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Eucalyptol-based green extraction of brown alga Zonaria tournefortii
Sustainable Chemistry and Pharmacy 10 (2018) 97–102
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Sustainable Chemistry and Pharmacy journal homepage: www.elsevier.com/locate/scp
Eucalyptol-based green extraction of brown alga Zonaria tournefortii a
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S. Hamiche , N. Bouzidi , Y. Daghbouche , A. Badis , S. Garrigues , M. de la Guardia , ⁎ M. El Hattaba, a b
Laboratory of Natural Products Chemistry and Biomolecules (LNPCB), University of Blida 1, Road of Soumaâ, P.O. Box 270, 09000 Blida, Algeria Department of Analytical Chemistry, University of Valencia, Research Building, 50 Dr. Moliner Street, 46100 Burjassot, Valencia, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Green extraction Eucalyptol Zonaria tournefortii Total phenolic compound Antioxidant activity
A green extraction method, based on the use of 1,8-cineole (eucalyptol) as biosolvent, has been developed to prepare crude extracts from the brown alga Zonaria tournefortii characterized by chemical composition, particularly dominated by phenolic compounds derived from phloroglucinol. The main advantage of the developed technique are the recovery of eucalyptol, based on multistep liquid-liquid extraction with distilled water, followed by centrifugation and elimination of the aqueous phase, and the complete recycling of biosolvent by steam distillation. A comparative study between the proposed green extract and the conventional extract, prepared by solvent maceration using the mixture CH2Cl2/MeOH (1/1:v/v), was performed in terms of qualitative and quantitative determination of several parameters as:(i) the total phenolic content determined by the FolinCiocalteu assay, (ii) the presence of phenols determined by high performance liquid chromatography (HPLC), and (iii) the antioxidant activity assessed by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay. In short, eucalyptol provides a safe and selective extraction of phenolic compounds from Zonaria tournefortii with no environmental side effects and a good recovery of the solvent.
1. Introduction Green Chemistry is based on the design, development, and implementation of chemical products and processes to reduce or eliminate the use and generation of hazardous substances to human health and the environment (Anastas and Warner, 1998; Armenta et al., 2008). In natural product extraction, Green Chemistry will reduce energy consumption, allowing the use of alternative solvents and renewable natural products, and ensure a safe and high quality extract/product (Chemat et al., 2012). For greening extraction process at laboratory and industrial scales it is necessary to: (i) improve and optimize existing processes; (ii) use non-dedicated equipment; and (iii) search for alternative solvents, the so called green solvents. Sustainable solvents or biobased solvents have interesting advantages for the replacement of traditional solvents in the treatment of natural products (Chemat and Vian, 2014) and for sample preparation in Analytical Chemistry (Armenta et al., 2015; De la Guardia and Garrigues, 2012; Vian et al., 2017) thus providing safe extracts for consumers and safe treatments for operators and the environment. Ethanol, obtained by fermentation of sugar-rich materials, is the most common used bio-solvent due to its low price and biodegradability (Chemat et al., 2012). Additionally terpene compounds, as α-pinene
⁎
obtained from pine oils and d-Limonene from citrus oils have been successfully used (Tanzi et al., 2012), and also glycerol has been proposed as a green solvent (Wolfson et al., 2007; Ou et al., 2011; DiazAlvarez et al., 2011). Additionally, methyl esters of fatty acids (MEFA) of natural origin, known as biodiesel, were used as biosolvents (Gonzalez et al., 2007), in particular the methyl soyate, canolate and cocoate can replace conventional organic solvents. Ethyl lactate is an agrochemical alternative to traditional liquid solvent, it is fully biodegradable and was approved by the U.S. Food and Drug Administration (FDA) as pharmaceutical and food additive. The aforementioned characteristics have encouraged the use of natural products as biosolvents and several potential applications are reported in literature (Ishida and Chapman, 2009; Vicente et al., 2011). However, to our knowledge there is any previous work based on the use of eucalyptol (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane), also known as 1,8-cineole, as biosolvent. Eucalyptol is the main terpene obtained from several terrestrial plants such as Euclayptus globules, and has been evaluated in this study as an alternative to the use of chlorinated solvents and methanol for the extraction of brown alga Zonaria tournefortii. Previous studies on the lipid extract of brown alga revealed the
Corresponding author. E-mail address: [email protected] (M. El Hattab).
https://doi.org/10.1016/j.scp.2018.10.005 Received 19 May 2018; Received in revised form 14 October 2018; Accepted 14 October 2018 2352-5541/ © 2018 Elsevier B.V. All rights reserved.
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presence of phenolic compounds such as acylphloroglucinols and its derivatives (Amico et al., 1981, 1982; Tringali and Piattelli, 1982a, 1982b; Blackman et al., 1988). Further study on the same species has allowed the isolation and structural characterization of the eicosapentaenoic acid (El Hattab et al., 2009). The partial solubility in water of eucalyptol has been valorised in order to separate it from the extract by multistep liquid-liquid extraction. Additionally it was made a comparative study between the extracts obtained by the green extraction method proposed and those obtained by conventional extraction with a toxic organic solvents mixture as CH2Cl2 and methanol in terms of extraction yield, chemical composition, total phenolic content, and antioxidant activity.
25 g of brown algae finely chopped
Solvent maceration with 160 mL of eucalyptol at room temperature
Filtration of the mixture
Crude extract + eucalyptol (filtrate)
2. Material and methods Multistep liquid-liquid extraction of filtrate with water (1/20 : v/v)
2.1. Solvents and reagents All solvents used for chromatography were HPLC grade and were purchased from Sigma Aldrich (Lyon, France). Reagents and solvents were of analytical grade. Eucalyptol, phloroglucinol, Folin-Ciocalteu reagent, methanol, were supplied by Panreac (Barcelona, Spain). Ascorbic acid, butylatedhydroxyanisole (BHA), butylatedhydroxytoluene (BHT), methylene chloride and acetonitrile were purchased from Sigma Aldrich (Lyon, France). The 2, 2-diphenyl-1 picrylhydrazyl (DPPH) reagent, trichloroacetic acid and chloroform were provided by Fluka (Strasbourg, France).
Centrifugation 4000 rpm (10 min)
Pure extract
Yield: 0.45% m/v
Water + eucalyptol
Hydro-distillation Dean-stark
2.2. Plant material Plant materials were collected in the Mediterranean coast of Algeria on the west of Tipaza (36°37' 12 '' NR, 2°39' 00 '' E), in June 2012. These samples belongs to: Phaeophyceae class, Dictyotales order, Dictyotaceae family, Zonaria genus and tournefortii (Tipaza; June 2012; J.V. Lamouroux). The alga was identified by Dr. H. Seridi and a voucher specimen (H.S. N° 251) was deposited in the Herbarium of Biological Oceanography and Marine Environment Laboratory (USTHB-Algeria). Algae were manually sorted out to remove any trace of epiphytes. They were then air-dried under shade without any other pre-treatment before extraction.
Water
Eucalyptol
Fig. 1. Green extraction procedure using eucalyptol as biosolvent.
range from 4000 to 400 cm−1. The system was equipped with a temperature-stabilized detector with KBr beam splitter and precise digital signal processing. Spectra were obtained at 4 cm−1 nominal resolution and accumulating 20 scans per spectrum. All measurements were carried out using a micro-flow cell model 555 from Eurolabo (Paris, France) with NaCl windows and a path length of 0.5 mm. Connection tubes were made in PTFE with 0.8 mm internal diameter. All samples were diluted in chloroform and FTIR spectra were obtained in the same experimental conditions using a background of pure chloroform.
2.3. Extraction procedures 2.3.1. Conventional solvent extraction Air-dried algae finely chopped (25 g) were placed in 250 mL flasks, and macerated with 160 mL of a mixture of organic solvents CH2Cl2/ MeOH (1/1:v/v). The extraction was carried out on batch mode at room temperature for one week, followed by filtration and evaporation of the solvent. The dry residue obtained was subsequently treated with diethyl ether to give a crude extract.
2.5. HPLC analysis HPLC analysis was carried out using a chromatography system Agilent 1260 Infinity (Santa Clara, CA, USA), equipped with a quaternary pump G13UC, a standard autosampler G1329B and a diode array detector G1315CD. Separations were performed using the isocratic mode, at room temperature, employing an analytical Agilent RP 18column (250 mm × 4 mm ×5 µm). Extracts were analyzed using the eluent mixture MeCN/H2O (95/5:v/v) at a flow rate of 1 mL min−1. The detection wavelength was fixed at 220 nm. A semi-preparative Purospher STARRP 18 column (250 mm × 10 mm × 5 µm) (Molsheim, France) was used for isolation secondary metabolites from the conventional extract, which were mainly phenolic metabolites of phloroglucinol derivatives.
2.3.2. Alternative solvent extraction The developed extraction procedure (Fig. 1.) was based on the use of eucalyptol as an alternative solvent and was performed by the following three steps: i) solvent maceration, in the same conditions as for conventional solvent extraction (25 g algae with 160 mL eucalyptol), ii) solvent separation by filtration and a multistep liquid-liquid extraction with distilled water, followed by centrifugation and separation of the aqueous phase (the number of steps needed to completely recover eucalyptol was determined by using Fourier transform infrared (FTIR) analysis, and iii) solvent recycling by steam distillation using a DeanStark apparatus.
2.6. NMR analysis 2.4. Fourier transform infrared analysis NMR spectra were recorded using Bruker Avance 400 instrument (Strasbourg, France) (1H, 400 MHz and 13C, 100 MHz).1H NMR spectra were referenced internally on CDCl3 (δ1H 7.26 ppm), and 13C NMR
A Shimadzu (Beyne-Heusay, Germany) FTIR spectrometer model 8900 was employed to obtain infrared spectra in the wave number 98
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cyclic ether C(1)-O-C(8) are located between 1052 and 1169 cm−1. In the present study, the absorption band stretching in the finger print region at 1169 cm−1was used as a characteristic band in order to evaluate the number of water washing steps of extract required to separate quantitatively the biosolvent from the crude extract.
spectra were referenced internally on CDCl3 (δ13C 77.20 ppm). 2.7. Total phenolic content determination The total phenolic content was determined using the Folin–Ciocalteau method based on the colorimetric oxidation/reduction reaction of phenols, in accordance with the procedure previously described (Blanca et al., 2011) with some modifications. The crude extract (4 mg) was dissolved in 8 mL of the methanol/water mixture (1/3:v/v) at room temperature and then, 2 mL of each extract solution were mixed with 10 mL of water and 12 mL of sodium carbonate (29% w/v). 1 mL of the Folin–Ciocalteu reagent was added and the development of color was produced after 30 min in the dark at room temperature. The absorbance was measured at 760 nm with a Shimadzu UV-1700 spectrophotometer (Marne la Vallée, France) against the water blank prepared in the same way. A standard curve with phloroglucinol solutions (ranging from 0.008to 0.099 mg mL−1) was used for the calibration. Results were expressed as mg phloroglucinol equivalents/g extract and calculated by the following Eq. (1) (Adesegun et al., 2009):
T=
C×V M
3.2. Evaluation of the number of water washes The solution green extract with eucalyptol was subjected to liquidliquid extraction yielding two phases, an upper phase (extract+eucalyptol) and down phase (water+eucalyptol). After a chosen number of water washes, the upper solution was analyzed by FTIR and the down phase treated for eucalyptol recovery. Our interest was focused on the variation of the absorbance of the band of eucalyptol at 1169 cm−1 in the extract. The FTIR spectra after five, ten and twelve washes are given as inset in Fig. 2. The enlargement of the characteristic absorption band reveals, first, decrease of the absorption intensity versus the increasing of the number of water washes and that, after twelve washes, eucalyptol was totally removed or it remained as trace amounts. 3.3. FTIR analysis of the recycled eucalyptol
(1)
Where T is the total content of phenolic compounds expressed as milligram phloroglucinol equivalents per gram of extract (mg PGE g−1); C is the concentration of phloroglucinol established from the calibration curve (mg mL−1); V is the volume of extract (mL), M is the weight of extract (g).
The down phase of the liquid-liquid extraction process was subjected to steam distillation using a Dean stark set up. This operation allows a simple separation of eucalyptol from water. Through an FTIR analysis of the recycled eucalyptol (results not shown) it was confirmed that after water extraction by steam distillation, eucalyptol was not degraded and spectra of both, pure and recycled solvent, were identical.
2.8. Antioxidant activity determination by DPPH radical scavenging assay
3.4. Comparison of the extraction yield
Radical scavenging activity of extracts was assessed according to the procedure reported in the literature (Loo et al., 2008; Shimada et al., 1992). The method was based on the ability of extract to reduce the DPPH free radical, the activity being determined by spectrophotometry measurements. A volume of 1 mL of each sample, at different concentration levels, was mixed with 1 mL 0.004% solution of DPPH in methanol. The absorbance was recorded at 517 nm against water blank, prepared in the same way, over a period of 30 min. The percentage of inhibition was estimated using the following Eq. (2) (Keyrouz et al., 2011):
Where: Abs0corresponds to the absorbance of the reference which is free of phenols, and Absx is the absorbance of the sample. L-ascorbic acid, BHT, BHA and α-tocopherol were used as reference standards. Different concentrations of samples were tested to obtain a calibration curve giving the inhibition percentage according to the sample concentration. The EC50, deduced from the slope of the calibration curve, corresponds to the concentration of extract or standard antioxidant (mg mL−1) required to scavenge 50% of the DPPH in the reaction mixture.
The extraction yield obtained using eucalyptol lead to a low value (0.45% m/v) as compared to that obtained using conventional solvent extraction (2.16% m/w). This results can be interpreted by the low polarity of the green solvent in comparison to the mixture CH2Cl2/ MeOH characterized by a wide polarity range (Haynes, 2014; Reichardt, 1979). In fact, the biosolvent has a dielectric constant of the same order of that of chloroform and hexane. In contrast, the strong polarity of methanol favours the extraction of heavy polar molecules, such as tannins and dyes (Haynes, 2014; Reichardt, 1979). Through the use of terpenoids more polar than eucalyptol as biosolvent, the extraction yield could be increased. Further studies have reported a high yield extraction of natural products from marine and/or terrestrial organisms by the use of CH2Cl2/MeOH in comparison to other solvents as (Li et al., 2014; Breil et al., 2017; Stengel and Connan, 2015). Despite the low extraction yield obtained by using eucalyptol the most important advantages of extraction using biosolvent are the development of a process environmentally friendly based on avoiding toxic solvent and the obtention of high quality products, including the recovery of eucalyptol. In addition, due to the high selectivity of the biosolvent, the green extract contains very small undesirable products.
3. Results and discussion
3.5. HPLC analysis
3.1. FTIR spectra of eucalyptol
As mentioned through our previous study on the same species (El Hattab et al., 2009), the chemical composition of the crude extract is characterized by the presence of phenolic metabolites, phloroglucinol derivatives and eicosapentaenoic acid as major compounds (see Fig. 3.). In order to compare the chemical composition of the crude extracts obtained by both extraction methods, the phenolic secondary metabolites previously described from a crude extract obtained by conventional solvent extraction were isolated by semi-preparative HPLC. The structure of the isolated compounds was unambiguously elucidated by comparing their 1H and 13C NMR data with the literature (Amico et al., 1981, 1982).
Inhibiton percentage% =
Abs0 × Abs x ×100 Abs0
(2)
The infrared absorbance spectrum of eucalyptol in CHCl3 is indicated in Fig. 2. Methyl (2985 cm−1, 2968 cm−1) and methylene groups (2924 cm−1 and 2881 cm−1) stretching vibrations provided strong bands. The methyne bonds were identified by the absorption bands at 1466 cm−1, 1375 cm−1and 1052 cm−1. The literature reports that the characteristic absorption bands of cyclic ether (epoxide) is ranged from 1070 cm−1 to 1140 cm−1 for a symmetric stretch and could be extended to 1247 cm−1 for an asymmetric stretch (Easton et al., 2009). Concerning the eucalyptol, the stretching bands of the 99
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Fig. 2. FTIR spectra of pure eucalyptol in CHCl3 (green) and after washing the crude extract + eucalyptol (the filtrate) with water several times: 5 - blue, 10 – red and 12 - pink) (22 mg mL−1). Inset: Decrease in absorbance of the characteristic band of eucalyptol at 1169 cm−1 as function of the number of water washes (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
conventional extract is more complex and contain, in addition to the phenolic compounds described (Fig. 3C.), other compounds of phenolic nature such as tannins. It must be emphasized that phenolic compounds in marine algae are correlated with antioxidant activity and also depend on the solvent and algal species used for extraction (Movahedinia and Heydari, 2012).
The chromatography profiles of conventional and green extracts (Fig. 3.) obtained by analytical HPLC have a good similarity. Examination of the two chromatograms reveals the presence of higher levels of polar compounds in conventional extract than in the green extract. Furthermore, we notice the high content of compound (1) and a very low content of compounds (3) and (5) in the green extract. In contrast, the conventional extract shows low content in compounds (1) and (3) (see Fig. 3C.). These results may be related to the relative solubility of pure phenolic metabolites in both solvents. It must be pointed out the large scale of antimicrobial activities of phenolic compounds of brown algae (Pérez et al, 2016) particularly that of the phenolic metabolites isolated from the Zonaria genus (Wisespongpand and Kuniyoshi, 2003). The phenolic metabolites contents in both extracts, green and a conventional, are reported in Table 1.
3.7. Antioxidant activity The antioxidant activity of the obtained extracts, including standards, was assessed through its ability to scavenge the DPPH˙ free radical, and measured in the term of EC50 (efficient concentration providing 50% inhibition) calculated graphically using a non-linear fitting curve by plotting inhibition percentage according to sample dilution level. Results obtained for both kind of extracts, are summarized in Table 3. As it can be seen the activity of extracts was lower than for all the controls. In addition, DPPH˙ scavenging activity of conventional extracts was strongest than that of green ones. So there is a close relationship between the results of the total phenolic content and those of antioxidant activity, probably due to the antioxidant activity of phenolic secondary metabolites and other reductant agents. Because of the different extraction and measurement methods and units used in the antioxidant activity studies on seaweed reported in the literature, direct comparison of present results on radical scavenging activity of extracts with other studies is not feasible. However, many researchers have shown that phenolic compounds derived from marine brown algae have strong antioxidant activities against free radical mediated oxidation damage (Li et al., 2011).
3.6. Total phenolic content determination The total phenolic content in the green and conventional extracts was calculated from the calibration curve established with phloroglucinol as reference. The calibration curve was represented by the analytical features shown in Table 2. Once the calibration realized, the absorbance of samples can be normalized to a phloroglucinol equivalent (PEG) concentration by interpolation of absorbance values in the calibration line. The experimental results revealed that total phenolic content of conventional and green extracts measured in terms of mg PEG g−1 of crude extract were 725.0 ± 1.9 and 362.5 ± 1.4 mg (standard deviation of 5 independent measurements), respectively. The total phenolic content of the conventional extract is in good agreement with the one obtained by volumetric determination on the same extract (El Hattab et al., 2009). The high phenolic compound content of the conventional extract in comparison to green extract can be explained by the low selectivity of the solvent mixture in comparison to the green solvent and confirms the high yield obtained by conventional extraction using the mixture CH2Cl2/MeOH, which is characterized by a wide polarity range, in comparison to green extraction. Accordingly, the
4. Conclusion In the present study it has been developed a green extraction technique, based on the use of eucalyptol as biosolvent for extraction of phenolic compounds from Zonaria tournefortii alga. Green extracts were prepared and compared with conventional extracts obtained by 100
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Fig. 3. HPLC chromatogram of the crude extracts obtained by conventional solvent extraction using CH2Cl2/MeOH (1/1:v/v) (A) and alternative solvent extraction using eucalyptol (B). Chemical structure of secondary metabolites isolated from brown algae Zonaria tournefortii (C).
Table 1 Percentage content of phenolic metabolites in conventional and green extracts. Compounds
1 2 3 4 5
Table 2 Analytical features of the Folin-Ciocalteau determination of phloroglucinol.
Content % (w/w) Conventional extract
Green extract
15.28 3.57 7.64 24.27 15.11
20.17 6.36 1.81 6.30 1.72
Regression line
A= 6.512 C+0.082
Correlation coefficient Linear range (mg/mL) L.O.Q. (µg mL−1) L.O.D. (µg mL−1) R.S.D. % (n = 10)
0.9995 0.008–0.099 5.2 1.57 0.54 (for 0.045 mg mL−1)
Notes: R.S.D. %: Relative standard deviation, in percentage; L.O.Q.: Limit of quantification expressed in µg mL−1; L.O.D.: Limit of detection expressed in µg mL−1.
classical solvent extraction using the mixture of CH2Cl2/MeOH. The conventional extraction offers higher extraction yield than green extraction. Both extracts have been submitted to chemical analyses (total phenolic content, HPLC analysis), and antioxidant activities. The HPLC analysis shows a great similarity between extracts but with and increased amount of non-polar phenols when using eucalyptol. In
contrast, the total phenolic content and the antioxidant activities have indicated that the conventional extract has a strongest effect as compared to the green extract. The additional advantage of the proposed procedure is the recovery of eucalyptol after extraction.
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Table 3 EC50 Values of extracts and antioxidants standards. Samples
EC50 (µg mL−1)
L-ascorbic
4.9 6.0 8.5 58 140
acid BHA BHT Conventional extract Green extract
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Sustainable Chemistry and Pharmacy journal homepage: www.elsevier.com/locate/scp
Eucalyptol-based green extraction of brown alga Zonaria tournefortii a
a
a
a
b
T
b
S. Hamiche , N. Bouzidi , Y. Daghbouche , A. Badis , S. Garrigues , M. de la Guardia , ⁎ M. El Hattaba, a b
Laboratory of Natural Products Chemistry and Biomolecules (LNPCB), University of Blida 1, Road of Soumaâ, P.O. Box 270, 09000 Blida, Algeria Department of Analytical Chemistry, University of Valencia, Research Building, 50 Dr. Moliner Street, 46100 Burjassot, Valencia, Spain
A R T I C LE I N FO
A B S T R A C T
Keywords: Green extraction Eucalyptol Zonaria tournefortii Total phenolic compound Antioxidant activity
A green extraction method, based on the use of 1,8-cineole (eucalyptol) as biosolvent, has been developed to prepare crude extracts from the brown alga Zonaria tournefortii characterized by chemical composition, particularly dominated by phenolic compounds derived from phloroglucinol. The main advantage of the developed technique are the recovery of eucalyptol, based on multistep liquid-liquid extraction with distilled water, followed by centrifugation and elimination of the aqueous phase, and the complete recycling of biosolvent by steam distillation. A comparative study between the proposed green extract and the conventional extract, prepared by solvent maceration using the mixture CH2Cl2/MeOH (1/1:v/v), was performed in terms of qualitative and quantitative determination of several parameters as:(i) the total phenolic content determined by the FolinCiocalteu assay, (ii) the presence of phenols determined by high performance liquid chromatography (HPLC), and (iii) the antioxidant activity assessed by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay. In short, eucalyptol provides a safe and selective extraction of phenolic compounds from Zonaria tournefortii with no environmental side effects and a good recovery of the solvent.
1. Introduction Green Chemistry is based on the design, development, and implementation of chemical products and processes to reduce or eliminate the use and generation of hazardous substances to human health and the environment (Anastas and Warner, 1998; Armenta et al., 2008). In natural product extraction, Green Chemistry will reduce energy consumption, allowing the use of alternative solvents and renewable natural products, and ensure a safe and high quality extract/product (Chemat et al., 2012). For greening extraction process at laboratory and industrial scales it is necessary to: (i) improve and optimize existing processes; (ii) use non-dedicated equipment; and (iii) search for alternative solvents, the so called green solvents. Sustainable solvents or biobased solvents have interesting advantages for the replacement of traditional solvents in the treatment of natural products (Chemat and Vian, 2014) and for sample preparation in Analytical Chemistry (Armenta et al., 2015; De la Guardia and Garrigues, 2012; Vian et al., 2017) thus providing safe extracts for consumers and safe treatments for operators and the environment. Ethanol, obtained by fermentation of sugar-rich materials, is the most common used bio-solvent due to its low price and biodegradability (Chemat et al., 2012). Additionally terpene compounds, as α-pinene
⁎
obtained from pine oils and d-Limonene from citrus oils have been successfully used (Tanzi et al., 2012), and also glycerol has been proposed as a green solvent (Wolfson et al., 2007; Ou et al., 2011; DiazAlvarez et al., 2011). Additionally, methyl esters of fatty acids (MEFA) of natural origin, known as biodiesel, were used as biosolvents (Gonzalez et al., 2007), in particular the methyl soyate, canolate and cocoate can replace conventional organic solvents. Ethyl lactate is an agrochemical alternative to traditional liquid solvent, it is fully biodegradable and was approved by the U.S. Food and Drug Administration (FDA) as pharmaceutical and food additive. The aforementioned characteristics have encouraged the use of natural products as biosolvents and several potential applications are reported in literature (Ishida and Chapman, 2009; Vicente et al., 2011). However, to our knowledge there is any previous work based on the use of eucalyptol (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane), also known as 1,8-cineole, as biosolvent. Eucalyptol is the main terpene obtained from several terrestrial plants such as Euclayptus globules, and has been evaluated in this study as an alternative to the use of chlorinated solvents and methanol for the extraction of brown alga Zonaria tournefortii. Previous studies on the lipid extract of brown alga revealed the
Corresponding author. E-mail address: [email protected] (M. El Hattab).
https://doi.org/10.1016/j.scp.2018.10.005 Received 19 May 2018; Received in revised form 14 October 2018; Accepted 14 October 2018 2352-5541/ © 2018 Elsevier B.V. All rights reserved.
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presence of phenolic compounds such as acylphloroglucinols and its derivatives (Amico et al., 1981, 1982; Tringali and Piattelli, 1982a, 1982b; Blackman et al., 1988). Further study on the same species has allowed the isolation and structural characterization of the eicosapentaenoic acid (El Hattab et al., 2009). The partial solubility in water of eucalyptol has been valorised in order to separate it from the extract by multistep liquid-liquid extraction. Additionally it was made a comparative study between the extracts obtained by the green extraction method proposed and those obtained by conventional extraction with a toxic organic solvents mixture as CH2Cl2 and methanol in terms of extraction yield, chemical composition, total phenolic content, and antioxidant activity.
25 g of brown algae finely chopped
Solvent maceration with 160 mL of eucalyptol at room temperature
Filtration of the mixture
Crude extract + eucalyptol (filtrate)
2. Material and methods Multistep liquid-liquid extraction of filtrate with water (1/20 : v/v)
2.1. Solvents and reagents All solvents used for chromatography were HPLC grade and were purchased from Sigma Aldrich (Lyon, France). Reagents and solvents were of analytical grade. Eucalyptol, phloroglucinol, Folin-Ciocalteu reagent, methanol, were supplied by Panreac (Barcelona, Spain). Ascorbic acid, butylatedhydroxyanisole (BHA), butylatedhydroxytoluene (BHT), methylene chloride and acetonitrile were purchased from Sigma Aldrich (Lyon, France). The 2, 2-diphenyl-1 picrylhydrazyl (DPPH) reagent, trichloroacetic acid and chloroform were provided by Fluka (Strasbourg, France).
Centrifugation 4000 rpm (10 min)
Pure extract
Yield: 0.45% m/v
Water + eucalyptol
Hydro-distillation Dean-stark
2.2. Plant material Plant materials were collected in the Mediterranean coast of Algeria on the west of Tipaza (36°37' 12 '' NR, 2°39' 00 '' E), in June 2012. These samples belongs to: Phaeophyceae class, Dictyotales order, Dictyotaceae family, Zonaria genus and tournefortii (Tipaza; June 2012; J.V. Lamouroux). The alga was identified by Dr. H. Seridi and a voucher specimen (H.S. N° 251) was deposited in the Herbarium of Biological Oceanography and Marine Environment Laboratory (USTHB-Algeria). Algae were manually sorted out to remove any trace of epiphytes. They were then air-dried under shade without any other pre-treatment before extraction.
Water
Eucalyptol
Fig. 1. Green extraction procedure using eucalyptol as biosolvent.
range from 4000 to 400 cm−1. The system was equipped with a temperature-stabilized detector with KBr beam splitter and precise digital signal processing. Spectra were obtained at 4 cm−1 nominal resolution and accumulating 20 scans per spectrum. All measurements were carried out using a micro-flow cell model 555 from Eurolabo (Paris, France) with NaCl windows and a path length of 0.5 mm. Connection tubes were made in PTFE with 0.8 mm internal diameter. All samples were diluted in chloroform and FTIR spectra were obtained in the same experimental conditions using a background of pure chloroform.
2.3. Extraction procedures 2.3.1. Conventional solvent extraction Air-dried algae finely chopped (25 g) were placed in 250 mL flasks, and macerated with 160 mL of a mixture of organic solvents CH2Cl2/ MeOH (1/1:v/v). The extraction was carried out on batch mode at room temperature for one week, followed by filtration and evaporation of the solvent. The dry residue obtained was subsequently treated with diethyl ether to give a crude extract.
2.5. HPLC analysis HPLC analysis was carried out using a chromatography system Agilent 1260 Infinity (Santa Clara, CA, USA), equipped with a quaternary pump G13UC, a standard autosampler G1329B and a diode array detector G1315CD. Separations were performed using the isocratic mode, at room temperature, employing an analytical Agilent RP 18column (250 mm × 4 mm ×5 µm). Extracts were analyzed using the eluent mixture MeCN/H2O (95/5:v/v) at a flow rate of 1 mL min−1. The detection wavelength was fixed at 220 nm. A semi-preparative Purospher STARRP 18 column (250 mm × 10 mm × 5 µm) (Molsheim, France) was used for isolation secondary metabolites from the conventional extract, which were mainly phenolic metabolites of phloroglucinol derivatives.
2.3.2. Alternative solvent extraction The developed extraction procedure (Fig. 1.) was based on the use of eucalyptol as an alternative solvent and was performed by the following three steps: i) solvent maceration, in the same conditions as for conventional solvent extraction (25 g algae with 160 mL eucalyptol), ii) solvent separation by filtration and a multistep liquid-liquid extraction with distilled water, followed by centrifugation and separation of the aqueous phase (the number of steps needed to completely recover eucalyptol was determined by using Fourier transform infrared (FTIR) analysis, and iii) solvent recycling by steam distillation using a DeanStark apparatus.
2.6. NMR analysis 2.4. Fourier transform infrared analysis NMR spectra were recorded using Bruker Avance 400 instrument (Strasbourg, France) (1H, 400 MHz and 13C, 100 MHz).1H NMR spectra were referenced internally on CDCl3 (δ1H 7.26 ppm), and 13C NMR
A Shimadzu (Beyne-Heusay, Germany) FTIR spectrometer model 8900 was employed to obtain infrared spectra in the wave number 98
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cyclic ether C(1)-O-C(8) are located between 1052 and 1169 cm−1. In the present study, the absorption band stretching in the finger print region at 1169 cm−1was used as a characteristic band in order to evaluate the number of water washing steps of extract required to separate quantitatively the biosolvent from the crude extract.
spectra were referenced internally on CDCl3 (δ13C 77.20 ppm). 2.7. Total phenolic content determination The total phenolic content was determined using the Folin–Ciocalteau method based on the colorimetric oxidation/reduction reaction of phenols, in accordance with the procedure previously described (Blanca et al., 2011) with some modifications. The crude extract (4 mg) was dissolved in 8 mL of the methanol/water mixture (1/3:v/v) at room temperature and then, 2 mL of each extract solution were mixed with 10 mL of water and 12 mL of sodium carbonate (29% w/v). 1 mL of the Folin–Ciocalteu reagent was added and the development of color was produced after 30 min in the dark at room temperature. The absorbance was measured at 760 nm with a Shimadzu UV-1700 spectrophotometer (Marne la Vallée, France) against the water blank prepared in the same way. A standard curve with phloroglucinol solutions (ranging from 0.008to 0.099 mg mL−1) was used for the calibration. Results were expressed as mg phloroglucinol equivalents/g extract and calculated by the following Eq. (1) (Adesegun et al., 2009):
T=
C×V M
3.2. Evaluation of the number of water washes The solution green extract with eucalyptol was subjected to liquidliquid extraction yielding two phases, an upper phase (extract+eucalyptol) and down phase (water+eucalyptol). After a chosen number of water washes, the upper solution was analyzed by FTIR and the down phase treated for eucalyptol recovery. Our interest was focused on the variation of the absorbance of the band of eucalyptol at 1169 cm−1 in the extract. The FTIR spectra after five, ten and twelve washes are given as inset in Fig. 2. The enlargement of the characteristic absorption band reveals, first, decrease of the absorption intensity versus the increasing of the number of water washes and that, after twelve washes, eucalyptol was totally removed or it remained as trace amounts. 3.3. FTIR analysis of the recycled eucalyptol
(1)
Where T is the total content of phenolic compounds expressed as milligram phloroglucinol equivalents per gram of extract (mg PGE g−1); C is the concentration of phloroglucinol established from the calibration curve (mg mL−1); V is the volume of extract (mL), M is the weight of extract (g).
The down phase of the liquid-liquid extraction process was subjected to steam distillation using a Dean stark set up. This operation allows a simple separation of eucalyptol from water. Through an FTIR analysis of the recycled eucalyptol (results not shown) it was confirmed that after water extraction by steam distillation, eucalyptol was not degraded and spectra of both, pure and recycled solvent, were identical.
2.8. Antioxidant activity determination by DPPH radical scavenging assay
3.4. Comparison of the extraction yield
Radical scavenging activity of extracts was assessed according to the procedure reported in the literature (Loo et al., 2008; Shimada et al., 1992). The method was based on the ability of extract to reduce the DPPH free radical, the activity being determined by spectrophotometry measurements. A volume of 1 mL of each sample, at different concentration levels, was mixed with 1 mL 0.004% solution of DPPH in methanol. The absorbance was recorded at 517 nm against water blank, prepared in the same way, over a period of 30 min. The percentage of inhibition was estimated using the following Eq. (2) (Keyrouz et al., 2011):
Where: Abs0corresponds to the absorbance of the reference which is free of phenols, and Absx is the absorbance of the sample. L-ascorbic acid, BHT, BHA and α-tocopherol were used as reference standards. Different concentrations of samples were tested to obtain a calibration curve giving the inhibition percentage according to the sample concentration. The EC50, deduced from the slope of the calibration curve, corresponds to the concentration of extract or standard antioxidant (mg mL−1) required to scavenge 50% of the DPPH in the reaction mixture.
The extraction yield obtained using eucalyptol lead to a low value (0.45% m/v) as compared to that obtained using conventional solvent extraction (2.16% m/w). This results can be interpreted by the low polarity of the green solvent in comparison to the mixture CH2Cl2/ MeOH characterized by a wide polarity range (Haynes, 2014; Reichardt, 1979). In fact, the biosolvent has a dielectric constant of the same order of that of chloroform and hexane. In contrast, the strong polarity of methanol favours the extraction of heavy polar molecules, such as tannins and dyes (Haynes, 2014; Reichardt, 1979). Through the use of terpenoids more polar than eucalyptol as biosolvent, the extraction yield could be increased. Further studies have reported a high yield extraction of natural products from marine and/or terrestrial organisms by the use of CH2Cl2/MeOH in comparison to other solvents as (Li et al., 2014; Breil et al., 2017; Stengel and Connan, 2015). Despite the low extraction yield obtained by using eucalyptol the most important advantages of extraction using biosolvent are the development of a process environmentally friendly based on avoiding toxic solvent and the obtention of high quality products, including the recovery of eucalyptol. In addition, due to the high selectivity of the biosolvent, the green extract contains very small undesirable products.
3. Results and discussion
3.5. HPLC analysis
3.1. FTIR spectra of eucalyptol
As mentioned through our previous study on the same species (El Hattab et al., 2009), the chemical composition of the crude extract is characterized by the presence of phenolic metabolites, phloroglucinol derivatives and eicosapentaenoic acid as major compounds (see Fig. 3.). In order to compare the chemical composition of the crude extracts obtained by both extraction methods, the phenolic secondary metabolites previously described from a crude extract obtained by conventional solvent extraction were isolated by semi-preparative HPLC. The structure of the isolated compounds was unambiguously elucidated by comparing their 1H and 13C NMR data with the literature (Amico et al., 1981, 1982).
Inhibiton percentage% =
Abs0 × Abs x ×100 Abs0
(2)
The infrared absorbance spectrum of eucalyptol in CHCl3 is indicated in Fig. 2. Methyl (2985 cm−1, 2968 cm−1) and methylene groups (2924 cm−1 and 2881 cm−1) stretching vibrations provided strong bands. The methyne bonds were identified by the absorption bands at 1466 cm−1, 1375 cm−1and 1052 cm−1. The literature reports that the characteristic absorption bands of cyclic ether (epoxide) is ranged from 1070 cm−1 to 1140 cm−1 for a symmetric stretch and could be extended to 1247 cm−1 for an asymmetric stretch (Easton et al., 2009). Concerning the eucalyptol, the stretching bands of the 99
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Fig. 2. FTIR spectra of pure eucalyptol in CHCl3 (green) and after washing the crude extract + eucalyptol (the filtrate) with water several times: 5 - blue, 10 – red and 12 - pink) (22 mg mL−1). Inset: Decrease in absorbance of the characteristic band of eucalyptol at 1169 cm−1 as function of the number of water washes (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
conventional extract is more complex and contain, in addition to the phenolic compounds described (Fig. 3C.), other compounds of phenolic nature such as tannins. It must be emphasized that phenolic compounds in marine algae are correlated with antioxidant activity and also depend on the solvent and algal species used for extraction (Movahedinia and Heydari, 2012).
The chromatography profiles of conventional and green extracts (Fig. 3.) obtained by analytical HPLC have a good similarity. Examination of the two chromatograms reveals the presence of higher levels of polar compounds in conventional extract than in the green extract. Furthermore, we notice the high content of compound (1) and a very low content of compounds (3) and (5) in the green extract. In contrast, the conventional extract shows low content in compounds (1) and (3) (see Fig. 3C.). These results may be related to the relative solubility of pure phenolic metabolites in both solvents. It must be pointed out the large scale of antimicrobial activities of phenolic compounds of brown algae (Pérez et al, 2016) particularly that of the phenolic metabolites isolated from the Zonaria genus (Wisespongpand and Kuniyoshi, 2003). The phenolic metabolites contents in both extracts, green and a conventional, are reported in Table 1.
3.7. Antioxidant activity The antioxidant activity of the obtained extracts, including standards, was assessed through its ability to scavenge the DPPH˙ free radical, and measured in the term of EC50 (efficient concentration providing 50% inhibition) calculated graphically using a non-linear fitting curve by plotting inhibition percentage according to sample dilution level. Results obtained for both kind of extracts, are summarized in Table 3. As it can be seen the activity of extracts was lower than for all the controls. In addition, DPPH˙ scavenging activity of conventional extracts was strongest than that of green ones. So there is a close relationship between the results of the total phenolic content and those of antioxidant activity, probably due to the antioxidant activity of phenolic secondary metabolites and other reductant agents. Because of the different extraction and measurement methods and units used in the antioxidant activity studies on seaweed reported in the literature, direct comparison of present results on radical scavenging activity of extracts with other studies is not feasible. However, many researchers have shown that phenolic compounds derived from marine brown algae have strong antioxidant activities against free radical mediated oxidation damage (Li et al., 2011).
3.6. Total phenolic content determination The total phenolic content in the green and conventional extracts was calculated from the calibration curve established with phloroglucinol as reference. The calibration curve was represented by the analytical features shown in Table 2. Once the calibration realized, the absorbance of samples can be normalized to a phloroglucinol equivalent (PEG) concentration by interpolation of absorbance values in the calibration line. The experimental results revealed that total phenolic content of conventional and green extracts measured in terms of mg PEG g−1 of crude extract were 725.0 ± 1.9 and 362.5 ± 1.4 mg (standard deviation of 5 independent measurements), respectively. The total phenolic content of the conventional extract is in good agreement with the one obtained by volumetric determination on the same extract (El Hattab et al., 2009). The high phenolic compound content of the conventional extract in comparison to green extract can be explained by the low selectivity of the solvent mixture in comparison to the green solvent and confirms the high yield obtained by conventional extraction using the mixture CH2Cl2/MeOH, which is characterized by a wide polarity range, in comparison to green extraction. Accordingly, the
4. Conclusion In the present study it has been developed a green extraction technique, based on the use of eucalyptol as biosolvent for extraction of phenolic compounds from Zonaria tournefortii alga. Green extracts were prepared and compared with conventional extracts obtained by 100
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Fig. 3. HPLC chromatogram of the crude extracts obtained by conventional solvent extraction using CH2Cl2/MeOH (1/1:v/v) (A) and alternative solvent extraction using eucalyptol (B). Chemical structure of secondary metabolites isolated from brown algae Zonaria tournefortii (C).
Table 1 Percentage content of phenolic metabolites in conventional and green extracts. Compounds
1 2 3 4 5
Table 2 Analytical features of the Folin-Ciocalteau determination of phloroglucinol.
Content % (w/w) Conventional extract
Green extract
15.28 3.57 7.64 24.27 15.11
20.17 6.36 1.81 6.30 1.72
Regression line
A= 6.512 C+0.082
Correlation coefficient Linear range (mg/mL) L.O.Q. (µg mL−1) L.O.D. (µg mL−1) R.S.D. % (n = 10)
0.9995 0.008–0.099 5.2 1.57 0.54 (for 0.045 mg mL−1)
Notes: R.S.D. %: Relative standard deviation, in percentage; L.O.Q.: Limit of quantification expressed in µg mL−1; L.O.D.: Limit of detection expressed in µg mL−1.
classical solvent extraction using the mixture of CH2Cl2/MeOH. The conventional extraction offers higher extraction yield than green extraction. Both extracts have been submitted to chemical analyses (total phenolic content, HPLC analysis), and antioxidant activities. The HPLC analysis shows a great similarity between extracts but with and increased amount of non-polar phenols when using eucalyptol. In
contrast, the total phenolic content and the antioxidant activities have indicated that the conventional extract has a strongest effect as compared to the green extract. The additional advantage of the proposed procedure is the recovery of eucalyptol after extraction.
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Table 3 EC50 Values of extracts and antioxidants standards. Samples
EC50 (µg mL−1)
L-ascorbic
4.9 6.0 8.5 58 140
acid BHA BHT Conventional extract Green extract
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