A}}_{750}\right)(\pm 0.0020)$
- $\mathrm{Chl}b\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=-1.2825\times \left({\mathrm{A}}_{630}{-\mathrm{A}}_{750}\right)-19.8839\times \left({\mathrm{A}}_{647}{-\mathrm{A}}_{750}\right)-4.8860\times \left({\mathrm{A}}_{664}{-\mathrm{A}}_{750}\right)-2.3416\times \left({\mathrm{A}}_{691}{-\mathrm{A}}_{750}\right)(\pm 0.0076)$
- $\mathrm{Chl}c\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=23.5902\times \left({\mathrm{A}}_{630}{-\mathrm{A}}_{750}\right)-7.8516\times \left({\mathrm{A}}_{647}{-\mathrm{A}}_{750}\right)-1.5214\times \left({\mathrm{A}}_{664}{-\mathrm{A}}_{750}\right)-1.7443\times \left({\mathrm{A}}_{691}{-\mathrm{A}}_{750}\right)(\pm 0.0075)$
- $\mathrm{Chl}d\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=-0.5881\times \left({\mathrm{A}}_{630}{-\mathrm{A}}_{750}\right)+0.0902\times \left({\mathrm{A}}_{647}{-\mathrm{A}}_{750}\right)-0.1564\times \left({\mathrm{A}}_{664}{-\mathrm{A}}_{750}\right)+11.0473\times \left({\mathrm{A}}_{691}{-\mathrm{A}}_{750}\right)(\pm 0.0030)$
- $\mathrm{Total}\mathrm{Chl}\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=\mathrm{Chl}a+\mathrm{Chl}b+\mathrm{Chl}c+\mathrm{Chl}d$

#### 2.3.4. 100%. N,N-Dimethylformamide (DMF)

- $\mathrm{Chl}a\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=12.70\times \left({\mathrm{A}}_{664.5}{-\mathrm{A}}_{750}\right)-2.79\times \left({\mathrm{A}}_{647}{-\mathrm{A}}_{750}\right)$
- $\mathrm{Chl}b\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=20.70\times \left({\mathrm{A}}_{647}{-\mathrm{A}}_{750}\right)-4.62\times \left({\mathrm{A}}_{664.5}{-\mathrm{A}}_{750}\right)$
- $\mathrm{Total}\mathrm{Chl}\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=\mathrm{Chl}a+\mathrm{Chl}b$

#### 2.3.5. DMSO:-Water (4:1, v/v)

- $\mathrm{Fucoxanthin}\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=7.69\times \left({\mathrm{A}}_{480}{-\mathrm{A}}_{750}\right)-5.55\times [\left({\mathrm{A}}_{631}{-\mathrm{A}}_{750}\right)+\left({\mathrm{A}}_{582}{-\mathrm{A}}_{750}\right)-0.297\times \left({\mathrm{A}}_{665}{-\mathrm{A}}_{750}\right)]-0.377\times \left({\mathrm{A}}_{665}{-\mathrm{A}}_{750}\right)$

#### 2.3.6. Phosphate Buffer (pH = 6.8)

- $\mathrm{Phycoerythrin}\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=\frac{{\mathrm{A}}_{565}{-\mathrm{A}}_{750}}{{2.41\times 10}^{6}}{\times \mathrm{240,000}\times 10}^{3}$
- $\mathrm{Phycocyanin}\left(\mathsf{\mu}\mathrm{g}/\mathrm{mL}\right)=\frac{{\mathrm{A}}_{618}{-\mathrm{A}}_{750}}{{1.90\times 10}^{6}}{\times \mathrm{264,000}\times 10}^{3}$

#### 2.4. Statistical Analysis

## 3. Results and Discussion

^{2+}), resulting in pheophytin, or may uptake oxygen, resulting in allomerization products [63,64]. These degradation products are spectrally different from chlorophylls, thus interfering with all chlorophyll determinations. This leads to an enlarged peak of chl a and, consequently, in lower and wider peaks for the remaining chlorophylls (chls b, c and d) [41,47]. Moreover, although pheophytin and allomerization products might be present in both acetone and alcohol extracts, its presence is severely and negatively notorious in methanol extracts, and, particularly, in chl b determination. However, if these solvents are neutralized, especially methanol, the formation of these products can be avoided [41,47]. For this reason, in this study, the extraction of chlorophylls from the different algae was made using both methanol and neutralized methanol (acid-free methanol) in order to compare the efficiency of both solvents. Hence, it was possible to observe that, in general, the content of chl a was inferior in methanol acid-free extracts, being significantly lower in Laminaria ochroleuca acid-free methanol extracts. Contrariwise, the contents of chls b, c and d, when detected, were higher in the acid-free methanol extracts obtained from all the algae studied, with significant differences between the two solvents found for almost all of the samples. Therefore, these results show that, effectively, the acidity of methanol may have led to the formation of pheophytin and allomerization products, resulting in false higher values of chl a counterbalanced with false lower values of chls b, c and d in methanol extracts. In particular, Laminaria ochroleuca appeared to be the seaweed most affected by the acidity of methanol, since significant differences were found for all chlorophyll pigments (a, b, c and d). N,N-dimethylformamide (DMF) also seems to be a good extraction solvent for all the samples since the extracts presented the second higher contents of chlorophylls. This can be explained by the fact that chlorophyll is quite stable in this solvent [41], thus not occurring the degradation verified with the other solvents. However, the total content of chlorophylls in DMF extracts might be underestimated once only chls a and b were determined. To the best of our knowledge, no equation has been developed for the determination of chls c and d. Therefore, in further studies it would be interesting to ascertain if it is possible to develop equations that allow the simultaneous determination of chls a, b, c and d in DMF extracts. This would allow comparing results obtained using the different solvents in a more accurate manner. In contrast to the higher results observed for DMF extracts, the ethanolic ones presented the lowest content in chlorophylls for all the algae, except for Himanthalia elongata, which had results that were slightly higher (but not significantly different, p > 0.05) than methanol and acid-free methanol extracts. This might be related to the fact that ethanol is a weak inhibitor of chlorophyllase activity [41]. Thus, the hydrolysis of chlorophylls to the corresponding chlorophyllides and the subsequent degradation products might have led to the lowering of chlorophylls content in the sample, and, consequently, to the low results observed for ethanol extracts. However, the acidity of ethanol might have also influenced the results [41]. As already aforementioned, the acidity of alcohols may lead to the formation of pheophytin and allomerization products of chlorophylls which spectrally interfere with chlorophyll determination. Although these interferents are described to be more present in methanolic extracts, they might also have been formed in the ethanolic ones, possibly affecting the results [41]. In future studies, it would be interesting to investigate if any of these factors effectively affect the extraction of chlorophylls by ethanol. For instance, the chlorophyllase activity assay or the removal of chlorophyllase from the extracts through methodologies already described in the literature (e.g., filtration, centrifugation or denaturation at 60 °C) could be performed to explore the influence of this enzyme in the obtained results [41]. Moreover, it would be of interest to evaluate the influence of ethanol acidity on chlorophylls extraction as well, by comparing an ethanolic acid-free extract with an ethanolic extract, as herein performed for methanol.

## 4. Conclusions

## Author Contributions

## Funding

## Acknowledgments

## Conflicts of Interest

## References

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**Figure 1.**The chemical structure of the chlorophylls present in marine algae: (

**a**) Chlorophyll a; (

**b**) Chlorophyll b; (

**c**) Chlorophyll c1; (

**d**) Chlorophyll c2; (

**e**) Chlorophyll d.

**Figure 3.**Chemical structure of the phycobilins present in phycocyanin and phycoerythrin, respectively: (

**a**) Phycocyanobilin; (

**b**) Phycoerythrobilin.

**Table 1.**Content of chlorophylls and carotenoids extracted from three brown edible algae (Himanthalia elongata, Laminaria ochroleuca (Kombu), Undaria pinnatifida (Wakame)), a red one (Porphyra spp. (Nori)), and microalgae (Spirulina spp.) using different solvents. The results are expressed in μg/g of algae.

Methanol | Methanol Acid Free | Ethanol | Acetone | DMF | ||
---|---|---|---|---|---|---|

Himanthalia elongata | Chl a | 63.3 ± 4.7 ^{b} | 60.3 ± 5.9 ^{b} | 67.6 ± 3.2 ^{b} | 156.7 ± 15.3 ^{a} | 75.6 ± 6.2 ^{b} |

Chl b | n.d. | n.d. | n.d. | n.d. | n.d. | |

Chl c | n.d. | n.d. | n.d. | n.d. | n.d. | |

Chl d | 2.7 ± 0.2 ^{c} | 4.5 ± 0.5 ^{b} | 3.4 ± 0.3 ^{b,c} | 11.5 ± 0.4 ^{a} | n.d. | |

Total chls | 66.0 ± 4.8 ^{b} | 64.8 ± 5.5 ^{b} | 71.0 ± 3.3 ^{b} | 168.2 ± 15.0 ^{a} | 75.6 ± 6.2 ^{b} | |

Total carotenoids | 2.3 ± 0.0 ^{b} | 2.9 ± 0.3 ^{a} | ||||

Undaria pinnatifida | Chl a | 349.0 ± 7.6 ^{c} | 331.4 ± 28.8 ^{c} | 321.3 ± 19.2 ^{c} | 542.5 ± 32.7 ^{a} | 436.5 ± 26.8 ^{b} |

Chl b | n.d. | n.d. | n.d. | n.d. | n.d. | |

Chl c | n.d. | n.d. | n.d. | 15.98 ± 1.60 | n.d. | |

Chl d | 3.27 ± 0.45 ^{c} | 3.35 ± 0.37 ^{c} | 4.32 ± 0.23 ^{b} | 15.64 ± 0.83 ^{a} | n.d. | |

Total chls | 352.2 ± 8.0 ^{c} | 334.7 ± 29.1 ^{c} | 325.6 ± 19.4 ^{c} | 574.1 ± 33.2 ^{a} | 436.5 ± 26.8 ^{b} | |

Total carotenoids | 54.6 ± 1.3 ^{a} | 54.2 ± 3.4 ^{a} | ||||

Laminaria ochroleuca | Chl a | 143.1 ± 12.0 ^{bc} | 111.2 ± 3.0 ^{d} | 114.0 ± 4.5 ^{cd} | 183.5 ± 14.8 ^{a} | 160.5 ± 5.6 ^{ab} |

Chl b | 7.2 ± 0.4 ^{c} | 12.3 ± 0.1 ^{b} | n.d. | 14.1 ± 0.5 ^{b} | 22.4 ± 1.4 ^{a} | |

Chl c | 6.7 ± 0.6 ^{c} | 12.7 ± 0.5 ^{b} | 3.8 ± 0.3 ^{d} | 17.9 ± 0.7 ^{a} | n.d. | |

Chl d | 12.0 ± 0.4 ^{b} | 20.7 ± 0.6 ^{a} | 9.8 ± 0.2 ^{b} | 19.8 ± 0.3 ^{a} | n.d. | |

Total chls | 168.9 ± 13.1 ^{b} | 156.8 ± 4.1 ^{bc} | 127.7 ± 4.4 ^{c} | 235.3 ± 15.4 ^{a} | 182.9 ± 7.0 ^{b} | |

Total carotenoids | 27.0 ± 2.4 ^{a} | 24.4 ± 2.2 ^{a} | ||||

Porphyra spp. | Chl a | 504.5 ± 22.9 ^{ab} | 533.4 ± 20.3 ^{a} | 431.9 ± 19.8 ^{b} | 489.2 ± 7.8 ^{ab} | 538.4 ± 39.3 ^{a} |

Chl b | n.d. | n.d. | n.d. | 7.2 ± 0.4 | n.d. | |

Chl c | n.d. | n.d. | n.d. | n.d. | n.d. | |

Chl d | 5.20 ± 0.2 ^{c} | 8.83 ± 0.8 ^{b} | 2.67 ± 0.2 ^{d} | 16.25 ± 2.9 ^{a} | n.d. | |

Total chls | 509.7 ± 28.0 ^{a} | 542.2 ± 25.8 ^{a} | 434.6 ± 24.5 ^{b} | 512.7 ± 13.4 ^{a} | 538.40 ± 39.30 ^{a} | |

Totalcarotenoids | 70.8 ± 2.5 ^{a} | 74.5 ± 3.8 ^{a} | ||||

Spirulina spp. | Chl a | 9872 ± 499 ^{a} | 9388 ± 191 ^{a} | 996.0 ± 15.0 ^{d} | 3766 ± 96 ^{c} | 5179 ± 447 ^{b} |

Chl b | 105.0 ± 5.6 ^{c} | 168.6 ± 14.9 ^{b} | n.d. | 123.5 ± 4.2 ^{bc} | 373.7 ± 30.3 ^{a} | |

Chl c | 121.9 ± 12.7 ^{b} | 275.3 ± 33.12 ^{a} | 15.34 ± 0.84 ^{c} | n.d. | n.d. | |

Chl d | 155.4 ± 11.1 ^{a} | 159.9 ± 8.7 ^{a} | 27.2 ± 1.8 ^{c} | 84.6 ± 4.5 ^{b} | n.d. | |

Total chls | 10253 ± 503 ^{a} | 9991.4 ± 186.6 ^{a} | 1038.5 ± 15.9 ^{d} | 3974.3 ± 97.4 ^{c} | 5553.0 ± 476.3 ^{b} | |

Totalcarotenoids | 1263.9 ± 54.0 ^{a} | 1238.6 ± 42.6 ^{a} |

**Table 2.**Total chlorophylls and carotenoids contents and best extraction solvent for the analyzed samples (results expressed in μg/g of algae).

Total Chlorophylls | Total Carotenoids | |
---|---|---|

Himanthalia elongata | 168.2 ± 15.0 ^{b} (Acetone) | 2.9 ± 0.3 ^{b} (Methanol acid-free) |

Undaria pinnatifida | 574.1 ± 33.2 ^{b} (Acetone) | 54.6 ± 1.3 ^{b} (Methanol) |

Laminaria ochroleuca | 235.3 ± 15.4 ^{b} (Acetone) | 27.0 ± 2.4 ^{b} (Methanol) |

Porphyra spp. | 542.2 ± 25.8 ^{b} (Methanol acid-free) | 74.5 ± 3.8 ^{b} (Methanol acid-free) |

Spirulina spp. | 10253 ± 503 ^{a} (Methanol) | 1263.9 ± 54.0 ^{a} (Methanol) |

**Table 3.**Fucoxanthin content of brown edible algae (Himanthalia elongata, Laminaria ochroleuca (Kombu), Undaria pinnatifida (Wakame)) extracted using DMSO-water (4:1, v/v) as solvent. The results are expressed in μg/g of algae.

Fucoxanthin | |
---|---|

Himanthalia elongata | 2.79 ± 0.31 ^{c} |

Undaria pinnatifida | 26.81 ± 0.79 ^{a} |

Laminaria ochroleuca | 14.21 ± 0.31 ^{b} |

**Table 4.**Phycoerythrin and phycocyanin contents of Porphyra spp. and Spirulina spp. extracted using phosphate buffer (Ph = 6.8). The results are expressed in μg/g of algae.

Phycoerythrin | Phycocyanin | |
---|---|---|

Porphyra spp. | 8319 ± 288 ^{a} | 5305 ± 193 ^{b} |

Spirulina spp. | 8180 ± 301 ^{a} | 20732 ± 846 ^{a} |

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**MDPI and ACS Style**

Osório, C.; Machado, S.; Peixoto, J.; Bessada, S.; Pimentel, F.B.; C. Alves, R.; Oliveira, M.B.P.P.
Pigments Content (Chlorophylls, Fucoxanthin and Phycobiliproteins) of Different Commercial Dried Algae. *Separations* **2020**, *7*, 33.
https://doi.org/10.3390/separations7020033

**AMA Style**

Osório C, Machado S, Peixoto J, Bessada S, Pimentel FB, C. Alves R, Oliveira MBPP.
Pigments Content (Chlorophylls, Fucoxanthin and Phycobiliproteins) of Different Commercial Dried Algae. *Separations*. 2020; 7(2):33.
https://doi.org/10.3390/separations7020033

**Chicago/Turabian Style**

Osório, Catarina, Susana Machado, Juliana Peixoto, Sílvia Bessada, Filipa B. Pimentel, Rita C. Alves, and M. Beatriz P. P. Oliveira.
2020. "Pigments Content (Chlorophylls, Fucoxanthin and Phycobiliproteins) of Different Commercial Dried Algae" *Separations* 7, no. 2: 33.
https://doi.org/10.3390/separations7020033