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. 2020 Jul 28;10(47):27995-28006.
doi: 10.1039/d0ra04378h. eCollection 2020 Jul 27.

Matrix solid-phase dispersion as a greener alternative to obtain bioactive extracts from Haematococcus pluvialis. Characterization by UHPLC-QToF

Aly Castillo  1 , Simón Pereira  2 , Ana Otero  3 , Sarah Fiol  4 , Carmen Garcia-Jares  1 , Marta Lores  1
Affiliations

Matrix solid-phase dispersion as a greener alternative to obtain bioactive extracts from Haematococcus pluvialis. Characterization by UHPLC-QToF

Aly Castillo et al. RSC Adv. .

Abstract

So far, research on the microalga Haematococcus pluvialis has been focused mainly on the exploitation of its high astaxanthin content, leaving aside the use of other bioactive compounds present. This study is focused on obtaining and characterizing extracts enriched in bioactive compounds from this microalga red aplanospores. This is performed by means of Matrix Solid-Phase Dispersion (MSPD) extraction process, in an environmentally friendly way with low energy consumption and GRAS solvents. The effects of extraction parameters, particularly the extraction solvents (ethanol, ethyl lactate and water) are studied, in order to obtain maximum recovery of the main antioxidant compounds of interest (carotenoids, fatty acids and derivatives). Characterization of extracts is carried out by HPLC-DAD (High Performance Liquid Chromatography Diode Array Detector) and UHPLC-QToF (Ultra High-Performance Liquid Chromatography Quadrupole Time-of-Flight). The results show that MSPD produced extracts with higher bioactive compound recoveries than conventional cell disruption extractions. At the same time, a novel untargeted characterization for this species is performed, identifying compounds not previously dated in H. pluvialis, which include 10-phenyldecanoic acid and the -oxo and -hydroxy derivatives of palmitic acid. This approach, first applied to a freshwater microalgae, characterized by rigid and resistant aplanospores, provided a synergistic and sustainable extract, giving a broader focus on the use of this microalga.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1. Scheme of the extraction process using the MSPD technique applied to red stage biomass of the microalga H. pluvialis.
Fig. 2
Fig. 2. Microscope images of H. pluvialis red. (a) Non-disruptive 10×; (b) non-disruptive 40×; (c) disruption with cell disruptor 10×; (d) disruption with cell disruptor 40×; (e) first step MSPD (disruption with mortar) 40×; (f) second step MSPD (extract obtained) 40×.
Fig. 3
Fig. 3. Relative efficiency of major carotenoids extraction (astaxanthin, lutein–zeaxanthin, β-carotene) in ethanol by two methods: MSPD and cell disruption. The following “extraction volume/sample size” ratios were used: (a) 5 mL/0.4 mg, (b) 5 mL/0.2 mg, (c) 5 mL/0.1 mg, (d) 10 mL/0.2 mg.
Fig. 4
Fig. 4. Untargeted and targeted compound identification algorithm using UHPLC-QToF.
Fig. 5
Fig. 5. Overlay of chromatography and mass profiles (negative and positive ionization) of the bioactive compounds identified in MSPD extract of H. pluvialis red aplanospores by UHPLC-QTOF.
Fig. 6
Fig. 6. Analysis of UHPLC-QToF response of carotenoids to modifications in extraction solvents. (a) Astaxanthin. (b) Canthaxanthin. (c) Neoxanthin–violaxanthin. (d) Violaxanthin–neoxanthin. (e) Diadinoxanthin. (f) Echinenone. (g) AME C18:1. (h) AME C18:2. (i) AME C18:4.
Fig. 7
Fig. 7. Analysis of UHPLC-QToF response of target fatty acids to modifications in extraction solvents composition. (a) EPA. (b) Linolenic acid (α + γ). (c) Arachidonic acid. (d) Linoleic acid. (e) Palmitic acid. (f) Oleic acid. (g) Eicosadienoic acid.
Fig. 8
Fig. 8. Analysis of UHPLC-QToF response of untargeted fatty acids to modifications in extraction solvents. (a) N-oxopalmitic acid. (b) N-hydroxypalmitic acid (c) 10-phenyldecanoic acid.
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