Efficient Solvent Selection Approach for High Solubility of Active

Research Article pubs.acs.org/journal/ascecg

Efficient Solvent Selection Approach for High Solubility of Active Phytochemicals: Application for the Extraction of an Antimalarial Compound from Medicinal Plants Souhila Laboukhi-Khorsi,†,‡ Kamel Daoud,‡ and Smain Chemat*,† †

Extraction and Separation Techniques Team, Centre de Recherches Scientifique et Technique en Analyses Physico-Chimiques (CRAPC), BP 384, Zone Industrielle de Bousmail, RP 42004 Tipaza, Algeria ‡ Faculté de Génie des Procédés et de Génie Mécanique, Université Houari Boumediene (USTHB), BP 32, El-Alia, 16112 Bab-Ezzouar, Alger, Algeria ABSTRACT: The proposed approach unveils an efficient solvent selection manner for the extraction of active compounds from medicinal plants based on their high solvency for the target compound while considering green engineering principles. Rationalization of resources is achieved through theoretical screening using the Hansen solubility parameters in practice (HSPiP) approach of 26 solvent candidates toward the solvency of the antimalarial drug “artemisinin”. Solvent selection is challenged to meet some green engineering principles, from which four solvents are identified and then confirmed using gravimetric tests. The highest solubility at 20 °C is attributed to dimethyl sulfoxide with 103.7 mg·mL−1, while isopropanol recorded 8.45 mg·mL−1. Despite being a polar solvent, propylene glycol gave a very low solubility of 0.6 mg·mL−1. Being a food grade with very low global warming potential score, isopropanol stands as a green alternative to substitute n-hexane, where solubility measurements using changes of turbidity measurement indicate good solvency for artemisinin with 39.92 mg·mL−1 at 40 °C, a temperature at which generally extraction is conducted. Application of isopropanol to artemisinin extraction from Artemesia annua L. indicates a good artemisinin yield of 65% in only a single batch sequence at which the recovered crystals have a purity of more than 67%. KEYWORDS: Solubility, Artemisinin, Extraction, Green solvent, Process development, Active compound

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INTRODUCTION An increasing demand for sustainable approaches in process design is opening a new window to develop solutions that contribute to a green shift of industrial processes. With an expected annual market growth of 6−8%,1 production of medicinal food and drugs from botanical sources should embrace green processing routes to ensure sustainability and keep momentum. For instance, extraction and purification processes to recover high value compounds from plants require the use of large amounts of hazardous solvents entailing potential hazards while producing sometimes low extraction and/or selectivity yields. For example, residual vegetable oils are usually recovered using volatile solvents such as n-hexane despite its safety and security concerns for industry and consumers.2 Extraction of artemisinin, a sesquiterpenoid lactone peroxide known for its potent antimalarial activity, is currently performed using petroleum ether or hexane.3 This process gives a relatively low overall yield and selectivity due to low solubility of artemisinin in hexane, where only 60% of available artemisinin in the dried biomass (Artemesia annua L.) can be recovered as a consequence to artemisinin decomposition in hot conditions and losses encountered during purification steps. © 2017 American Chemical Society

In industry, these nonpolar solvents hold low dielectric constants and require addition of polar additives, typically 5% v/v of ethanol or ethyl acetate, to avoid explosion risk through static discharges.3 However, this polar fraction acts as solubility enhancers for waxy and glycosidic fractions present in biomass.4 This fact calls for new approaches that aim to select solvents with higher solubility and selectivity for target compound(s) from plant material like artemisinin. The scientific community agreed on 12 standardized principles of green engineering to establish a sustainable approach that minimizes impact on the environment and reduces burden associated with safety and security.5 Calls to develop green solvent alternatives to hexane must adhere to important requisites during the selection process, like minimizing the release of hazardous material to air by selecting solvents with higher boiling points like ionic liquids (inherent rather than circumstantial). Besides, the selectivity of a solvent for the target analyte compared to side components is an important factor, as this will have a paramount influence on Received: February 7, 2017 Revised: March 22, 2017 Published: March 24, 2017 4332

DOI: 10.1021/acssuschemeng.7b00384 ACS Sustainable Chem. Eng. 2017, 5, 4332−4339

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ACS Sustainable Chemistry & Engineering

based on gravimetric measurements such as the shake flask method described by Higuchi and Connors14 or solvent removal method for pressurized solvents applied for fluid gases R134a and R1234yf.13 Although being credited as the most reliable experimental drug solubility measurement methods, their applications are too inherent, require large quantities of pure analyte, and may engender a lot of errors due to manipulation, recording the mass of rapidly evaporating solvents, and rapid drop of temperature during sampling. In addition to issues pertained to limits of detection and quantification, some analytes like those without chromophores require specific detectors. According to Lapkin et al.,3 great differences are noted between their findings and earlier reported figures15 for artemisinin solubilities in n-hexane and ethyl acetate. They obtained mixed results for toluene and acetonitrile. The solubility of artemisinin in toluene is significantly lower than the figure reported previously by Liu et al.,15 while its solubility value in acetonitrile is much higher. Turbidity change measurements have been largely used for the estimation of metastable zone width of pharmaceutical intermediates, particularly mineral compounds, and also for optimization of crystallization processes of many pharmaceutical products and have the merit to give accurate and reproductive solubility data at different temperatures.16,17 However, this concept is barely known in the area of natural products, probably owing to high prices of commercially available turbidity-based systems. Using theoretical data such as HSP parameters as a support tool to comprehend solubility behavior reduces significantly the number of experiments and solvent candidates, enabling the concentration of resources and time to refine solubility determination using turbidity change measurements only for best solvent candidates. This approach adheres to green engineering principles aiming at maximizing efficiency and meeting the need to minimize excess. Knowing that artemisinin has very low solubility in water,18 screening solvents that are soluble in water such as isopropanol, propylene glycol, and ionic liquids can intuitively make them suitable antisolvents for the recovery of artemisinin and reduce purification steps. In an attempt to meet green engineering principles, this work profits from theoretical calculations using HSP parameters for artemisinin in different solvents for solubility determination and supports the selection of best solvents. Then, experimental solubility measurements using the shake flask method are conducted for the selected solvents to depict the best solvent system. The latter is screened at a large temperature range using a turbidity-based setup to record highly accurate data for artemisinin solubility and metastable zone width. This study is complemented by an application of the selected solvent for the extraction of artemisinin from Artemesia annua L. biomass and confirms its suitability for high yields of artemisinin.

further purification steps through selecting solvents with high solubility potential for target compounds (meeting need to minimize excess). In addition, the separation procedure must be planned to reduce energy required to recover the final product, and the solvent system should lead to optimize time, space, and energy consumption (maximize efficiency), like in the case of supercritical CO2 extraction of caffeine from coffee beans.6,7 Signatory countries of the Montreal and Kyoto protocols engaged to phase out compounds with ozone depleting (ODP) and global warming potential (GWP),8,9 thus it is critical to avoid in situ degradation, determine end-oflife cycle, and consider end-of-life fate of selected solvents through reuse or recycle in order to conserve complexity and concretize durability rather than immortality. Although holding low boiling points, crude oil derived solvents such as isopropanol, ethyl acetate, ethanol, and 2butanone stand as potential eco-friendly candidates, they are less likely to be rapidly released into our environment due to their low vapor pressure and low volatility making them accepted in industry. Unlike ethanol or methanol, isopropyl alcohol is not miscible with salt solutions, so adding salts like sodium chloride lead to its separation from aqueous solutions.10 Clearly, regulatory constraints and consumer ethics in pharmaceutical and food products have to be integrated into the selection process paradigm, which limits further the number of applicable solvents drastically. Nonetheless, screening the extractability of an active compound from plant material in large number of solvents seems to be nonproductive as it requires availability of bulky amounts of solvents to perform experiments and optimize extraction conditions. Recently, the calculation of Hansen solubility parameters (HSP) based on Hildebrand’s theory allowed good interpretation of dispersion, polar, and hydrogen-bonding forces of a solute toward a range of solvents, aiming to describe and to explain the interactions between them. For example, it was used to predict the tendency of solubility of volatile aroma compounds in vegetable oils.11 In another study, predictive HSP results gave good consistency with extraction yields for biosourced solvents α-pinene and methyl tetrathydrofuran (Me-THF) of aroma from blackcurrant buds and may represent good alternatives to hexane despite a different flavor profile.12 Likewise, few reports started to expand the applicability of COSMO-RS for solubility calculations of solids in green solvents.3,13 However, the difference of Gibbs energies of the analyte at a given temperature of its pure solid status and its a pure liquid status known as Gibbs energy of fusion is required to make calculations. Generally, this value can be estimated by quantitative structure−property relationship approach, but it would introduce considerable error. Therefore, it is important to be cautious when taking solubility data into consideration. Suberu et al.13 reported a deviation of the absolute values of the predicted solubilities of artemisinin in fluorinated solvents R1234yf and R134a by COSMO-RS compared to those obtained experimentally but insisted these are still in the expected range of quantitative accuracy. HSP and COSMO/RS orientations avoid the use of bulky solvents and reduce the number of solvent candidates to only those with high solubility potential for the target compound(s) and, thus, represent a good starting point that meets many green engineering principles like meeting the need to minimize excess. In parallel, experimental solubility measurements can be used to prospect its extractability, but most of the available data are

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EXPERIMENTAL SECTION

Material. Crushed leaves of Artemesia annua L. stored under dark and cool conditions originating from Madagascar (BIONEX: Lot 1A1152) was kindly provided by Prof. Alexei LAPKIN (University of Cambridge). Artemisinin analytical standard (98%) in the form of a white crystalline powder was purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). Isopropyl alcohol (isopropanol), ethyl acetate, dimethyl sulfoxide (DMSO), propylene glycol, and HPLC grade acetonitrile were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Ultra pure water was obtained using Direct-Q UV3 (Millipore) system. 4333


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temperature at 293 K, then 20 μL of the supernatant is sampled using a Hamilton HPLC syringe and injected instantly into HPLC. Turbidity-Based Solubility Studies. The solvent exhibiting higher solubility for artemisinin is selected to record solubility data including clear and cloud points to determine metastable zones. Runs are performed using a system designed to measure turbidity change as a function of temperature in E1207s Crystal-EYES process control system (HEL Ltd., UK). The system uses a turbidity sensor with builtin near-infrared (NIR) light source and detector, which is made of hard-wearing hastelloy for excellent chemical compatibility. The light source sends light through the solution and back to a detector via a mirror. Turbidity is measured as a function of the light intensity; as particles increase, light intensity drops and then turbidity increases. The setup includes 150 mL jacketed glass reactor in which a platinum resistance thermometer and a turbid metric fiber-optic probes are inserted into the solution to record solubility and temperature changes data. The system is controlled by means of a PC running WinISO version 2.3.122.1 software (HEL Ltd., UK), while the temperature of the jacketed reactor is controlled by a HAAKE thermo-stated bath. A scheme for the experimental setup is shown in Figure 1.

Methods. Hansen Solubility Parameters. The HSP are considered as important properties of various substances and can be used for prediction of their behavior in numerous applications. Dr. Charles M. Hansen embraced Hildebrand’s work by characterizing the total molecular force associated with a solvent (or soil) as the sum of three component forces, then suggested separating the Hildebrand solubility parameter into three components.19 In other terms, the total cohesive energy density is approximated to be equal to the sum of the energy densities required to overcome intermolecular dispersion forces δd2 (quantifies the effect of those intermolecular forces between molecules upon adjacent molecules), molecular polar forces arising from dipole moments δp2, and forces pertained to hydrogen bonds between molecules δh2 (exchange of electrons, proton donor/acceptor), as given eq 1:12,19 δtotal 2 = δd 2 + δp2 + δ h 2

(1)

where δtotal corresponds to the Hansen total solubility parameter and consists of three parameters in terms of dispersion (δd), polar (δp), and hydrogen-bonding (δh) that are used to construct the Hansen solubility spheres. For instance, the separation distance between sphere centers of the solute and the solvent are referred to as Ra and are calculated using the following eq 2:

R a 2 = 4 × [(δdA − δdB)2 + (δpA − δpB)2 + (δ hA − δ hB)2 ]

(2)

where A refers to the solute and B corresponds to the solvent. The constant number 4 is based on Prigogine’s corresponding states theory, which has proved to effectively expand the dimensions and gives spherical plots. In order to assess the affinity of a solute toward a solvent, hence its potential dissolution, the relative energy difference (RED) number is calculated as follows (eq 3):12,19 RED = R a /R 0

(3)

where R0 is the radius of the Hansen solubility sphere, and Ra is the distance of a solvent from the center of the Hansen solubility sphere. In general, these parameters follow the classical ‘“like dissolve like”’ rule: the smaller Ra is, the greater the affinity is between solute and solvent. It means that potentially good solvents exhibit RED numbers smaller than 1, while inappropriate solvents will have progressively higher RED numbers larger than 1. In this study, 26 solvents ranging from conventional petroleum solvents, ionic liquids, and biosourced solvents were screened using Hansen solubility parameters in practice (HSPiP) software (version 4.1.07 developed by Abbott and Yamamoto) in order to calculate the solubility spheres coordinates of artemisinin (center and radius). The software uses a correlation method based on a quality-to-fit function to ensure that most solvents solubilizing the solute are located inside, in contrast to solvents that can not solubilize the solute and would be located outside the solubility sphere. The simplified molecular input line entry syntax (SMILES) notations for each compound is inserted into the software, which can break SMILES into corresponding functional groups using the Yamamoto-molecular break (Y-MB) method and then estimate their HSP parameters. These solubility parameters are further modeled into a three-dimensional HSP sphere for easier illustration of the solute/solvent interaction. Scores of the solubility tests are computed within HSPiP software by assigning a score of 0 for nonsoluble solvents or 1 for soluble solvents. Gravimetric-Based Solubility by Means of HPLC. To confirm HSPiP data, four solvents namely water, DMSO, propylene glycol, and isopropanol are selected to estimate their solubility for artemisinin by means of the gravimetric method. Generally, solubilities of artemisinin are reported in the literature13,20 at room temperature (293 or 298 K), therefore the solubility is evaluated at room temperature 293 K using the shake flask method. This method consists of adding an excess of artemisinin to 5 mL of solvent into a 25 mL glass vial containing a magnetic stirrer. The vial is sealed and placed in a thermo-stated water bath kept at 293 K and stirred at around 400 rpm for 2 h to achieve equilibrium with the solid phase. Thereafter, stirring is stopped, and the vial is left to settle over 2 h inside the water bath while keeping the

Figure 1. Experimental setup scheme for solubility measurements using turbidity changes in function of temperature. To record turbidity versus temperature changes, the following steps are indicated: an excess of artemisinin is added to the solvent (in case of isopropanol: 1.5 g of artemisinin in 60 mL of solvent is suggested corresponding to a concentration of 25 mg·mL−1), and the mixture is stirred gently using a magnetic stirrer at room temperature. The solution is heated at a constant rate of (1 ± 0.05) °C/min to bring it to complete dissolution of the analyte, then cooled down at a constant rate of (2 ± 0.1) °C/min to a temperature at which crystals are formed and visible. These steps are repeated several times to reproduce the solubility curves. Three dilutions of artemisinin 15, 20, and 25 mg· mL−1 are tested in order to get corresponding clear (solubility or dissolution) and cloud (supersaturation or crystallization) points and construct the metastable zone width that is very important to control the shape and the size of final crystalline products.21 Extraction from Biomass. The maximum amount of artemisinin present in biomass is evaluated according to the procedure reported earlier by Chemat et al.22 It consists of extracting 50 g of biomass over 6 h with 300 mL of ethyl acetate under magnetic stirring at 40 °C for three times, where each time fresh portions of solvent are added. These extracts are evaporated to dryness in a rotary evaporator, and the residue is dissolved in 20 mL of acetonitrile. After filtration through 0.45 μm membrane, the solution is injected into HPLC. For the selected solvent, 20 g of dry Artemesia annua L. is extracted using 120 mL of solvent following the same sample preparation indicated above. Extraction yields are calculated as a percentage relative to the maximum amount of artemisinin evaluated earlier. To achieve crystallization, an excess of water, playing a role of an antisolvent, is added gradually while mixing the solution with a magnetic stirrer. Gradually, the solution became turbid and is left to 4334


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ACS Sustainable Chemistry & Engineering Table 1. Solubility Behavior of Artemisinin in Different Solventsa no. 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

solvent hexane acetonitrile dichloromethane dimethyl sulfoxide (DMSO) 2,3-butylene carbonate dimethyl carbonate toluene cyclohexane chloroform propylene glycolb ethanol waterb methanolb isopropanol isopropyl acetate methyl-t-butyl ether (MTBE) heptane methyl ethyl ketone (MEK) n-butyl amine ethyl acetate 1-butanol 2-butanol 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) N,N-dimethylethanolamine (DMEA) 1,3-dimethyl-2-imidazolidinone (DMI) acetone

δd (MPa1/2)

δp (MPa1/2)

δh (MPa1/2)

Ra (MPa1/2)

RED

molar volume (cm3· mol−1)

14.90 15.30 17.00 18.40 18.00 15.50 18.00 16.80 17.80 16.80 15.80 15.50 14.70 15.80 14.90 14.80 15.30 16.00 16.20 15.80 16.00 15.80 17.90

0.10 18.00 7.30 16.40 16.80 8.60 1.40 0.00 3.10 10.40 8.80 16.00 12.30 6.10 4.50 4.30 0.00 9.00 4.50 5.30 5.70 5.70 8.40

0.10 6.10 7.10 10.20 3.10 9.70 2.00 0.20 5.70 21.30 19.40 42.30 22.30 16.40 8.20 5.00 0.00 5.10 8.00 7.20 15.80 14.50 7.50

11.30 10.85 2.07 10.17 11.25 2.24 9.48 11.07 6.07 13.44 11.27 35.13 15.14 8.32 3.90 5.29 11.27 3.48 3.02 2.53 7.79 6.55 3.70

1.000 0.960 0.183 0.900 0.996 0.198 0.839 0.980 0.537 1.190 0.997 3.109 1.340 0.736 0.345 0.468 0.997 0.308 0.267 0.224 0.689 0.580 0.327

127.5 52.9 64.4 71.3 105.5 84.7 106.6 108.9 80.5 73.7 58.6 18.0 40.6 76.9 117.6 119.8 147.0 90.2 98.8 98.6 92.0 92.0 131.5

14.50 17.60 15.50

2.90 7.10 10.40

3.90 7.50 7.00

7.13 3.03 3.40

0.631 0.268 0.301

103.0 150.1 73.8

Artemisinin trial values: δd = 16.14 MPa1/2, δp = 7.50 MPa1/2 and δh = 8.23 MPa1/2; R0 = 11.3 MPa1/2; molar volume = 239.7 cm3·mol−1). bOut of solubility sphere.

a

precipitate overnight at 4 °C. The obtained precipitate is recovered by simple filtration and left to dry in a desiccator. The residue is weighted and dissolved in 5 mL of acetonitrile, then submitted for a purity check by HPLC. The purity yield is calculated as a percentage of artemisinin mass per residue sample mass. HPLC Analysis. Analysis of artemisinin is achieved using HP-Agilent Technologies 1100 HPLC system equipped with a UV−vis detector. To this end, an hypersil 120 Å ODS 3 column (250 × 5.4 mm; id: 4.6 mm) set at a temperature of 40 °C is used with an acetonitrile:water (65:35%v/v) mobile phase operating in an isocratic mode at 0.8 mL min−1 flow rate, where detection is set at a wavelength of 220 nm. Calibration curve of artemisinin is calculated at different concentrations varying from 0.125 to 5 mg/mL.

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RESULTS AND DISCUSSION Hansen Solubility Parameters. The solubility sphere of artemisinin against a set of 26 solvents is realized. Using a quality-to-fit function, HSPs of artemisinin are obtained (δd = 17.4 MPa1/2, δp = 7.4 MPa1/2 and δh = 7.4 MPa1/2), where the radius of its solubility sphere is indicated as R0 = 13.4 MPa1/2 (Table 1). The high value of the dispersive interaction parameter, δd, for artemisinin is explained by the large molecular volume it occupies and its functionalized groups (hydroxyl and peroxide groups) that enhance the dispersed intermolecular forces throughout the entire molecule’s volume. These HSP data are used to construct the 3D solubility sphere of artemisinin, in which green dots located at the center of the sphere represent the theoretical optimum HSP for ideal solvency, and blue dots located inside the sphere are regarded as good solvents, while red cubes indicate those with relatively limited solubility for artemsinin (Figure 2).

Figure 2. General three-dimensional Hansen solubility sphere (blue dots: good solubility; red cubes: poor solubility).

Among the tested solvents, 24 candidates are found to be inside the artemisinin solubility sphere, so presumably good solvents, whereas only 3 candidates (water, propylene glycol, and methanol) are outside, thus not likely to dissolve artemisinin. However, methanol is pointed out incorrectly as outside of the solubility sphere, as artemisinin is known to be soluble in methanol, so this data is corrected. This misfit is due to the mathematical method of calculation that assumes the solubility volume to be spherical with a correlation factor of around 71%.19,23,24 4335


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ACS Sustainable Chemistry & Engineering Table 2. Gravimetric Solubility Measurements of Artemisinin in Selected Solvents isopropanol propylene glycol DMSO water DMPU DMI ethyl acetate dimethyl carbonate dichloromethane

solubility 20 °C (mg·mL−1)

RED

BP (°C)

GWP (kgCO2 eq/kg)

flammability score (NFPA)

8.45 0.60 103.70 0.06 15.963 16.203 39.4031 9.973 1195.2020

0.736 1.190 0.900 3.109 0.327 0.268 0.224 0.198 0.183

66.7 186.5 189.0 100.0 246.5 225.0 77.10 91.00 39.6

1.8528 4.1429 1.2730 − − − 3.0528 3.2032 8.7033

3 1 2 − 1 2 3 5 1

approach at 25 °C are summarized and compared to solubility data reported in the literature (Table 2). The GWP of these solvents is very low, where DMSO and isopropanol record lowest values with 1.2730 and 1.8528 kgCO2 eq/kg, respectively. Dichloromethane (DCM) exhibits the highest solubility for artemisinin but has several hazard and environmental issues. According to the International Association for Research on Cancer (IARC), there is suf f icient evidence in experimental animals and limited evidence in humans to categorize DCM as likely to be carcinogenic to humans.34 Also, it is classified as a VOC and recently shown to be an ODP agent.35 In parallel, the limited solubility values of water and propylene glycol corroborate with their low RED values. The literature reports acceptable solubilities for DMI and DMPU, however, ionic liquids are too viscous and have very low vapor pressures, therefore additional operations are required to recover the analyte from the crude extracts. High boiling point solvents require high energy input, which make them unpractical to be recycled through vacuum solvent distillation. DMSO presents a very high solubility for artemisinin (103.7 mg·mL−1), but it holds stability issues at high temperatures, and it would necessitate high energy to be recovered by distillation. Although DMSO is considered as a greener option in synthesis, it is classified as only a “usable” solvent in the Pfizer Medicinal Chemistry Solvent Selection Guide.36 Ethyl acetate may profile as a good alternative (39.4 mg·mL−1) with its low boiling point and GWP score, but it is known to solubilize other co-metabolites and requires partition with other organic solvents; hence, it would practically complicate purification steps for the recovery of artemisinin, so relatively it is not as green as it seems to be. Dimethyl carbonate gives moderate solubility for artemisinin (9.97 mg· mL−1) despite its very low RED value (0.198), though it is unlikely to be adopted in the extraction industry with its dissuasive flammability attribute (NFPA: 5). Isopropanol gave a moderate solubility for artemisinin of 8.45 mg·mL−1 and holds a major lead as a promising solvent. It is recognized as a safe solvent (GRAS, class 3) and is not known as a human health hazard at levels normally accepted in pharmaceuticals.37 Compared to DMSO, isopropanol has a low boiling point facilitating its recovery by vacuum distillation and is categorized as a recommended solvent in the green solvent selection guide ranking.38 It is important to note that an isopropanol solubility value at 20 °C is very low compared to the value reported by Qu et al.20 of 20.1 mg·mL−1 at 24.5 °C. Conflicting results of RED values and solubilities evaluated gravimetrically call for the adoption of a more precise method to confirm the solubility range. In these terms, solubility evaluation using turbidity change measure-

Nonpolar solvents like n-hexane, heptane, and cyclohexane have similar RED values equivalent to 1 (i.e., at the limit of solubility sphere), though different from artemisinin HSP values predicting low solubility for artemisinin. In parallel, polar solvents display similar HSP values to artemisinin, as both hold hydroxyl or carboxyl groups which resulted in a higher δp and δh values. Interestingly, dimethyl carbonate and ionic liquids like 1,3-dimethyl-2-imidazolidinone (DMI) and 1,3-dimethyl3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) exhibited very low RED values, envisaging good solvency for artemisinin. Indeed, studies of Lapkin et al.3 on solubility estimation of artemisinin in a range of conventional and novel solvents using the COSMO-RS approach reported that the most interesting solvents are dimethyl carbonate, dimethylformamide, DMI, DMPU, and γ-valerolactone. According to their low RED values, dichloromethane, ethyl acetate, isopropyl acetate, and butylamine are credited to have good solvency for artemisinin. Nonetheless, acetate solvents are known to solubilize phenolic compounds and pigments like chlorophylle, therefore require additional steps for the recovery of analyte.25 Butylamine has a distinct unpleasant odor and low vapor pressure and requires high energy input to break the intermolecular forces, therefore slashing its green adoption. Dichloromethane has a very low boiling point and is classified as a volatile organic compound (VOC), which does not make it the solvent of predilection in industry. It is recently suspected of causing cancer and is included in REACH restrictions H351.26,27 Other solvents with low RED values include isopropanol (0.736) and dimethyl sulfoxide (0.900). The structure of artemisinin suggests that it is weakly polar and therefore should have relatively high solubility in the medium polarity solvent following the rule of thumb “like dissolves like”. Hence, DMSO and isopropanol at a lower extent are expected to deliver high solubility values for artemisinin. On top, they are miscible with water, so they can introduce the latter as an antisolvent to accomplish crystallization. Betting on these solvents will contribute to optimizing time and process steps, aligning with the concept of maximizing efficiency. Therefore, isopropanol and DMSO are considered as good green candidates and are selected to evaluate their artemisinin solubility using the gravimetric approach. Albeit being appreciated as food-grade solvents, water and propylene glycol disappoint in contrast with very high RED values and are unlikely to dissolve much artemsinin. Despite their low probability, solubilities of water and propylene glycol are evaluated to confirm their low solubility for an eventual use as antisolvents during crystallization. Gravimetric Solubility Studies. Solubility measurements conducted for the four selected solvents by the gravimetric 4336


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ACS Sustainable Chemistry & Engineering ments is proposed to extract precise solubility values at different temperatures for the selected solvent “isopropanol”. Turbidity-Based Solubility Studies. Figure 3 regroups the measurement plots of turbidity changes as a function of temperature at three dilutions of artemisinin. At very low temperatures, high turbidity is recorded, indicating very low solubility of artemsinin. By increasing the temperature, the

turbidity value drops gradually, and the solid starts to dissolve at approximately 6−7 min. At 11 min, all of the artemisinin crystals disappear at which a constant turbidity value is registered. When initiating another decrease in temperature, the turbidity value rises steeply again, and the solute precipitates out after approximately 20 min. Data of clear (solubility) and cloud (supersaturation) points at different concentrations can be depicted easily as indicated in Figure 3 and are used to draw solubility and supersaturation curves (Figure 4), where very good reproducibility is obtained for clear and cloud points (±0.3 °C). Artemisinin is totally soluble in isopropanol at conditions below the solubility curve, while crystallization occurs at conditions above the supersaturation curve. Figure 4 shows that artemisinin has a wide metastable zone width (MSZW) stretching from 6 to 9 °C, which denotes the region between the solubility curve and the onset of nucleation (i.e., crystallization). At the same concentration, MSZW decreases when temperature drops leaving a narrow window between solubility and supersaturation, which favors a rapid aggregation of the analyte and induces quick crystallization. In order to avoid co-crystallization of co-metabolites with the target compound, it is, therefore, not advised to favor crystallization under very low temperatures.16 Solubility values at different temperatures can be easily deducted from the solubility curve and are reported in Table 2. In fact, the correct value for artemisinin solubility in isopropanol at 20 °C is 14.54 mg·mL−1 and is evaluated at 25 °C as 18.71 mg·mL−1. Solubility differences with the shake flask method are attributed to manipulation errors and drop in temperature at the sampling and/or weighing stage in gravimetric measurements. Solubility determination using turbidity changes measurement is more robust and precise and uses very low amount of solvent and solute. It requires the preparation of a single concentrated stock solution that can be used for other dilutions where no loss is incurred. It offers a clear advantage to record a precise solubility value at any desired temperature while gaining crucial information about MSZW to optimize the crystallization process for active compounds recovery from natural extracts. The proposed approach can be easily applied to extract solubility information and develop greener options of solvents or solvent mix for other high-value phytochemicals (active compound and/or standardized fraction or extract). Biomass Extraction with Isopropanol. To follow, isopropanol is tested for the extraction of artemisinin from Artemesia annua L. Generally, extraction is conducted at 40 °C at which the solubility of artemisinin in isopropanol is expected to be 39.92 mg·mL−1 (Table 3). The results indicate that the maximum artemisinin content is evaluated at 9.98 mg·g−1 of Artemesia annua L. (∼1%), which is in the range reported in the literature of 0.1−1.4%.3,13,20,22 When using isopropanol as a solvent in a single batch, an extraction yield of 6.19 ± 0.25 mg·g−1 is obtained (∼65% of total content), which is a good result compared to the low yield obtained by hexane of 60% requiring cycles of 8 h of processing.39 Furthermore, crystallization yielded 4 g which represents 60% of total extract, and the purity check indicates that crystals contains 67% of artemisinin which represents an acceptable result considering that the extract has not followed any prior purification, which we suggest to add using adequate adsorbents. Qu et al.21 used dichloromethane as an extracting

Figure 3. Turbidity versus temperature changes at different concentrations of artemisinin in isopropanol (temperature changes (°C): dash line; turbidity changes: straight line). 4337


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Figure 4. Metastable zone width for artemisinin in isopropanol.

study complies with today’s shift for sustainability and calls for innovative industry leaders to seize the environmental and regulatory incentives that it will unleash. This work identified isopropanol as practically viable and environmentally benign solvent substitute for n-hexane in the recovery of artemisinin from Artemesia annua L. Turbidity change measurements provided precise solubility data at different temperatures and enriched our knowledge about the stability of the solution at the equilibrium point vicinity, as indicated by the metastable zone width, which is essential information for optimized process development and scale-up of artemisinin crystallization.

Table 3. Solubility of Artemisinin in Isopropanol at Different Temperatures temperature (°C)

solubility (mg·mL−1)

5 10 15 20 25 30 35 40

6.81 8.77 11.29 14.54 18.71 24.09 31.01 39.92

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solvent and proposed a hybrid technique that includes evaporation of the solvent to 1/5, then acetonitrile is added as antisolvent. Another evaporation is performed, followed by an antisolvent precipitation feeding water to obtain 95% artemisinin purity but only a 30% yield. It is noted that other co-metabolites and impurities are extracted with artemisinin, which is by the way not exclusive to isopropanol. The presence of waxes is discarded, as isopropanol holds a very low solubility for oils and fatty acids,40 while the solubility of free sugars in isopropanol is the lowest among other alcohols according to Montanes et al.41 Nonetheless, separation of sugars (nonionized substances) can be achieved by ion exchange chromatography which is worth future investigation to increase crystallization efficiency. We believe that our method uses a greener approach that reduces the number of solvents and processing steps and contributes to minimize or use of innocuous auxiliary agents while selecting safer reagents (solvents), minimizing the potential for accidents,42 however an optimization of the crystallization step is required to achieve higher purity.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Smain Chemat: 0000-0003-2123-9603 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are highly grateful to the Ministry of Higher Education and Scientific Research in Algeria (MESRS) for funding this project via a PURAQ grant. Special thanks goes also to Prof. Farid CHEMAT of Université d’Avignon et Pays de Vaucluse (France) for HPSiP calculations.

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REFERENCES

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CONCLUSION This study succeeded to fulfill green engineering principles during solvent selection for artemisinin extraction through selecting solvents that maximize efficiency and meet the need to minimize excess. This is achieved by combining theoretical estimations of solute−solvent affinities of a large number of candidates using Hansen solubility parameters and then concentrating on solubility evaluations of the best solvents by means of turbidity change measurements. To some extent, the more efficient and rational use of resources we applied in this 4338


Research Article

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Efficient Solvent Selection Approach for High Solubility of Active

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