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Article
Polyol-based deep eutectic solvents for extraction of
natural polyphenolic antioxidants from Chlorella vulgaris
WAN M. ASYRAF WAN MAHMOOD, Atiwich Lorwirachsutee,
Constantinos Theodoropoulos, and Maria Gonzalez-Miquel
ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/
acssuschemeng.8b05642 • Publication Date (Web): 08 Feb 2019
Downloaded from http://pubs.acs.org on February 8, 2019

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Page 1 of 15 ACS Sustainable Chemistry & Engineering

1
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7 Polyol-based deep eutectic solvents for extraction of natural
8 polyphenolic antioxidants from Chlorella vulgaris.
9
10 Wan M. Asyraf Wan Mahmood,a,b Atiwich Lorwirachsutee,a,b Constantinos Theodoropoulos,a,b
11
Maria Gonzalez-Miquel*a,b,c.
12
13 aSchool of Chemical Engineering and Analytical Sciences, Faculty of Science and Engineering, The
14
15 University of Manchester, The Mill, Sackville Street, Manchester M1 3AL, UK.
bBiochemical and Bioprocess Engineering Group, The University of Manchester, The Mill, Sackville
16
17 Street, Manchester M1 3AL, UK.
18 cDepartamento de Ingeniería Química Industrial y del Medio Ambiente, ETS Ingenieros Industriales.
19 Universidad Politécnica de Madrid, C/ José Gutiérrez Abascal 2, Madrid 28006, Spain.
20
*Email: maria.gonzalezmiquel@upm.es
21
22
23 KEYWORDS. Polyphenolic antioxidants, microalgae, extraction, Chlorella vulgaris, deep eutectic solvents.
24 ABSTRACT: Due to increasing demand of natural antioxidants for pharmaceutical, food and cosmetic applications, extraction
25 of polyphenols from natural resources has received enormous attention. In this regard, microalgae biomass exhibits great
26 potential for target bioactive compounds accumulation. Conventionally, petroleum-derived volatile organic solvents (VOCs)
27 and water have been used to recover polyphenols from biomass; however, VOCs are hazardous, non-environmentally friendly
28 solvents while water suffers from co-extraction of other impurities. Therefore, the goal of this work is to evaluate renewable
29 deep eutectic solvents (DES) as alternative to conventional solvents for recovering polyphenols from microalgal biomass. In
30 particular, Chlorella vulgaris was subjected to solvent extraction using twelve DES systems composed of choline chloride
31 (ChCl) and polyols including glycerol (Gly), ethylene glycol (EG), 1,3-propanediol (PDO) and 1,4-butanediol (BDO) and two
benchmark conventional solvents, namely ethyl acetate and water. Initially, the extraction efficiency was assessed based on
32
total phenolic content (TPC) via Folin-Ciocalteu method, as the most favorable operating conditions were determined (i.e.
33
temperature of 60°C, extraction time of 100 minutes and 20:1 solvent to biomass ratio). Afterwards, solvent extracts were
34 analyzed for their antioxidant activity via DPPH free radical scavenging method and their polyphenolic profiles were
35 characterized via chromatographic analysis, with major phenolic compounds being gallic acid, caffeic acid, p-coumaric acid,
36 and ferulic acid. Furthermore, biomass surface characterization was performed via scanning electron microscopy (SEM) to
37 further understand the effect of the solvents during the extraction process. Overall results support that polyol-based DES
38 outperformed conventional solvents in terms of polyphenolic extraction efficiency, antioxidant activity of the extracts and
39 selectivity of target antioxidants from Chlorella vulgaris, setting the grounds for developing more sustainable extraction
40 processes for recovering natural antioxidants from microalgae biomass.
41
42
43
44 INTRODUCTION In fact, the global polyphenolic market
45 exceeded USD 700 million in 2015 and is projected
Polyphenolic compounds are bioactive substances to reach USD 1.33 billion by 2024 4 due to favorable
46
47
widely occurring as secondary metabolites from food and safety regulations (GRAS status) and
48 plants, which are constituted by aromatic rings growing consumer awareness of health benefits
49 attached to hydroxyl substituents.1 They are well related to polyphenol consumption. Natural
50 known for their anti-oxidant, anti-inflammatory, polyphenolic compounds can be extracted from a
51 anti-microbial and anti-tumoral properties for variety of biomass sources, including plants, food
52 which are highly demanded by pharmaceutical, waste5 and microalgae.6-8 In recent years,
53 food and cosmetic industries.2, 3 microalgae have received particular attention due
54 to its ease of cultivation, high growth rate and
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accumulation capacity of target biomolecules and solvent that can be efficiently used to extract
1 bioactive compounds, such as lipids 9, carotenoids bioactive compounds from natural sources.5, 20
2
and polyphenols, mainly from Chlorella sp. and In the present study, the feasibility of using
3
some other species like Nannochloropsis sp. and renewable DES in extracting polyphenolic
4
5 Spirulina platensis.8-10 compounds from Chlorella vulgaris microalgae
6 Conventionally, volatile organic compounds biomass was explored as a means to develop
7 (VOCs) such as ethyl acetate were used in the sustainable processes for natural antioxidants
8 extraction of phenolic compounds from biomass6, production. In particular, twelve DES systems
11; however, these solvents are usually derived from composed of choline chloride as HBA (a readily
9
10 petrochemical sources and present different available food additive from the group of
11 drawbacks including high volatility, flammability provitamins) and a variety of polyols including
12 and toxicity, being hazardous towards the glycerol, ethylene glycol, 1,3-propanediol and 1,4-
13 environment and human health; moreover, their butanediol as HBD (which can be obtained as
14 use is being extensively limited by different glycerol surplus derivatives from biofuel
15 international regulations including REACH (EC processing) were systematically evaluated as
16
1907/2006; i.e. registration, evaluation, extraction solvents for microalgae biomass, and
17
authorisation and restriction of chemicals). Water compared to ethyl acetate and water acting as
18
19
has been also proposed for the extraction of benchmark conventional solvents. The operating
20 polyphenolic compounds from microalgae since its conditions such as temperature, time and solvent to
21 physico-chemical properties complies with U.S. biomass ratio were evaluated for extracting
22 Food and Drug Administration (FDA) and European polyphenolic compounds from microalgae biomass
23 regulations, being a non-toxic and environmentally- in terms of total phenolic content (TPC) yielded by
24 friendly solvent12; nevertheless, water suffers from the different solvents. Afterwards, the antioxidant
25 co-extraction of other water-soluble impurities activity of the polyphenolic extracts was further
26 which can lead to lower extraction yield and it is analysed via DPPH free radical scavenging method
27 only specific towards hydrophilic and polar and the polyphenolic profile of the extracts were
28 compounds, making it less effective towards non- characterized through high-performance liquid
29 polar and hydrophobic compounds. chromatography (HPLC) analysis. Lastly, surface
30 characterization of Chlorella vulgaris before and
31 Ionic liquids, low melting point organic salts
with inherent properties including negligible after the extraction was performed via scanning
32
33 vapour pressure, low flammability, excellent electron microscopy (SEM) to provide insights on
34 thermal and chemical stability and tuneability13, 14, the effect of the solvents for further biomass
35 were then successfully proposed as novel designer processing.
36 solvents for extracting lipids15, 16 and other
37 bioactive compounds17 from microalgae biomass; METHODOLOGY
38 yet concerns related to high cost of synthesis and
39 unknown toxicity still need to be tackled for further Microalgae biomass
40 process scale-up and implementation.5 Dried Chlorella vulgaris was purchased from
41 Algae4Future where it was cultivated in a closed
42 In this regard, deep eutectic solvents (DES)
photobioreactor autotrophically, supplied with A4F
43 have recently emerged as a new class of solvents
proprietary culture medium (A4F-M1) and dried
44 that possess similar features to ionic liquids6, 18 with
via spray drying method. The biomass was stored in
45 the additional properties of biodegradability, lower
a dark room at room temperature prior to use, and
46 toxicity and easiness of preparation from readily
its composition is shown in Table 1.
47 available materials 6, 12. DES are mixtures of
48 Brønsted or Lewis acids and bases, and can be Table 1 Chlorella vulgaris biomass composition
49 commonly obtained by the complexation of Microalgae Chlorella vulgaris
50 hydrogen bond acceptors (HBA) and hydrogen Moisture (%)a 5-10
51 bond donors (HBD) resulting a solvent with lower
52 Ash content (%)a 6-12
melting point than its individual constituents.19 The Total protein (%)a 47-53
53
combination of a large number of possible HBA and Total lipid content (%)b 24-27
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HBD will lead to the formation of task-specific aAlgae4Future

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bBligh and Dyer method21 supplier and purity or concentration. Choline

1 chloride, glycerol, ethylene glycol, 1,3-propanediol
2 Chemicals and
3 All chemicals used in this study were analytical
4 grade as shown in Table 2 with their respective
5 1,4-butanediol were used for preparing the deep Choline chloride (ChCl), glycerol (Gly), ethylene
6
eutectic solvents (DES). The extraction efficiency of glycol (EG), 1,3-propanediol (PDO) and 1,4-
7
all of the synthesized DES were compared to ethyl butanediol (BDO) were dried in Thermo Scientific
8
9
acetate (EA) and water (H2O), which were taken as vacutherm oven at 50 °C and 150 mBar for 24 hours
10 conventional benchmark solvents. Gallic acid, to remove its water content. Specified molar ratios
11 sodium bicarbonate solution and Folin-Ciocalteu between the hydrogen bond acceptor (HBA)
12 reagent were used to analyze the total phenolic (choline chloride) and hydrogen bond donor (HBD)
13 content (TPC) of the extracts. Antioxidant activities (glycerol, ethylene glycol, 1,3-propanediol and 1,4 -
14 of each extract were determined as per DPPH free butanediol) were synthesised as tabulated in Table
15 radical scavenging method. Standards of gallic acid, 3. The mixtures were heated to 50°C and stirred
16 p-coumaric acid, ferulic acid and caffeic acid were around 4 to 6 hours until the mixture becomes
17 used for identification and quantification purposes. homogenous or a transparent liquid. 5, 20
18 Formic acid and acetonitrile were used as one of the
19 mobile phases in high-performance liquid Table 3 DES components and molar ratios
20 Name Component Component Molar
chromatography (HPLC) analysis.
21 1 (HBA) 2 (HBD) ratio
22
Table 2 List of chemicals, supplier and purity or DES1 ChCl Gly 1:2
23
concentration DES2 ChCl Gly 1:3
24
25
Chemicals Supplier Purity/Concentration DES3 ChCl Gly 1:4
26 Choline Sigma- 98% DES4 ChCl EG 1:2
27 chloride Aldrich DES5 ChCl EG 1:3
28 Glycerol Sigma- 95% DES6 ChCl EG 1:4
29 Aldrich DES7 ChCl PDO 1:2
30 Ethylene Sigma- 99.8% DES8 ChCl PDO 1:3
31 glycol Aldrich DES9 ChCl PDO 1:4
32 1,3- Sigma- 99% DES10 ChCl BDO 1:2
33 propanediol Aldrich DES11 ChCl BDO 1:3
34 1,4- Sigma- 99% DES12 ChCl BDO 1:4
35 butanediol Aldrich
36
Ethyl acetate VWR 99% Polyphenolic compounds extraction
37
Sodium Sigma- Prior to the extraction process, the biomass was
38
39 bicarbonate Aldrich 7.5% subjected to pre-treatment via liquid nitrogen
40 Folin- Sigma- followed by mortar and pestle to promote cell
41 ciocalteu Aldrich 1.9-2.1 N destruction to enhance polyphenolic yield.
42 DPPH Sigma- Extraction of polyphenolic compounds was
43 radicals Aldrich 97% performed using water, ethyl acetate and the 12
44 Gallic acid Sigma- DES systems. For the base case extraction, 10:1 of
45 Aldrich 97.5-102.5% solvent to biomass ratio was used as reported by
46 p-coumaric Sigma- Goiris et al.,22 and Choochote et al.,23. Then, the
47 acid Aldrich 98% mixtures were placed in a Labnet VorTemp 1550
48 Sigma- shaking incubator at 700 rpm, for 100 minutes, at
49 Ferulic acid Aldrich 99% 40 °C as suggested by Bucic-Kojic et al.,24, 25. After
50 Sigma- 100 minutes, the supernatant and biomass were
51
Caffeic acid Aldrich 98% separated using a Labnet Spectrafuge 6c centrifuge
52
53 for 10 minutes at 3000 rpm. Lastly, the supernatant
54 Deep eutectic solvent (DES) synthesis was filtered through Sartorius RC 0.45 μm filter to
55 remove any large precipitate. To improve the
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extraction process, various operating parameters

1 such as time, temperature, and solvent to biomass Characterization of polyphenolic profile via
2
ratio were examined. The aforementioned High-Performance Liquid Chromatographic
3
parameters have been shown to have significant (HPLC) analysis
4
5 impact on the polyphenolic extraction efficiency as The separation of phenolic compounds extracted
6 indicated by with Bucic-Kojic et al.,24 , who studied from Chlorella vulgaris via conventional solvents
7 the solvent to biomass ratio and temperature for and DES was achieved on a Nucleosil 100-5 C18
8 solid-liquid extraction and Bubalo et al.,7 who column (15cm x 4.6mm, 5um) and analysed by
9 optimised the extraction time and temperature. In HPLC with DAD detector. 20 uL of extracts were
10 this study, the extraction temperature was firstly separated by the use of two mobile phases, 0.1% of
11 evaluated at 40, 50, 60 and 70 °C for 100 minutes formic acid in distilled water (A) and 0.1% of formic
12 and compared against the base case at 40 °C, to acid in acetonitrile (B), at 0.5 mL/min. The
13 determine the best extraction temperature for separation was observed at 300 nm. The column
14 polyphenolic compound extraction. By using the temperature was maintained at 25°C. Gradient
15 best extraction temperature, extraction times of 25, elution of 50 minutes of analysis was shown in
16
55, 75, 100 and 125 minutes were used to Table 4. Identification of phenolic compounds was
17
determine the best extraction time which yielded performed by comparing its retention time to those
18
19
the highest TPC content. Finally 20:1, 10:1 and 5:1 standards. External standard calibration curve was
20 of solvent to biomass ratio were evaluated at the employed for quantification purposes.
21 best extraction temperature and extraction time.
22 Table 4 HPLC gradient elution
23 Total polyphenolics content (TPC) Time (minutes) Mobile phase A
24 The total phenolic contents of all extracts were 0 – 30 90%
25 determined by Folin-Ciocalteu method as per ISO 30 – 33 10%
26 14502-1:2005, the most common and globally 35 - 50 90%
27 accepted.8, 26 In a centrifuge tube, 100 μl of extract
28 was added, followed by 1 mL of distilled water and Evaluation of antioxidant activity via DPPH free
29 200 μl of Folin-Ciocalteu reagent. The tube was radical scavenging method
30 shaken vigorously for 1 minute before adding 300 0.1 mM of DPPH in methanol was prepared, and one
31
μl of 7.5% sodium bicarbonate solution which was mL of the solution was used onto three mL of each
32
33
freshly prepared and filtered through Sartorius RC extract. The mixture was incubated for 30 minutes
34 0.45 μm filter to remove any remaining sodium and observed using UV analysis at 517 nm. The
35 bicarbonate crystals. The Folin-Ciocalteu reagent color changes and its absorbance were recorded. A
36 should be added and mixed with the sodium formula of % discoloration (Eq. 2.2)29 was
37 bicarbonate solution to prevent the air-oxidation of employed to determine the antioxidant activity of
38 phenols.27 Then, the centrifuge tube was wrapped the phenolic compounds in all extracts.
39 in aluminium foil as phenolic compounds are very
40 sensitive to light. The solution was then shaken % 𝑑𝑖𝑠𝑐𝑜𝑙𝑜𝑢𝑟𝑎𝑡𝑖𝑜𝑛 =
𝐴0 ― 𝐴1
𝑥 100%-----------Eq. 2
41 again for 1 minute and left in darkness for 1 hour at 𝐴1
42 room temperature. Each extract was analysed by
43 UV and monitored at 675 nm and calculated as A0 =Absorbance of blank DPPH reagent
44 gallic acid equivalent (GAE)/g biomass (Eq. 1)28. A1 =Absorbance of DPPH reagent and extracts
45
46 (𝐶 𝑥 𝑉) Biomass surface characterization via Scanning
47 𝑇𝑃𝐶 = 𝑀 ---------------------------------------Eq. 1 Electron Microscope (SEM) analysis
48 The dried biomass samples were coated with
49 TPC = total phenolic content (mg GAE/g carbon (7.5mm) prior to SEM analysis to improve
50 biomass) the sample conductivity. SEM images were
51 C = concentration of gallic acid equivalent obtained using ZEISS EVO 60 model microscope,
52
(mg/L) and the condition was set as the resolution of 1 nm
53
54
V = volume of extracts used for analysis (L) and an acceleration voltage of 5kV.
55 M = mass of the extracts used for analysis (mg)
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RESULTS AND DISCUSSION acetate6, 11 and 12 DES systems. All of the solvents
1 used were diluted with 30% water. The
2 The feasibility of using sustainable deep eutectic performance of the individual components of DES
3 solvents (DES) as an alternative to conventional were also assessed, i.e. ChCl, Gly, EG, PDO and BDO.
4 volatile organic solvents (VOCs) in the extraction of
5 The extraction efficiency was monitored in terms of
natural polyphenolic antioxidants from microalgal total phenolic content (TPC) via Folin-Ciocalteu
6
biomass was investigated herein. In particular, 12 method and UV analysis at 675 nm as depicted in
7
8 DES systems and two conventional solvents, i.e. EA Figure 1.
9 as benchmark VOC and H2O as globally accepted The TPC provided by water and ethyl
10 green universal solvent, were evaluated to study acetate extracts were at 2.02 and 0.91 mg GAE/g
11 their feasibility in extracting polyphenolic biomass. These trends are in agreement with those
12 compounds from Chlorella vulgaris. ChCl salts as reported by with Hajimahmoodi et al.,10 where
13 hydrogen bond acceptor (HBA) and a variety of extracted 3.69 and 0.02 mg GAE/g biomass with
14 polyols including Gly, EG, PDO and BDO as water and ethyl acetate respectively at 80°C for 30
15 hydrogen bond donor (HBD) were chosen as minutes with 20:1 solvent to biomass ratio. Based
16 starting materials due to their wide availability as on the TPC of DES individual components, glycerol
17 well as their renewable, non-toxic and shows the lowest TPC at 0.43 mg GAE/g biomass,
18 biodegradable nature. 5, 19
19
followed by choline chloride at 0.45 mg GAE/g
Moreover, the polarity of the chosen DES biomass. All other polyols, namely ethylene glycol,
20
play an important role in increasing the extraction 1,3-propanediol and 1,4-butanediol yielded much
21
22
efficiency of bioactive compounds based on the higher TPC at 2.6 mg GAE/g biomass, 2.0 mg GAE/g
23 principle of ‘like-dissolve-like’, while it can be tuned biomass, and 2.09 mg GAE/g biomass respectively.
24 and by choosing a variety of range of HBA and HBD The high viscosity of Gly and ChCl may be one of the
25 or by addition of water. 5, 12, 20 Chlorella vulgaris has contributing factors for the low TPC extracted,
26 been selected as the microalgae biomass reference while a lower viscosity and higher polarity of
27 in this study due to its widely proven capacity to polyols contributed to much higher TPC extracted
28 accumulate target biomolecules and bioactive from microalgal biomass.
29 compounds, including polyphenolic antioxidants 8, 9, Lower viscosity of polyols can enhance the
30 30. Hence, it acts as a benchmark in assessing the
mass transfer of phenolic compounds into the
31 performance of the proposed solvents regarding solvents. In contrast, the high polarity of water was
32 total phenolic content (TPC), antioxidant activity
33
not beneficial as water-soluble contaminants, and
and polyphenolic profile of the extracts. Based on other compounds can be co-extracted thus
34
the TPC via colourimetric analysis using Folin- reducing phenolic compound content.12 The
35
Ciocalteu reagent, the best performing DES and two polyphenol extraction efficiency provided by
36
37 conventional solvents (EA and H2O) were further polyol-based DES is generally higher that obtained
38 evaluated on their antioxidant activity via DPPH with individual counterparts, although the HBA:
39 method along polyphenolic identification and HBD molar ratio of DES play an important role.
40 quantification through HPLC analysis. Structural Glycerol-based DES have much higher TPC
41 analysis using SEM was also used to determine the at lower ratio of Gly to ChCl. TPC decreases from 2.5
42 effect of pre-treatment and solvents onto the to 1.14 to 1.01 mg GAE/ g biomass for 1:2, 1:3 and
43 cellular structure of Chlorella vulgaris before and 1:4 of ChCl to Gly respectively. Higher ratio of
44 after extraction. glycerol increases the solvent viscosity which
45
minimizing the mass transfer of polyphenolic
46 Phenolic extraction via conventional and DES compounds into the extracting solvent. The other
47 Base extraction conditions comprising of extraction
48 three polyol-based DES, ethylene glycol, 1,3-
temperature (T), extraction time (t) and solvent to propanediol and 1,4-butanediol had much higher
49
biomass ratio (S/F) at 40°C, 100 minutes and 10:1 TPC at 1:4 ratio of HBA to HBD, which were about
50
51
respectively were employed for extracting 3.87 mg GAE/g biomass, followed by 3.22 mg
52 polyphenolic compounds from microalgal biomass, GAE/g biomass, and 3.60 mg GAE/g biomass
53 Chlorella vulgaris. The solid-liquid extraction respectively. A higher ratio of polyols (ethylene
54 method was performed on the biomass using two glycol, 1,3-propanediol and 1,4-butanediol) to
55 conventional solvents, namely water and ethyl
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choline chloride reduces the surface tension and relative to conventional solvents which can be
1 viscosity of DES12. Overall results support that ascribed to favorable electrostatic and hydrogen
2
polyol-based DES have the ability to enhance the bond interactions with the target solutes 5, 12, 31.
3
extraction efficiency of polyphenolic compounds
4
5
6 4.5
7
4
TPC (mg GAE/g biomass)

8
9 3.5
10 3
11
2.5
12
13 2
14 1.5
15 1
16
17 0.5
18 0
19
O

EA

Cl

S1

S2

S3

S4

S5

S6

EG

S7

S8

S9

2
O

O
20
Gl

S1

S1

S1
H2

PD

BD
Ch

DE

DE

DE

DE

DE

DE

DE

DE

DE

DE

DE

DE
21 Solvents
22
Figure 1 Total phenolic content (TPC) of Chlorella vulgaris extracted with proposed solvents at base case extraction
23
conditions (T=40 °C, t=100 min, S/F = 10:1). [DES1 = ChCl:Gly (1:2), DES2 = ChCl:Gly (1:3), DES3 = ChCl:Gly (1:4), DES4
24
= ChCl:EG (1:2), DES5 = ChCl:EG (1:3), DES6 = ChCl:EG (1:4), DES7 = ChCl:PDO (1:2), DES8 = ChCl:PDO (1:3), DES9 =
25
ChCl:PDO (1:4), DES10 = ChCl:BDO (1:2), DES11 = ChCl:BDO (1:3), DES12 = ChCl:BDO (1:4)].
26
27
28
29 Effect of operating conditions (temperature, extraction essays of polyphenolic compounds from
30 time, and solvent to biomass ratio) on Chlorella vulgaris. Increasing temperature
31 microalgal polyphenol extraction promotes higher solubility and diffusion coefficient
32 To determine the influence of temperature on the of polyphenolic compounds into the extracting
33 extraction of polyphenolic compounds from solvents, as supported by previous works.5, 32
34 Chlorella vulgaris, 40 °C, 50 °C, 60 °C and 70 °C were Higher temperatures also enhance penetration of
35 employed for 100 minutes using 10:1 solvent to solvents into the cell matrix hence increasing the
36 biomass ratio. The extracts were analysed TPC of the extracts8. However, an increase in
37 colourimetrically using Folin-Ciocalteu reagent and temperature for the extraction of polyphenolic
38 monitored using UV at 670 nm to obtain the total compounds can lead to degradation33. In fact, Volt
39 phenolic content (TPC). et al. 34 reported that the percentage of gallic acid
40
As shown on Figure 2, 60 °C has provided degradation increased to 25% at 80 °C as compared
41
42
the highest TPC for the majority of DES, such as DES to only 15% degradation at 60°C. To determine the
43 1 (3.23 mg GAE/g biomass), DES 2 (1.76 mg GAE/g most suitable extraction time, a range between 25
44 biomass), DES 5 (1.93 mg GAE /g biomass), DES 7 minutes and 125 minutes was evaluated for the
45 (2.18 mg GAE/g biomass), and DES 8-12 (2.16-4.77 extraction of polyphenolic compounds from
46 mg GAE/g biomass). Only DES 3 and DES 6 have Chlorella vulgaris at the temperature of 60°C using
47 shown the highest TPC at 50 °C at 1.02 mg GAE/g 10:1 solvent to biomass ratio. As shown in Figure
48 biomass and 4.21 mg GA equivalent/g biomass 3, the majority of DES yielded the highest TPC at
49 respectively. Conventional solvents, EA and H2O, 100 minutes, such as DES 1-2 (1.76-3.23 mg GAE/g
50 have shown the highest TPC at 70 °C (1.40 mg biomass), DES 5-7 (1.93-4.15 mg GAE/g biomass),
51 GAE/g biomass) and 50 °C (2.53 mg GAE/g DES 9 (3.88 mg GAE/g biomass), and DES 11-12
52 biomass) respectively. As most of the solvents have (3.31-4.77 mg GAE/g biomass). Only DES 4, 8 and
53 provided the highest TPC at 60 °C, thus such 10 provided the highest TPC at 125 minutes with
54
temperature was selected for the subsequent 1.15 mg GAE/g biomass, 2.98 mg GAE/g biomass,
55
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and 2.74 mg GAE/g biomass respectively. As for compounds from Chlorella vulgaris. Generally,
1 conventional solvents, EA and H2O have the highest increasing extraction time will lead to higher
2
TPC at 75 minutes (2.43 mg GAE/g biomass) and extraction yield due to the longer exposure of
3
125 minutes (2.55 mg GAE/g biomass) solvent to the compound of interest, although there
4
5 respectively. As most of the solvents yielded the is a maximum time after which the TPC content of
6 highest TPC at 100 minutes, such extraction time the extracts decreases due oxidation of
7 was selected for extracting polyphenolic polyphenolic compounds8.
8
9
10
11
12 6 40°C 50°C 60°C 70°C
13
TPC (mg GAE/g biomass)

14
5
15
16
4
17
18
19 3
20
21 2
22
23 1
24
25 0
26 H2O EA ChCl DES1 DES2 DES3 Gly DES4 DES5 DES6 EG DES7 DES8 DES9 PDO DES10DES11DES12 BDO
27 Solvents
28
29
30
Figure 2 Total phenolic content (TPC) of Chlorella vulgaris extracted with proposed solvents at t=100 and S/F = 10:1
31
as a function of temperature. [DES1 = ChCl:Gly (1:2), DES2 = ChCl:Gly (1:3), DES3 = ChCl:Gly (1:4), DES4 = ChCl:EG (1:2),
32 DES5 = ChCl:EG (1:3), DES6 = ChCl:EG (1:4), DES7 = ChCl:PDO (1:2), DES8 = ChCl:PDO (1:3), DES9 = ChCl:PDO (1:4),
33 DES10 = ChCl:BDO (1:2), DES11 = ChCl:BDO (1:3), DES12 = ChCl:BDO (1:4)].
34
35
36
37 A) 3 25 min 50 min 75 min 100 min 125 min
TPC (mg GAE/g biomass)

38
2.5
39
40 2
41
42 1.5
43
44 1
45
46 0.5
47
48 0
49 H2O EA ChCl Gly EG PDO BDO
50 Solvents
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1 B) 6 25 min 50 min 75 min 100 min 125 min

2 TPC (mg GAE/g biomass)
3 5
4
4
5
6
3
7
8
2
9
10 1
11
12 0
13
DES1 DES2 DES3 DES4 DES5 DES6 DES7 DES8 DES9 DES10 DES11 DES12
14
15 Solvents
16 Figure 3 Total phenolic content (TPC) of Chlorella vulgaris extracted with A) conventional solvents and individual DES
17 compounds and B) DES at T=60 °C and S/F = 10:1 as a function of extraction time. [DES1 = ChCl:Gly (1:2), DES2 =
18 ChCl:Gly (1:3), DES3 = ChCl:Gly (1:4), DES4 = ChCl:EG (1:2), DES5 = ChCl:EG (1:3), DES6 = ChCl:EG (1:4), DES7 =
19 ChCl:PDO (1:2), DES8 = ChCl:PDO (1:3), DES9 = ChCl:PDO (1:4), DES10 = ChCl:BDO (1:2), DES11 = ChCl:BDO (1:3),
20 DES12 = ChCl:BDO (1:4)].
21
22
23 12 5:1 10:1 20:1
24
25 10
TPC (mg GAE/g biomass)

26
27 8
28
29
6
30
31
32 4
33
34 2
35
36 0
37
O

EA

Cl

S1

S2

S3

S4

S5

S6

EG

S7

S8

S9

2
O

O
Gl

S1

S1

S1
38
H2

PD

BD
Ch

DE

DE

DE

DE

DE

DE

DE

DE

DE

DE

DE

39 Solvents DE
40 Figure 4 Total phenolic content (TPC) of Chlorella vulgaris extracted with proposed solvents at T=60 °C and t=100
41 min as a function of solvent to biomass ratio. [DES1 = ChCl:Gly (1:2), DES2 = ChCl:Gly (1:3), DES3 = ChCl:Gly (1:4),
42 DES4 = ChCl:EG (1:2), DES5 = ChCl:EG (1:3), DES6 = ChCl:EG (1:4), DES7 = ChCl:PDO (1:2), DES8 = ChCl:PDO (1:3),
43 DES9 = ChCl:PDO (1:4), DES10 = ChCl:BDO (1:2), DES11 = ChCl:BDO (1:3), DES12 = ChCl:BDO (1:4)].
44
45
46
47
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The next parameter to evaluate was solvent to HPLC analysis and biomass structural analysis via
1 biomass ratio. In particular, three ratios of 5:1, 10:1 SEM.
2
and 20:1 were used for extraction at 60 °C of
3
temperature during 100 minutes. As shown in Characterization of polyphenolic profile via
4
5 Figure 4, the TPC for all extracts increased High-Performance Liquid Chromatographic
6 significantly from 5:1 to 20:1 solvent to biomass (HPLC) analysis
7 ratio, since increasing the amount of solvent HPLC analysis was employed to identify and
8 promote contact between both phases and quantify the polyphenolic extracts yielded by the
9 penetration of the extraction solvent into the cell four best performing DES (i.e. DES1, DES6, DES9,
10 matrix. At 20:1, EA and H2O extracts yielded TPC DES12) and conventional solvents (EA and H2O,)
11 about 3.62 mg GAE/ g biomass and 1.89 mg GAE/ g obtained at the most favorable operating conditions
12 biomass respectively; meanwhile DES1, DES6, (temperature of 60 °C, 100 minutes and 20:1
13 DES9, and DES12 extracts provided the highest TPC solvent to biomass ratio).
14 at 5.27 mg GAE/ g biomass, 7.19 mg GAE/ g The HPLC chromatographic profiles
15 biomass, 9.19 mg GAE/ g biomass, and 9.87 mg (Figure 5 and Supplementary Information)
16
GAE/ g biomass respectively. Thus, 20:1 of solvent indicated that the four major phenolic compounds8,
17
to biomass ratio was selected as suitable ratio for i.e. gallic acid (peak at 6.475 min), caffeic acid (peak
18
19
extracting phenolic compounds from Chlorella at 11.533 min), p-coumaric acid (peak at 13.598
20 vulgaris. min), and ferulic acid (peak at 14.001 min) were
21 Based on the obtained results, it can be present in the extracts. Based on Table 5, the
22 concluded that the most favorable conditions for highest content of polyphenolic compounds yielded
23 extraction of polyphenolic compounds from by DES was provided by DES1 (0.86 mg/g biomass),
24 Chlorella vulgaris were 60 °C of extraction followed by DES 12 (0.77 mg/g biomass), DES6
25 temperature, 100 minutes of extraction time and (0.64 mg/g biomass), and DES9 (0.55 mg/g
26 20:1 of solvent to biomass ratio. Moreover, DES1, biomass); meanwhile, EA and H2O provided lower
27 DES6, DES9 and DES12 were the solvents yielding content of target polyphenolic compounds in the
28 the highest polyphenol content; therefore, such extracts at 0.61 mg/g biomass and 0.33 mg/g
29 best performing DES along with H2O and EA were biomass respectively (Figures S1-S6 in the ESI
30 chosen for further assessments on the antioxidant represent the HPLC chromatograms of
31
activity via DPPH free radical scavenging method, polyphenolic compounds from Chlorella vulgaris
32
33
characterization of polyphenolic profile through using all 6 extraction solvents).
34
35 Table 5 Selected phenolic compounds content via HPLC analysis
36 Solvent EA H2O DES1 DES6 DES9 DES12
37 Gallic acid 0.13 0.03 0.67 0.38 0.032 0.35
38 Caffeic acid 0.17 0.11 ND ND 0.2 0.12
39 p-Coumaric acid 0.17 ND 0.16 0.14 0.16 0.19
40 Ferulic acid 0.14 0.19 0.03 0.12 0.16 0.11
41
42 Total (mg/g biomass) 0.61 0.33 0.86 0.64 0.55 0.77
43
44 favorable operating conditions of T=60 °C, t=100 min
45 and S/F = 20:1.
46
47 The presence of these phenolic compounds
48 corroborates with Safafar et al.30 and Onofrejova et
49 al. 35. Furthermore, our results support that the
50 proposed polyol-based DES are able to provide a
51 higher selectivity towards target antioxidants,
52 especially gallic acid, than conventional solvents.
53 Three main methods have been reported to recover
54 Figure 5 HPLC profile of polyphenolic compounds from
Chlorella vulgaris extracted with DES1 under most the polyphenolic compounds from DES extracts,
55
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namely precipitation by addition of anti-solvents,
1 liquid−liquid extraction (LLE) using another
2
solvent, and solid−liquid extraction (SLE) using
3
resin or molecular sieves1. However, note that the
4
5 use of DES composed of GRAS (Generally Recognize
6 as Safe) and food–grade chemicals as extracting
7 solvents can minimise the cost of separating the
8 compounds from the extraction solvent, since they
9 could be potentially employed in food industry, fine
10 chemical formulations and pharmaceutical
11 purposes1.
12
13 The preliminary assessment of the capacity
14 of the different solvents to extract polyphenolic
15 compounds was initially performed in terms of TPC
16
via Folin-Ciocalteu method by using UV-Vis.
17
However, the precise value of TPC may be subject
18
19
to slight over or under estimations due to light
20 scattering by viscous sample or absorption of other
21 impurities. Therefore, HPLC analysis was employed
22 to further identify and quantify the concentration of
23 those specific polyphenols present in the extracts.
24 An important factor affecting the extraction
25 efficiency is the structure of the HBD employed to
26 form each DES, which affects not only the overall
27 solvent viscosity but also the solvent capacity to
28 interact through hydrogen bonding with the
29 different polyphenols. It should be noted that
30 glycerol has three -OH groups as compared with the
31
two –OH groups contained in the structure of the
32
33
other assessed polyols; this enhances the hydrogen
34 bonding interactions within the neat Gly-based
35 DES, increasing solvent viscosity but also enhancing
36 the solvent ability to form specific interactions with
37 the solutes, hence increasing the recovery of target
Evaluation of antioxidant activity via DPPH free
radical scavenging method
The antioxidant activity of the polyphenolic
compounds extracted was determined via the fast
and reliable 2,2-diphenyl-1-picrylhydrazyl (DPPH)
assay8. The six solvent extracts (EA, H2O, DES1,
DES6, DES9, and DES12) were subjected to DPPH
assay in which its radical scavenging activity was
determined via UV analysis at 517nm8, 36 by
monitoring its percentage discoloration.
Percentage discoloration of DPPH reagent is based
on the ability of the phenolic compounds to lose its
hydrogen to DPPH37 forming DPPH-H
(diphenylhydrazine); hence, the discoloration is
caused by the decreasing DPPH radicals and
increasing DPPH-H molecules36. As presented in
Figure 6, DES1 and DES12 extracts exhibits the
highest percentage discoloration at 68% and 65%
respectively, which corresponds to a higher ability
to scavenge DPPH radicals, hence indicating higher
antioxidant properties. The extracts provided by
the conventional solvents of EA and H2O present
much lower antioxidant activity at 55% and 40% of
DPPH discoloration. As determined in HPLC
analysis, DES1 and DES12 extracts show a high
content of the phenolic compounds, especially gallic
acid. This high content of phenolic compounds
contributes to the high percentage discolouration
of DPPH radicals. This corroborates with Zakaria et
al.8, in which higher phenolic content will
contribute to a higher antioxidant activity.
80
70
% discolouration

60
38 polyphenols. As for the diols, EG-based DES present
39 lower viscosity overcoming potential mass transfer 50
40 limitations during the extraction process, although 40
41 the vicinal position of the –OH in its structure may 30
42 cause steric hindrance within the molecule, hence 20
43 limiting at some extent the ability of the solvent to 10
44 easily interact with the solutes. However, diols with 0
45
longer alkyl chains presenting –OH groups at EA H2O DES1 DES6 DES9 DES12
46
terminal positions may overcome the steric effects Figure 6 Antioxidant capacity of Chlorella vulgaris
47
48 enhancing extraction of target solutes, as it is the polyphenolic extracts via best performing solvents
case of BDO-based DES. Therefore, the structure of under most favorable operating conditions of T=60 °C,
49
the HBD is a vital parameter to consider since it may t=100 min and S/F = 20:1.
50
51 lead to specific solvent-solute interactions to isolate
52 the bioactive molecules of interest. Biomass surface characterization via Scanning
53 Electron Microscope (SEM) analysis
54 Prior to solvent extraction of polyphenolic
55 compounds, Chlorella vulgaris biomass was
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subjected to pre-treatment by liquid nitrogen cellulose after extraction, hence releasing the
1 followed by mortar and pestle to promote cell natural products into the surroundings.
2
disruption to increase the extraction yield38. During In addition, the results provided by these
3
this process, the cells were fractured and burst as SEM images also agrees with those reported by Nur
4
5 ice crystals formed inside the cells due to the use of Atikah Md Noor et al.,41 where they pre-treated oil
6 liquid nitrogen and force was imposed by the palm empty fruit bunch with DES for sugar
7 mortar and pestle39. The states of the cells before production. DES had shown the ability to impact the
8 and after extraction via DES (DES1 and DES12) and fiber, promoting cavities and roughness onto the
9 conventional solvents (ethyl acetate and water) surface where the structural lignin seemed to be
10 were observed via Scanning Electron Microscope broken. Thus, results suggest that, besides
11 (SEM) images as shown in Figure 7. increasing the polyphenol extraction efficiency,
12 DES solvents may also be advantageous in
13 disrupting the microalgae cell walls which could be
14 beneficial to facilitate further biomass processing.
15
16
CONCLUSIONS
17
18 This study demonstrates the feasibility of using
19 renewable polyol-based DES in extracting phenolic
20 compounds from microalgae biomass, Chlorella
21 vulgaris, as a means to develop sustainable
22 processes for natural antioxidants manufacturing.
23 In particular, the polyphenol extraction efficiency
24
provided by twelve DES systems composed of
25
26
choline chloride (ChCl) and a variety of polyols
27 including glycerol (Gly), ethylene glycol (EG), 1,3-
28 propanediol (PDO) and 1,4-butanediol (BDO) was
29 evaluated and their performances were compared
30 to benchmark conventional solvent such as ethyl
31 acetate and water in terms of total phenolic content
32 (TPC via Folin-Ciocalteau method), antioxidant
33 activity (DPPH free radical scavenging method),
34 and polyphenolic profile of the extracts (via HPLC
35 analysis). Overall results support that the proposed
36 DES are able to yield extracts with higher total
37 polyphenolic content than conventional solvents,
Figure 7 SEM images of Chlorella vulgaris (A) before
38
extraction (B) conventional solvent [1=H2O, 2=EA] and mainly DES1 (ChCl:Gly 1:2) DES6 (ChCl:EG 1:4),
39
(C) DES [1=DES1, 2=DES12] DES9 (ChCl: PDO 1:4), and DES12 (ChCl:BDO 1:4)
40
41 which provided up to two-fold extraction efficiency
42
Untreated biomass (A) shows that the cells were under the most favorable operating conditions of
43 uniform and exhibited a circular shape. Meanwhile, 60°C of extraction temperature, 100 minutes of
44 the treated biomass (B1, B2, C1 and C2), exhibit extraction time and 20:1 solvent to biomass ratio.
45 more deconstructed cells, which appear to be Moreover, DES 1 and DES 12 were proved to be
46 shrunken and wrinkled indicating that the significantly more selective towards target
47 intercellular matter has been exposed to the polyphenolic compounds, especially gallic acid,
48 environment and have been in contact with the while providing polyphenolic extracts with higher
49 extracting solvents. Biomass extracted by DES show antioxidant capacity than conventional solvents.
50 agglomerated cells which can be ascribed to the Furthermore, microscopy surface characterization
51 higher viscosity of the extracting solvents. This of biomass before and after the extraction
52 agrees with a previous study40 reporting that DES suggested the ability of DES solvents to disrupt the
53 (ChCl: 1,2-butanediol) formed porous slice on microalgae cell walls which could be beneficial for
54
Salvia miltiorrhiza Bunge due to the dissolution of further biomass processing.
55
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ASSOCIATED CONTENT 10. Hajimahmoodi, M.; Faramarzi, M. A.; Mohammadi, N.;

1 Supporting Information: HPLC profiles of Soltani, N.; Oveisi, M. R.; Nafissi-Varcheh, N., Evaluation of
2 antioxidant properties and total phenolic contents of some strains
polyphenolic compounds from Chlorella vulgaris of microalgae. Journal of Applied Phycology 2009, 22 (1), 43-50,
3
extracted with ethyl acetate, water, DDES1, DES6, 10.1007/s10811-009-9424-y.
4
5 DES9 and DES 12 under most favorable operating 11. Khoddami, A.; Wilkes, M. A.; Roberts, T. H., Techniques
6 conditions of T=60 °C, t=100 min and S/F = 20:1. for analysis of plant phenolic compounds. Molecules 2013, 18 (2),
2328-75, 10.3390/molecules18022328.
7
AUTHOR INFORMATION 12. Zainal-Abidin, M. H.; Hayyan, M.; Hayyan, A.;
8 Jayakumar, N. S., New horizons in the extraction of bioactive
9 Corresponding Author: compounds using deep eutectic solvents: A review. Analytica
10 Dr. María Gonzalez-Miquel chimica acta 2017, 979, 1-23, 10.1016/j.aca.2017.05.012.
11 E-mail address: maria.gonzalezmiquel@upm.es 13. Marrucho, I. M.; Branco, L. C.; Rebelo, L. P., Ionic liquids
12 in pharmaceutical applications. Annual review of chemical and
13 ACKNOWLEDGEMENTS biomolecular engineering 2014, 5, 527-46, 10.1146/annurev-
14 chembioeng-060713-040024.
Wan Mohd Asyraf Wan Mahmood would like to
15 14. Ventura, S. P. M.; FA, E. S.; Quental, M. V.; Mondal, D.;
acknowledge the Ministry of Higher Education Freire, M. G.; Coutinho, J. A. P., Ionic-Liquid-Mediated Extraction
16
(MOHE) Malaysia for financially supporting this and Separation Processes for Bioactive Compounds: Past, Present,
17
project and granting the pre-doctoral scholarship. and Future Trends. Chemical reviews 2017, 117 (10), 6984-7052,
18
10.1021/acs.chemrev.6b00550.
19 15. Orr, V. C. A.; Plechkova, N. V.; Seddon, K. R.; Rehmann,
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6 Table of Contents
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24 Synopsis: Renewable polyol-based Deep Eutectic Solvents for extraction of phenolic compounds from
25 microalgae biomass to develop sustainable processes for natural antioxidants manufacturing.
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ACS Paragon Plus Environment