Polyol-Based Deep Eutectic Solvents for Extraction of Natural

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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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ACS Sustainable Chemistry & Engineering

Polyol-based deep eutectic solvents for extraction of natural polyphenolic antioxidants from Chlorella vulgaris. Wan M. Asyraf Wan Mahmood,a,b Atiwich Lorwirachsutee,a,b Constantinos Theodoropoulos,a,b Maria Gonzalez-Miquel*a,b,c. aSchool

of Chemical Engineering and Analytical Sciences, Faculty of Science and Engineering, The University of Manchester, The Mill, Sackville Street, Manchester M1 3AL, UK. bBiochemical and Bioprocess Engineering Group, The University of Manchester, The Mill, Sackville Street, Manchester M1 3AL, UK. cDepartamento de Ingeniería Química Industrial y del Medio Ambiente, ETS Ingenieros Industriales. Universidad Politécnica de Madrid, C/ José Gutiérrez Abascal 2, Madrid 28006, Spain. *Email: [email protected] KEYWORDS. Polyphenolic antioxidants, microalgae, extraction, Chlorella vulgaris, deep eutectic solvents. ABSTRACT: Due to increasing demand of natural antioxidants for pharmaceutical, food and cosmetic applications, extraction of polyphenols from natural resources has received enormous attention. In this regard, microalgae biomass exhibits great potential for target bioactive compounds accumulation. Conventionally, petroleum-derived volatile organic solvents (VOCs) and water have been used to recover polyphenols from biomass; however, VOCs are hazardous, non-environmentally friendly solvents while water suffers from co-extraction of other impurities. Therefore, the goal of this work is to evaluate renewable deep eutectic solvents (DES) as alternative to conventional solvents for recovering polyphenols from microalgal biomass. In particular, Chlorella vulgaris was subjected to solvent extraction using twelve DES systems composed of choline chloride (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 total phenolic content (TPC) via Folin-Ciocalteu method, as the most favorable operating conditions were determined (i.e. temperature of 60°C, extraction time of 100 minutes and 20:1 solvent to biomass ratio). Afterwards, solvent extracts were analyzed for their antioxidant activity via DPPH free radical scavenging method and their polyphenolic profiles were characterized via chromatographic analysis, with major phenolic compounds being gallic acid, caffeic acid, p-coumaric acid, and ferulic acid. Furthermore, biomass surface characterization was performed via scanning electron microscopy (SEM) to further understand the effect of the solvents during the extraction process. Overall results support that polyol-based DES outperformed conventional solvents in terms of polyphenolic extraction efficiency, antioxidant activity of the extracts and selectivity of target antioxidants from Chlorella vulgaris, setting the grounds for developing more sustainable extraction processes for recovering natural antioxidants from microalgae biomass.

INTRODUCTION

In fact, the global polyphenolic market exceeded USD 700 million in 2015 and is projected to reach USD 1.33 billion by 2024 4 due to favorable food and safety regulations (GRAS status) and growing consumer awareness of health benefits related to polyphenol consumption. Natural polyphenolic compounds can be extracted from a variety of biomass sources, including plants, food waste5 and microalgae.6-8 In recent years, microalgae have received particular attention due to its ease of cultivation, high growth rate and

Polyphenolic compounds are bioactive substances widely occurring as secondary metabolites from plants, which are constituted by aromatic rings attached to hydroxyl substituents.1 They are well known for their anti-oxidant, anti-inflammatory, anti-microbial and anti-tumoral properties for which are highly demanded by pharmaceutical, food and cosmetic industries.2, 3

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solvent that can be efficiently used to extract bioactive compounds from natural sources.5, 20 In the present study, the feasibility of using renewable DES in extracting polyphenolic compounds from Chlorella vulgaris microalgae biomass was explored as a means to develop sustainable processes for natural antioxidants production. In particular, twelve DES systems composed of choline chloride as HBA (a readily available food additive from the group of provitamins) and a variety of polyols including glycerol, ethylene glycol, 1,3-propanediol and 1,4butanediol as HBD (which can be obtained as glycerol surplus derivatives from biofuel processing) were systematically evaluated as extraction solvents for microalgae biomass, and compared to ethyl acetate and water acting as benchmark conventional solvents. The operating conditions such as temperature, time and solvent to biomass ratio were evaluated for extracting polyphenolic compounds from microalgae biomass in terms of total phenolic content (TPC) yielded by the different solvents. Afterwards, the antioxidant activity of the polyphenolic extracts was further analysed via DPPH free radical scavenging method and the polyphenolic profile of the extracts were characterized through high-performance liquid chromatography (HPLC) analysis. Lastly, surface characterization of Chlorella vulgaris before and after the extraction was performed via scanning electron microscopy (SEM) to provide insights on the effect of the solvents for further biomass processing.

accumulation capacity of target biomolecules and bioactive compounds, such as lipids 9, carotenoids and polyphenols, mainly from Chlorella sp. and some other species like Nannochloropsis sp. and Spirulina platensis.8-10 Conventionally, volatile organic compounds (VOCs) such as ethyl acetate were used in the extraction of phenolic compounds from biomass6, 11; however, these solvents are usually derived from petrochemical sources and present different drawbacks including high volatility, flammability and toxicity, being hazardous towards the environment and human health; moreover, their use is being extensively limited by different international regulations including REACH (EC 1907/2006; i.e. registration, evaluation, authorisation and restriction of chemicals). Water has been also proposed for the extraction of polyphenolic compounds from microalgae since its physico-chemical properties complies with U.S. Food and Drug Administration (FDA) and European regulations, being a non-toxic and environmentallyfriendly solvent12; nevertheless, water suffers from co-extraction of other water-soluble impurities which can lead to lower extraction yield and it is only specific towards hydrophilic and polar compounds, making it less effective towards nonpolar and hydrophobic compounds. Ionic liquids, low melting point organic salts with inherent properties including negligible vapour pressure, low flammability, excellent thermal and chemical stability and tuneability13, 14, were then successfully proposed as novel designer solvents for extracting lipids15, 16 and other bioactive compounds17 from microalgae biomass; yet concerns related to high cost of synthesis and unknown toxicity still need to be tackled for further process scale-up and implementation.5 In this regard, deep eutectic solvents (DES) have recently emerged as a new class of solvents that possess similar features to ionic liquids6, 18 with the additional properties of biodegradability, lower toxicity and easiness of preparation from readily available materials 6, 12. DES are mixtures of Brønsted or Lewis acids and bases, and can be commonly obtained by the complexation of hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD) resulting a solvent with lower melting point than its individual constituents.19 The combination of a large number of possible HBA and HBD will lead to the formation of task-specific

METHODOLOGY Microalgae biomass Dried Chlorella vulgaris was purchased from Algae4Future where it was cultivated in a closed photobioreactor autotrophically, supplied with A4F proprietary culture medium (A4F-M1) and dried via spray drying method. The biomass was stored in a dark room at room temperature prior to use, and its composition is shown in Table 1. Table 1 Chlorella vulgaris biomass composition

Microalgae Moisture (%)a Ash content (%)a Total protein (%)a Total lipid content (%)b aAlgae4Future

2


Chlorella vulgaris 5-10 6-12 47-53 24-27

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bBligh

supplier and purity or concentration. Choline chloride, glycerol, ethylene glycol, 1,3-propanediol and

and Dyer method21

Chemicals All chemicals used in this study were analytical grade as shown in Table 2 with their respective 1,4-butanediol were used for preparing the deep eutectic solvents (DES). The extraction efficiency of all of the synthesized DES were compared to ethyl acetate (EA) and water (H2O), which were taken as conventional benchmark solvents. Gallic acid, sodium bicarbonate solution and Folin-Ciocalteu reagent were used to analyze the total phenolic content (TPC) of the extracts. Antioxidant activities of each extract were determined as per DPPH free radical scavenging method. Standards of gallic acid, p-coumaric acid, ferulic acid and caffeic acid were used for identification and quantification purposes. Formic acid and acetonitrile were used as one of the mobile phases in high-performance liquid chromatography (HPLC) analysis.

Choline chloride (ChCl), glycerol (Gly), ethylene glycol (EG), 1,3-propanediol (PDO) and 1,4butanediol (BDO) were dried in Thermo Scientific vacutherm oven at 50 °C and 150 mBar for 24 hours to remove its water content. Specified molar ratios between the hydrogen bond acceptor (HBA) (choline chloride) and hydrogen bond donor (HBD) (glycerol, ethylene glycol, 1,3-propanediol and 1,4 butanediol) were synthesised as tabulated in Table 3. The mixtures were heated to 50°C and stirred around 4 to 6 hours until the mixture becomes homogenous or a transparent liquid. 5, 20 Table 3 DES components and molar ratios

Name DES1 DES2 DES3 DES4 DES5 DES6 DES7 DES8 DES9 DES10 DES11 DES12

Table 2 List of chemicals, supplier and purity or concentration

Chemicals Choline chloride Glycerol Ethylene glycol 1,3propanediol 1,4butanediol Ethyl acetate Sodium bicarbonate Folinciocalteu DPPH radicals Gallic acid p-coumaric acid Ferulic acid Caffeic acid

Supplier SigmaAldrich SigmaAldrich SigmaAldrich SigmaAldrich SigmaAldrich VWR SigmaAldrich SigmaAldrich SigmaAldrich SigmaAldrich SigmaAldrich SigmaAldrich SigmaAldrich

Purity/Concentration 98% 95% 99.8% 99% 99% 99%

Component 1 (HBA) ChCl ChCl ChCl ChCl ChCl ChCl ChCl ChCl ChCl ChCl ChCl ChCl

Component 2 (HBD) Gly Gly Gly EG EG EG PDO PDO PDO BDO BDO BDO

Molar ratio 1:2 1:3 1:4 1:2 1:3 1:4 1:2 1:3 1:4 1:2 1:3 1:4

Polyphenolic compounds extraction Prior to the extraction process, the biomass was subjected to pre-treatment via liquid nitrogen followed by mortar and pestle to promote cell destruction to enhance polyphenolic yield. Extraction of polyphenolic compounds was performed using water, ethyl acetate and the 12 DES systems. For the base case extraction, 10:1 of solvent to biomass ratio was used as reported by Goiris et al.,22 and Choochote et al.,23. Then, the mixtures were placed in a Labnet VorTemp 1550 shaking incubator at 700 rpm, for 100 minutes, at 40 °C as suggested by Bucic-Kojic et al.,24, 25. After 100 minutes, the supernatant and biomass were separated using a Labnet Spectrafuge 6c centrifuge for 10 minutes at 3000 rpm. Lastly, the supernatant was filtered through Sartorius RC 0.45 μm filter to remove any large precipitate. To improve the

7.5% 1.9-2.1 N 97% 97.5-102.5% 98% 99% 98%

Deep eutectic solvent (DES) synthesis

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extraction process, various operating parameters such as time, temperature, and solvent to biomass ratio were examined. The aforementioned parameters have been shown to have significant impact on the polyphenolic extraction efficiency as indicated by with Bucic-Kojic et al.,24 , who studied the solvent to biomass ratio and temperature for solid-liquid extraction and Bubalo et al.,7 who optimised the extraction time and temperature. In this study, the extraction temperature was firstly evaluated at 40, 50, 60 and 70 °C for 100 minutes and compared against the base case at 40 °C, to determine the best extraction temperature for polyphenolic compound extraction. By using the best extraction temperature, extraction times of 25, 55, 75, 100 and 125 minutes were used to determine the best extraction time which yielded the highest TPC content. Finally 20:1, 10:1 and 5:1 of solvent to biomass ratio were evaluated at the best extraction temperature and extraction time.

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Characterization of polyphenolic profile via High-Performance Liquid Chromatographic (HPLC) analysis The separation of phenolic compounds extracted from Chlorella vulgaris via conventional solvents and DES was achieved on a Nucleosil 100-5 C18 column (15cm x 4.6mm, 5um) and analysed by HPLC with DAD detector. 20 uL of extracts were separated by the use of two mobile phases, 0.1% of formic acid in distilled water (A) and 0.1% of formic acid in acetonitrile (B), at 0.5 mL/min. The separation was observed at 300 nm. The column temperature was maintained at 25°C. Gradient elution of 50 minutes of analysis was shown in Table 4. Identification of phenolic compounds was performed by comparing its retention time to those standards. External standard calibration curve was employed for quantification purposes. Table 4 HPLC gradient elution

Time (minutes) 0 – 30 30 – 33 35 - 50

Total polyphenolics content (TPC) The total phenolic contents of all extracts were determined by Folin-Ciocalteu method as per ISO 14502-1:2005, the most common and globally accepted.8, 26 In a centrifuge tube, 100 μl of extract was added, followed by 1 mL of distilled water and 200 μl of Folin-Ciocalteu reagent. The tube was shaken vigorously for 1 minute before adding 300 μl of 7.5% sodium bicarbonate solution which was freshly prepared and filtered through Sartorius RC 0.45 μm filter to remove any remaining sodium bicarbonate crystals. The Folin-Ciocalteu reagent should be added and mixed with the sodium bicarbonate solution to prevent the air-oxidation of phenols.27 Then, the centrifuge tube was wrapped in aluminium foil as phenolic compounds are very sensitive to light. The solution was then shaken again for 1 minute and left in darkness for 1 hour at room temperature. Each extract was analysed by UV and monitored at 675 nm and calculated as gallic acid equivalent (GAE)/g biomass (Eq. 1)28. 𝑇𝑃𝐶 =

Evaluation of antioxidant activity via DPPH free radical scavenging method 0.1 mM of DPPH in methanol was prepared, and one mL of the solution was used onto three mL of each extract. The mixture was incubated for 30 minutes and observed using UV analysis at 517 nm. The color changes and its absorbance were recorded. A formula of % discoloration (Eq. 2.2)29 was employed to determine the antioxidant activity of the phenolic compounds in all extracts. % 𝑑𝑖𝑠𝑐𝑜𝑙𝑜𝑢𝑟𝑎𝑡𝑖𝑜𝑛 = A0 A1

𝐴0 ― 𝐴1 𝐴1

𝑥 100%-----------Eq. 2

=Absorbance of blank DPPH reagent =Absorbance of DPPH reagent and extracts

Biomass surface characterization via Scanning Electron Microscope (SEM) analysis The dried biomass samples were coated with carbon (7.5mm) prior to SEM analysis to improve the sample conductivity. SEM images were obtained using ZEISS EVO 60 model microscope, and the condition was set as the resolution of 1 nm and an acceleration voltage of 5kV.

(𝐶 𝑥 𝑉) 𝑀

Mobile phase A 90% 10% 90%

---------------------------------------Eq. 1

TPC = total phenolic content (mg GAE/g biomass) C = concentration of gallic acid equivalent (mg/L) V = volume of extracts used for analysis (L) M = mass of the extracts used for analysis (mg)

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acetate6, 11 and 12 DES systems. All of the solvents used were diluted with 30% water. The performance of the individual components of DES were also assessed, i.e. ChCl, Gly, EG, PDO and BDO. The extraction efficiency was monitored in terms of total phenolic content (TPC) via Folin-Ciocalteu method and UV analysis at 675 nm as depicted in Figure 1. The TPC provided by water and ethyl acetate extracts were at 2.02 and 0.91 mg GAE/g biomass. These trends are in agreement with those reported by with Hajimahmoodi et al.,10 where extracted 3.69 and 0.02 mg GAE/g biomass with water and ethyl acetate respectively at 80°C for 30 minutes with 20:1 solvent to biomass ratio. Based on the TPC of DES individual components, glycerol shows the lowest TPC at 0.43 mg GAE/g biomass, followed by choline chloride at 0.45 mg GAE/g biomass. All other polyols, namely ethylene glycol, 1,3-propanediol and 1,4-butanediol yielded much higher TPC at 2.6 mg GAE/g biomass, 2.0 mg GAE/g biomass, and 2.09 mg GAE/g biomass respectively. The high viscosity of Gly and ChCl may be one of the contributing factors for the low TPC extracted, while a lower viscosity and higher polarity of polyols contributed to much higher TPC extracted from microalgal biomass. Lower viscosity of polyols can enhance the mass transfer of phenolic compounds into the solvents. In contrast, the high polarity of water was not beneficial as water-soluble contaminants, and other compounds can be co-extracted thus reducing phenolic compound content.12 The polyphenol extraction efficiency provided by polyol-based DES is generally higher that obtained with individual counterparts, although the HBA: HBD molar ratio of DES play an important role. Glycerol-based DES have much higher TPC at lower ratio of Gly to ChCl. TPC decreases from 2.5 to 1.14 to 1.01 mg GAE/ g biomass for 1:2, 1:3 and 1:4 of ChCl to Gly respectively. Higher ratio of glycerol increases the solvent viscosity which minimizing the mass transfer of polyphenolic compounds into the extracting solvent. The other three polyol-based DES, ethylene glycol, 1,3propanediol and 1,4-butanediol had much higher TPC at 1:4 ratio of HBA to HBD, which were about 3.87 mg GAE/g biomass, followed by 3.22 mg GAE/g biomass, and 3.60 mg GAE/g biomass respectively. A higher ratio of polyols (ethylene glycol, 1,3-propanediol and 1,4-butanediol) to

RESULTS AND DISCUSSION The feasibility of using sustainable deep eutectic solvents (DES) as an alternative to conventional volatile organic solvents (VOCs) in the extraction of natural polyphenolic antioxidants from microalgal biomass was investigated herein. In particular, 12 DES systems and two conventional solvents, i.e. EA as benchmark VOC and H2O as globally accepted green universal solvent, were evaluated to study their feasibility in extracting polyphenolic compounds from Chlorella vulgaris. ChCl salts as hydrogen bond acceptor (HBA) and a variety of polyols including Gly, EG, PDO and BDO as hydrogen bond donor (HBD) were chosen as starting materials due to their wide availability as well as their renewable, non-toxic and biodegradable nature. 5, 19 Moreover, the polarity of the chosen DES play an important role in increasing the extraction efficiency of bioactive compounds based on the principle of ‘like-dissolve-like’, while it can be tuned and by choosing a variety of range of HBA and HBD or by addition of water. 5, 12, 20 Chlorella vulgaris has been selected as the microalgae biomass reference in this study due to its widely proven capacity to accumulate target biomolecules and bioactive compounds, including polyphenolic antioxidants 8, 9, 30. Hence, it acts as a benchmark in assessing the performance of the proposed solvents regarding total phenolic content (TPC), antioxidant activity and polyphenolic profile of the extracts. Based on the TPC via colourimetric analysis using FolinCiocalteu reagent, the best performing DES and two conventional solvents (EA and H2O) were further evaluated on their antioxidant activity via DPPH method along polyphenolic identification and quantification through HPLC analysis. Structural analysis using SEM was also used to determine the effect of pre-treatment and solvents onto the cellular structure of Chlorella vulgaris before and after extraction. Phenolic extraction via conventional and DES Base extraction conditions comprising of extraction temperature (T), extraction time (t) and solvent to biomass ratio (S/F) at 40°C, 100 minutes and 10:1 respectively were employed for extracting polyphenolic compounds from microalgal biomass, Chlorella vulgaris. The solid-liquid extraction method was performed on the biomass using two conventional solvents, namely water and ethyl

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relative to conventional solvents which can be ascribed to favorable electrostatic and hydrogen bond interactions with the target solutes 5, 12, 31.

choline chloride reduces the surface tension and viscosity of DES12. Overall results support that polyol-based DES have the ability to enhance the extraction efficiency of polyphenolic compounds 4.5 TPC (mg GAE/g biomass)

4 3.5 3 2.5 2 1.5 1 0.5

PD O DE S1 0 DE S1 1 DE S1 2 BD O

S9 DE

S8 DE

S7 DE

EG

S6 DE

S5 DE

S4

y

DE

Gl

Ch Cl DE S1 DE S2 DE S3

EA

0

H2 O

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Solvents Figure 1 Total phenolic content (TPC) of Chlorella vulgaris extracted with proposed solvents at base case extraction 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 = 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 = ChCl:PDO (1:4), DES10 = ChCl:BDO (1:2), DES11 = ChCl:BDO (1:3), DES12 = ChCl:BDO (1:4)].

Effect of operating conditions (temperature, time, and solvent to biomass ratio) on microalgal polyphenol extraction To determine the influence of temperature on the extraction of polyphenolic compounds from Chlorella vulgaris, 40 °C, 50 °C, 60 °C and 70 °C were employed for 100 minutes using 10:1 solvent to biomass ratio. The extracts were analysed colourimetrically using Folin-Ciocalteu reagent and monitored using UV at 670 nm to obtain the total phenolic content (TPC). As shown on Figure 2, 60 °C has provided the highest TPC for the majority of DES, such as DES 1 (3.23 mg GAE/g biomass), DES 2 (1.76 mg GAE/g biomass), DES 5 (1.93 mg GAE /g biomass), DES 7 (2.18 mg GAE/g biomass), and DES 8-12 (2.16-4.77 mg GAE/g biomass). Only DES 3 and DES 6 have shown the highest TPC at 50 °C at 1.02 mg GAE/g biomass and 4.21 mg GA equivalent/g biomass respectively. Conventional solvents, EA and H2O, have shown the highest TPC at 70 °C (1.40 mg GAE/g biomass) and 50 °C (2.53 mg GAE/g biomass) respectively. As most of the solvents have provided the highest TPC at 60 °C, thus such temperature was selected for the subsequent

extraction essays of polyphenolic compounds from Chlorella vulgaris. Increasing temperature promotes higher solubility and diffusion coefficient of polyphenolic compounds into the extracting solvents, as supported by previous works.5, 32 Higher temperatures also enhance penetration of solvents into the cell matrix hence increasing the TPC of the extracts8. However, an increase in temperature for the extraction of polyphenolic compounds can lead to degradation33. In fact, Volt et al. 34 reported that the percentage of gallic acid degradation increased to 25% at 80 °C as compared to only 15% degradation at 60°C. To determine the most suitable extraction time, a range between 25 minutes and 125 minutes was evaluated for the extraction of polyphenolic compounds from Chlorella vulgaris at the temperature of 60°C using 10:1 solvent to biomass ratio. As shown in Figure 3, the majority of DES yielded the highest TPC at 100 minutes, such as DES 1-2 (1.76-3.23 mg GAE/g biomass), DES 5-7 (1.93-4.15 mg GAE/g biomass), DES 9 (3.88 mg GAE/g biomass), and DES 11-12 (3.31-4.77 mg GAE/g biomass). Only DES 4, 8 and 10 provided the highest TPC at 125 minutes with 1.15 mg GAE/g biomass, 2.98 mg GAE/g biomass,

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compounds from Chlorella vulgaris. Generally, increasing extraction time will lead to higher extraction yield due to the longer exposure of solvent to the compound of interest, although there is a maximum time after which the TPC content of the extracts decreases due oxidation of polyphenolic compounds8.

and 2.74 mg GAE/g biomass respectively. As for conventional solvents, EA and H2O have the highest TPC at 75 minutes (2.43 mg GAE/g biomass) and 125 minutes (2.55 mg GAE/g biomass) respectively. As most of the solvents yielded the highest TPC at 100 minutes, such extraction time was selected for extracting polyphenolic

40°C

TPC (mg GAE/g biomass)

6

50°C

60°C

70°C

5 4 3 2 1 0 H2O

EA ChCl DES1 DES2 DES3 Gly DES4 DES5 DES6 EG DES7 DES8 DES9 PDO DES10DES11DES12 BDO Solvents

Figure 2 Total phenolic content (TPC) of Chlorella vulgaris extracted with proposed solvents at t=100 and S/F = 10:1 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), 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), DES10 = ChCl:BDO (1:2), DES11 = ChCl:BDO (1:3), DES12 = ChCl:BDO (1:4)].

A) 3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25 min

50 min

75 min

100 min

125 min

2.5 2 1.5 1 0.5 0 H2O

EA

ChCl

Gly Solvents

7


EG

PDO

BDO



B) 6

25 min

50 min

75 min

100 min

125 min

5 4 3 2 1 0 DES1

DES2

DES3

DES4

DES5

DES6 DES7 Solvents

DES8

DES9

DES10

DES11

DES12

Figure 3 Total phenolic content (TPC) of Chlorella vulgaris extracted with A) conventional solvents and individual DES 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 = 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 = ChCl:PDO (1:2), DES8 = ChCl:PDO (1:3), DES9 = ChCl:PDO (1:4), DES10 = ChCl:BDO (1:2), DES11 = ChCl:BDO (1:3), DES12 = ChCl:BDO (1:4)]. 5:1

12 TPC (mg GAE/g biomass)

10:1

20:1

10 8 6 4 2

PD O DE S1 0 DE S1 1 DE S1 2 BD O

S9 DE

S8 DE

S7 DE

EG

S6 DE

S5 DE

y

S4 DE

Gl

Ch Cl DE S1 DE S2 DE S3

EA

0

H2 O

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Solvents Figure 4 Total phenolic content (TPC) of Chlorella vulgaris extracted with proposed solvents at T=60 °C and t=100 min as a function of solvent to biomass ratio. [DES1 = ChCl:Gly (1:2), DES2 = 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 = ChCl:PDO (1:2), DES8 = ChCl:PDO (1:3), DES9 = ChCl:PDO (1:4), DES10 = ChCl:BDO (1:2), DES11 = ChCl:BDO (1:3), DES12 = ChCl:BDO (1:4)].

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HPLC analysis and biomass structural analysis via SEM.

The next parameter to evaluate was solvent to biomass ratio. In particular, three ratios of 5:1, 10:1 and 20:1 were used for extraction at 60 °C of temperature during 100 minutes. As shown in Figure 4, the TPC for all extracts increased significantly from 5:1 to 20:1 solvent to biomass ratio, since increasing the amount of solvent promote contact between both phases and penetration of the extraction solvent into the cell matrix. At 20:1, EA and H2O extracts yielded TPC about 3.62 mg GAE/ g biomass and 1.89 mg GAE/ g biomass respectively; meanwhile DES1, DES6, DES9, and DES12 extracts provided the highest TPC at 5.27 mg GAE/ g biomass, 7.19 mg GAE/ g biomass, 9.19 mg GAE/ g biomass, and 9.87 mg GAE/ g biomass respectively. Thus, 20:1 of solvent to biomass ratio was selected as suitable ratio for extracting phenolic compounds from Chlorella vulgaris. Based on the obtained results, it can be concluded that the most favorable conditions for extraction of polyphenolic compounds from Chlorella vulgaris were 60 °C of extraction temperature, 100 minutes of extraction time and 20:1 of solvent to biomass ratio. Moreover, DES1, DES6, DES9 and DES12 were the solvents yielding the highest polyphenol content; therefore, such best performing DES along with H2O and EA were chosen for further assessments on the antioxidant activity via DPPH free radical scavenging method, characterization of polyphenolic profile through

Characterization of polyphenolic profile via High-Performance Liquid Chromatographic (HPLC) analysis HPLC analysis was employed to identify and quantify the polyphenolic extracts yielded by the four best performing DES (i.e. DES1, DES6, DES9, DES12) and conventional solvents (EA and H2O,) obtained at the most favorable operating conditions (temperature of 60 °C, 100 minutes and 20:1 solvent to biomass ratio). The HPLC chromatographic profiles (Figure 5 and Supplementary Information) indicated that the four major phenolic compounds8, i.e. gallic acid (peak at 6.475 min), caffeic acid (peak at 11.533 min), p-coumaric acid (peak at 13.598 min), and ferulic acid (peak at 14.001 min) were present in the extracts. Based on Table 5, the highest content of polyphenolic compounds yielded by DES was provided by DES1 (0.86 mg/g biomass), followed by DES 12 (0.77 mg/g biomass), DES6 (0.64 mg/g biomass), and DES9 (0.55 mg/g biomass); meanwhile, EA and H2O provided lower content of target polyphenolic compounds in the extracts at 0.61 mg/g biomass and 0.33 mg/g biomass respectively (Figures S1-S6 in the ESI represent the HPLC chromatograms of polyphenolic compounds from Chlorella vulgaris using all 6 extraction solvents).

Table 5 Selected phenolic compounds content via HPLC analysis

Solvent Gallic acid Caffeic acid p-Coumaric acid Ferulic acid

EA 0.13 0.17 0.17 0.14

H2O 0.03 0.11 ND 0.19

DES1 0.67 ND 0.16 0.03

DES6 0.38 ND 0.14 0.12

DES9 0.032 0.2 0.16 0.16

DES12 0.35 0.12 0.19 0.11

Total (mg/g biomass)

0.61

0.33

0.86

0.64

0.55

0.77

favorable operating conditions of T=60 °C, t=100 min and S/F = 20:1.

The presence of these phenolic compounds corroborates with Safafar et al.30 and Onofrejova et al. 35. Furthermore, our results support that the proposed polyol-based DES are able to provide a higher selectivity towards target antioxidants, especially gallic acid, than conventional solvents. Three main methods have been reported to recover the polyphenolic compounds from DES extracts,

Figure 5 HPLC profile of polyphenolic compounds from Chlorella vulgaris extracted with DES1 under most

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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.

namely precipitation by addition of anti-solvents, liquid−liquid extraction (LLE) using another solvent, and solid−liquid extraction (SLE) using resin or molecular sieves1. However, note that the use of DES composed of GRAS (Generally Recognize as Safe) and food–grade chemicals as extracting solvents can minimise the cost of separating the compounds from the extraction solvent, since they could be potentially employed in food industry, fine chemical formulations and pharmaceutical purposes1. The preliminary assessment of the capacity of the different solvents to extract polyphenolic compounds was initially performed in terms of TPC via Folin-Ciocalteu method by using UV-Vis. However, the precise value of TPC may be subject to slight over or under estimations due to light scattering by viscous sample or absorption of other impurities. Therefore, HPLC analysis was employed to further identify and quantify the concentration of those specific polyphenols present in the extracts. An important factor affecting the extraction efficiency is the structure of the HBD employed to form each DES, which affects not only the overall solvent viscosity but also the solvent capacity to interact through hydrogen bonding with the different polyphenols. It should be noted that glycerol has three -OH groups as compared with the two –OH groups contained in the structure of the other assessed polyols; this enhances the hydrogen bonding interactions within the neat Gly-based DES, increasing solvent viscosity but also enhancing the solvent ability to form specific interactions with the solutes, hence increasing the recovery of target polyphenols. As for the diols, EG-based DES present lower viscosity overcoming potential mass transfer limitations during the extraction process, although the vicinal position of the –OH in its structure may cause steric hindrance within the molecule, hence limiting at some extent the ability of the solvent to easily interact with the solutes. However, diols with longer alkyl chains presenting –OH groups at terminal positions may overcome the steric effects enhancing extraction of target solutes, as it is the case of BDO-based DES. Therefore, the structure of the HBD is a vital parameter to consider since it may lead to specific solvent-solute interactions to isolate the bioactive molecules of interest.

% discolouration

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80 70 60 50 40 30 20 10 0

EA H2O DES1 DES6 DES9 DES12 Figure 6 Antioxidant capacity of Chlorella vulgaris polyphenolic extracts via best performing solvents under most favorable operating conditions of T=60 °C, t=100 min and S/F = 20:1.

Biomass surface characterization via Scanning Electron Microscope (SEM) analysis Prior to solvent extraction of polyphenolic compounds, Chlorella vulgaris biomass was

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subjected to pre-treatment by liquid nitrogen followed by mortar and pestle to promote cell disruption to increase the extraction yield38. During this process, the cells were fractured and burst as ice crystals formed inside the cells due to the use of liquid nitrogen and force was imposed by the mortar and pestle39. The states of the cells before and after extraction via DES (DES1 and DES12) and conventional solvents (ethyl acetate and water) were observed via Scanning Electron Microscope (SEM) images as shown in Figure 7.

cellulose after extraction, hence releasing the natural products into the surroundings. In addition, the results provided by these SEM images also agrees with those reported by Nur Atikah Md Noor et al.,41 where they pre-treated oil palm empty fruit bunch with DES for sugar production. DES had shown the ability to impact the fiber, promoting cavities and roughness onto the surface where the structural lignin seemed to be broken. Thus, results suggest that, besides increasing the polyphenol extraction efficiency, DES solvents may also be advantageous in disrupting the microalgae cell walls which could be beneficial to facilitate further biomass processing. CONCLUSIONS This study demonstrates the feasibility of using renewable polyol-based DES in extracting phenolic compounds from microalgae biomass, Chlorella vulgaris, as a means to develop sustainable processes for natural antioxidants manufacturing. In particular, the polyphenol extraction efficiency provided by twelve DES systems composed of choline chloride (ChCl) and a variety of polyols including glycerol (Gly), ethylene glycol (EG), 1,3propanediol (PDO) and 1,4-butanediol (BDO) was evaluated and their performances were compared to benchmark conventional solvent such as ethyl acetate and water in terms of total phenolic content (TPC via Folin-Ciocalteau method), antioxidant activity (DPPH free radical scavenging method), and polyphenolic profile of the extracts (via HPLC analysis). Overall results support that the proposed DES are able to yield extracts with higher total polyphenolic content than conventional solvents, mainly DES1 (ChCl:Gly 1:2) DES6 (ChCl:EG 1:4), DES9 (ChCl: PDO 1:4), and DES12 (ChCl:BDO 1:4) which provided up to two-fold extraction efficiency under the most favorable operating conditions of 60°C of extraction temperature, 100 minutes of extraction time and 20:1 solvent to biomass ratio. Moreover, DES 1 and DES 12 were proved to be significantly more selective towards target polyphenolic compounds, especially gallic acid, while providing polyphenolic extracts with higher antioxidant capacity than conventional solvents. Furthermore, microscopy surface characterization of biomass before and after the extraction suggested the ability of DES solvents to disrupt the microalgae cell walls which could be beneficial for further biomass processing.

Figure 7 SEM images of Chlorella vulgaris (A) before extraction (B) conventional solvent [1=H2O, 2=EA] and (C) DES [1=DES1, 2=DES12]

Untreated biomass (A) shows that the cells were uniform and exhibited a circular shape. Meanwhile, the treated biomass (B1, B2, C1 and C2), exhibit more deconstructed cells, which appear to be shrunken and wrinkled indicating that the intercellular matter has been exposed to the environment and have been in contact with the extracting solvents. Biomass extracted by DES show agglomerated cells which can be ascribed to the higher viscosity of the extracting solvents. This agrees with a previous study40 reporting that DES (ChCl: 1,2-butanediol) formed porous slice on Salvia miltiorrhiza Bunge due to the dissolution of

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ASSOCIATED CONTENT Supporting Information: HPLC profiles of polyphenolic compounds from Chlorella vulgaris extracted with ethyl acetate, water, DDES1, DES6, DES9 and DES 12 under most favorable operating conditions of T=60 °C, t=100 min and S/F = 20:1. AUTHOR INFORMATION Corresponding Author: Dr. María Gonzalez-Miquel E-mail address: [email protected] ACKNOWLEDGEMENTS Wan Mohd Asyraf Wan Mahmood would like to acknowledge the Ministry of Higher Education (MOHE) Malaysia for financially supporting this project and granting the pre-doctoral scholarship. REFERENCES 1. Ruesgas-Ramon, M.; Figueroa-Espinoza, M. C.; Durand, E., Application of Deep Eutectic Solvents (DES) for Phenolic Compounds Extraction: Overview, Challenges, and Opportunities. Journal of agricultural and food chemistry 2017, 65 (18), 35913601, 10.1021/acs.jafc.7b01054. 2. Naczk, M. S., Fereidoon, Extraction and analysis of phenolics in food. Journal of Chromatography A 2004, 1054 (1-2), 95-111, 10.1016/s0021-9673(04)01409-8. 3. Claudine Manach, A. S., Christine Morand, Christian Rémésy, and Liliana Jimenez, Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004, 79, 727–47. 4. RESEARCH, G. V. Tea Polyphenols Market Analysis By Application (Functional beverages, Functional food, Dietary supplements) By Product (Green tea, Olong tea, Black tea) And Segment Forecasts To 2020; 978-1-68038-002-6; 2015. 5. Ozturk, B.; Parkinson, C.; Gonzalez-Miquel, M., Extraction of polyphenolic antioxidants from orange peel waste using deep eutectic solvents. Separation and Purification Technology 2018, 206, 1-13, 10.1016/j.seppur.2018.05.052. 6. Peng, X.; Duan, M.-H.; Yao, X.-H.; Zhang, Y.-H.; Zhao, C.-J.; Zu, Y.-G.; Fu, Y.-J., Green extraction of five target phenolic acids from Lonicerae japonicae Flos with deep eutectic solvent. Separation and Purification Technology 2016, 157, 249-257, 10.1016/j.seppur.2015.10.065. 7. Cvjetko Bubalo, M.; Curko, N.; Tomasevic, M.; Kovacevic Ganic, K.; Radojcic Redovnikovic, I., Green extraction of grape skin phenolics by using deep eutectic solvents. Food Chem 2016, 200, 159-66, 10.1016/j.foodchem.2016.01.040. 8. Zakaria, S. M.; Kamal, S. M. M.; Harun, M. R.; Omar, R.; Siajam, S. I., Subcritical Water Technology for Extraction of Phenolic Compounds from Chlorella sp. Microalgae and Assessment on Its Antioxidant Activity. Molecules 2017, 22 (7), 10.3390/molecules22071105. 9. Wan Mahmood, W. M. A.; Theodoropoulos, C.; Gonzalez-Miquel, M., Enhanced microalgal lipid extraction using bio-based solvents for sustainable biofuel production. Green Chemistry 2017, 19 (23), 5723-5733, 10.1039/c7gc02735d.

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Table of Contents

Synopsis: Renewable polyol-based Deep Eutectic Solvents for extraction of phenolic compounds from microalgae biomass to develop sustainable processes for natural antioxidants manufacturing.

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Chlorella vulgaris

Polyol Deep Eutectic Solvents

Antioxidants


Polyol-Based Deep Eutectic Solvents for Extraction of Natural

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