(Horopito) Leaves Using Deep Eutectic Solvents - American Chemical

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An approach for polygodial extraction from Pseudowintera colorata (horopito) leaves using deep eutectic solvents Joanna Nadia, Kaveh Shahbaz, Marliya Ismail, and Mohammed Farid ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03221 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 8, 2017

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ACS Sustainable Chemistry & Engineering

A novel solvent based on TBAC and 1-dodecanol was prepared and examined to extract polygodial, an antifungi compound, from New Zealand native plant

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An approach for polygodial extraction from Pseudowintera colorata (horopito) leaves using deep eutectic solvents Joanna Nadia, Kaveh Shahbaz *, Marliya Ismail, Mohammed M. Farid* Department of Chemical and Materials Engineering University of Auckland, 2-6 Park Ave Auckland 1023, New Zealand

ABSTRACT Polygodial is a bioactive compound that is present in an ancient native plant known as “horopito” (Pseudowintera colorata) in New Zealand and it possesses antifungal, antibacterial, antifeedant, insecticidal, and antithelmintic properties. In this work, an approach for polygodial extraction using deep eutectic solvents (DESs) was presented. Two newly prepared DESs based on 1-dodecanol and polyethylene glycol were found to be comparable with ethanol in their polygodial extractability. However, they exposed a superior ability to protect polygodial from degradation and also a better solvent reusability for extraction of polygodial as compared to ethanol. The Box-Behnken design (BBD) in combination with response surface methodology (RSM) was used to design the experiment and optimization of polygodial extraction using the dodecanol-based DES. The optimal condition was acquired at 47.13 °C, for 1.03 h, and 5.01%w/v biomass, where 12.35 ± 0.05 mg polygodial/g dried horopito leaf was extracted.

Keywords: deep eutectic solvents; extraction; horopito leaves; polygodial

Corresponding authors: E-mail address: [email protected] (M.M. Farid), [email protected] (K. shahbaz)

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INTRODUCTION New Zealand is a country with a variety of unique flora and fauna. Its geographically located position allows the country to receive greater UVB light level, thus making the country abundant with “rich-in-bioactive” and “dense-in-nutrition” natural vegetation.1-3 One of them is horopito (Pseudowintera colorata), an ancient native plant that has survived against fungi and bacteria for more than 65 million years.4 Horopito was traditionally used for a range of medicinal purposes by both the native inhabitants of New Zealand, the Māori, and European settlers in New Zealand in the 19th century. The essential oil in horopito leaves contains a number of bioactive compounds, including polygodial.5 Polygodial is the main biologically active chemical constituent of horopito which has been proven in numerous scientific studies that it possesses antifungal, antibacterial, antifeedant, insecticidal, and antithelmintic properties.6-12 The antifungal activity is the most prominent among polyqodial properties and for this reason the extracted polygodial is being used in production of natural antifungal products in New Zealand.13 Polygodial (Figure 1) belongs to sesquiterpene dialdehydes group and it can be considered as a hydrogen bond acceptor due to its aldehyde oxygen atoms.14 The aldehydes in polygodial are highly reactive substances and they can readily react with biologically important nucleophiles, such as sulfhydryl, amino, and hydroxyl groups.7 Polygodial can be extracted from plant materials using conventional techniques, such as steam distillation,5 maceration (e.g. using acetonitrile, petroleum ether, dichloromethane, ethyl acetate and methanol),15-20 and Soxhlet extraction.21-22 However, techniques like steam distillation and Soxhlet extraction, which are operated at high temperature, became redundant due to the heat sensitivity of polygodial. In addition, although the use of organic solvents at lower temperature can avoid thermal degradation of the compound, they are toxic and require long extraction time and high amount of solvent.23-24 To overcome these limitations, innovative methods are being developed to enhance the yield of the extraction and reduce the environmental impact of the process. Supercritical carbon dioxide extraction is one of these technologies used to extract polygodial from horopito leaves, where liquefied carbon dioxide at high pressure (70-300 bar) and at certain temperature (20-60 °C) is used as the solvent. The method was reported to be able to remove 85-99% of active ingredients in the leaves with the purity of the polygodial in the extract laying between 15-59 %w/w.25 Despite of its good performance, the system is complicated and requires high capital costs. Pressurized hot water extraction (PHWE) is another emerging 3



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technique to extract essential oil containing polygodial from Tasmania lanceolata leaves, where an aqueous solution of ethanol was used as the solvent. The extraction was carried out in a portafilter of an espresso machine at 90°C, followed by subsequent liquid-liquid extraction of the essential oil with dichloromethane.14 Although PHWE has been shown to be effective for polygodial extraction, the process is conducted at a high temperature and utilizes dichloromethane which is unsafe chemical. Hence, there is a need to develop an economical and environmentally friendly technique for polygodial extraction. Interest in a new generation of solvents called deep eutectic solvents (DESs) has increased recently due to their potential application as “green solvents”.26 These designer solvents emerged to answer the challenge for the need of environmentally friendly solvents as alternatives to organic solvents, which are usually flammable, harmful to the environment, and toxic. Generally, a DES is prepared by mixing a quaternary ammonium or phosphonium halide salt as the hydrogen bond acceptor (HBA) with a hydrogen bond donor (HBD) which has the ability to form a hydrogen bond with the halide anion of the HBA, resulting in a eutectic mixture with a much lower melting point than that of the individual components.27-29 DESs possess very good physicochemical properties, such as negligible volatility, adjustable viscosity, a wide range of polarity and a high degree of solubilization strength for different compounds.26 From environmental perspective, DESs composed of natural primary metabolites fully represent green chemistry perspectives because they are biodegradable and sustainable. Additionally, they are of low costs due to the low cost of raw material and simple way of preparation.30 DESs offer a wide range of applications such as organic synthesis, extraction, electrochemistry, organic reactions and other applications.31-35 Presently, DESs are gaining a growing attention in extraction of bioactive compounds. DESs have been used to extract a wide range of bioactive compounds such as anthocyanins,36 hydrophilic phenolic acids and hydrophobic diterpenoid,37 phenolic compounds,38-41 flavonoids,42 and terpenoids.43 Furthermore, DESs have shown potential of enhancing the thermal stability of bioactive compounds.36 Lately, Cao et al. (2017) prepared new DESs based on methyltrioctylammonium chloride as HBA and alcohol and fatty alcohol as HBDs, which were successfully applied to extract artemisinin, a low-polarity compound.44 They reported that changes in the type and length of the alkyl chain of HBDs changed the polarity and viscosity of DESs, thus the polarity of DESs could be tuned to similar polarity of artemisinin, leading to higher extraction yield. 4


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To the best of our knowledge, there are no reported studies on extraction of polygodial from plant materials using such green solvents. Hence, the aim of this study is to introduce a more environmentally friendly, less profligate technique for extracting polygodial using DESs. In this study, different types of DESs were prepared and their applicability to extract polygodial from horopito leaves was investigated. Moreover, stability of extracted polygodial in the prepared DESs and the reusability of the DESs as solvents were investigated. MATERIALS AND METHODS Materials Horopito leaves were provided by Forest Herbs Research Ltd. in the form of dried and milled sample. The dried leaves were ground, sieved (≤ 200 µm), and stored under refrigerated conditions. Chemicals used in this work, together with their purity and source, are listed in Table 1. Preparation of DESs Fourteen DESs with different HBA: HBD combinations were prepared. All molar ratios of HBA: HBD to prepare the DESs were selected based on the lowest freezing temperature attained. To prepare the DESs, HBA was mixed with HBD at 90 °C with constant stirring until a homogeneous liquid formed and all particles had dissolved. Choline chloride (ChCl) and tetrabutylammonium chloride (TBAC) were used in this research to investigate the effect of HBA type to polygodial extraction efficiency of the DESs, meanwhile carboxylic acids, alcohols, and polyols were selected as the HBDs. Additionally, amino acid-based DESs were also prepared using L-proline either as the HBD or HBA. The compositions of the studied DESs along with their abbreviations are presented in Table 2. DESs Characterization Viscosity measurements were conducted at different temperatures (20 to 80 °C) using a controlled stress AR-G2 rheometer (TA Instruments Ltd, West Sussex, NB) equipped with an aluminium cone geometry. The uncertainty of the viscosity measurement was ±0.02 Pa.s. The melting point was measured using a Shimadzu Differential Scanning Calorimetry instrument (DSC-60). The DSC measurements were carried out at a heating rate of 5 °C/min. The uncertainty in melting point measurements was ± 0.05 °C. A thermogravimetric analyser (Shimadzu TGA-50) was used to measure the degradation temperature of the DESs. The measurements were carried out at atmospheric pressure and under a constant flow rate of 5



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Argon (50 mL/min). 10 mg of the sample was heated in an open aluminium pan at 10 °C/min until the temperature reached 400 °C. All measurements were repeated three times to ascertain measurements reliability. DESs Screening To screen the extraction performance of each DES, 0.25 g of horopito powder was mixed with 5 ml of DES at room temperature in a capped glass vial. The stirring speed was set to 1000 rpm to achieve perfect mixing. After one hour, the biomass was separated from the mixture using 0.45-µm PTFE microfilter. The ensuing clear liquid (4 mL) was then diluted with ethanol (1:1-v/v) for polygodial quantification using HPLC. The same procedure was repeated for screening the extraction performance of reference solvents (e.g. ethanol, methanol, water). HPLC Analysis The polygodial content in the sample was determined using a Shimadzu LC-20AT HPLC unit equipped with LabSolutions software for the data acquisition and analysis. A ZORBAX Eclipse XDB C-18 column (4.6 × 150 mm, 5 µm) along with a C-18 guard column (Eclipse XDB) was used for the compound separation. The Milli-QTM water and HPLC grade acetonitrile (MeCN) were used as mobile phases and pumped to the system at a flow rate of 1 mL/min. The column temperature was controlled at 25 °C. Peak areas of the samples (measured in UV absorbance units, AU) were monitored at 230 nm. The content of polygodial was determined by means of a calibration curve established with a regression equation y = 5.2062 × 107 x – 6.59946 × 105 (R2 = 0.9992), where y is the peak area and x is the concentration of polygodial standard solution in ethanol (mg/mL). Each sample was analyzed using HPLC with duplicate measurement. During HPLC analysis, it was observed that the presence of DESs in the samples did not affect the chromatograms obtained. Experimental Design and Statistical Analysis Design Expert software version 7.1 (Stat-Ease Inc., USA) was employed to perform the experimental design and optimization. Box-Behnken Design (BBD) in combination with response surface methodology (RSM) was applied to investigate the effect of three extraction parameters on the extraction yield of polygodial, namely: temperature (X1), stirring time (X2) and biomass percentage (X3) at three levels, with three replications of the center points. BBD was selected because designs of experiment using BBD are usually very efficient in reducing 6


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the number of required runs, compared to Central Composite Design (CCD). It also has been applied for optimization of many chemical and physical processes.45 Moreover, BBD technique is considered as the most suitable for evaluating the response surfaces involving factors having quadratic effect.46 RESULTS AND DISCUSSION Preparation of DESs In this work, fourteen DESs (DES1 to DES14) with various salts and HBDs were prepared and used for screening their performance in extracting polygodial from horopito powder. The selection of these DESs was made to assess two hypotheses: i) longer chain HBDs would form the DES with polarity close to polarity of polygodial, ii) DESs containing amino and hydroxyl groups can make strong hydrogen bond with polygodial.7 Among the prepared DESs, PEG300-based DES (DES2) and 1-dodecanol-based DES (DES4) were introduced for the first time as the newly prepared DESs in this work. Polyethylene glycol 300 (PEG300) as HBD was preferred as it has better biodegradability compared to high molecular weight PEGs.47 Hence, the important thermophysical properties of these two DESs were reported in Table 3 showing the melting temperature (Tm), degradation temperature (Td) and viscosity (η) of the newly introduced DES2 and DES4. The melting temperatures of the DES2 and DES4 were found to be lower than 23 °C, which means both DESs are in liquid phase at room temperature. Moreover, both DESs have lower melting temperatures than their constituting components. This depression can be associated with the strength of the interaction between the HBD and the HBA. The decomposition temperatures of both DESs are rather high, allowing the DESs to be used at elevated temperature. In combination with their low melting points, DES2 and DES4 have wide range of operating temperatures. Particularly for the extraction of heat sensitive bioactive compounds, the DESs can be used without any limitations at room temperature to avoid thermal degradation of the target compounds. Viscosity is another important property in the early design stage of any processes that employ new DESs. In this work, the viscosities of DES2 and DES4 were measured experimentally at a temperature range of 20 to 80 °C and their values were plotted against temperature in Figure 2. As expected, the viscosities of the DES2 and DES4 decreased with increasing temperature. The higher viscosity of DES2 compared to DES4 was most due to the existence of more hydroxyl groups in PEG300. The presence of hydroxyl groups increases the attractive forces between molecules by creating more hydrogen bonds with TBAC, thus 7



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making the resulted liquid more viscous.48 However, these measured values of viscosities are still considered as low (< 500 cP) and will be even lower at higher temperature.49 Their low viscosities make both DESs suitable for many purposes. Extraction of Polygodial Using DESs The screening extraction experiments were conducted at room temperature due to the heat sensitive nature of polygodial. The results obtained from a single run for each DES and reference solvents (ethanol, methanol, and water) are shown in Figure 3. In general, compared to water, all prepared DESs possess better affinity for polygodial; their extraction yield ranged from 1.9 to 31.1 times better than that of water. As expected, among other groups of DESs investigated in this study, alcohol-based and polyol-based DESs (DES1 to DES6) exhibited superior extraction efficiency, especially PEG-based and 1-dodecanol-based DESs. Based on the results, the viscosity and polarity of the DESs were found the main factors in determining the efficiency of polygodial extraction in this study. High viscosity is known as one of the major disadvantages when DES is used as an extraction solvent, 50 and based on visual observation, the amino acid-based and sugar-based DESs were the most viscous among the studied DESs in this work. The high viscosity of these DESs reduced the diffusivity of polygodial to the solvent, as reflected by Stokes-Einstein equation (‫= ܦ‬ ௞் ଺గఎோ

).51 However, although carboxylic acid-based-DESs have relatively low viscosity

compared to the amino acid-based and sugar-based DESs, they have similar polygodial extractability to the amino acid-based and sugar-based DESs. This suggests that the polarity of DESs also played important role in the extraction other than viscosity. In solvent extraction, “like-dissolve-like” is an important concept, where organic compounds dissolve in nonpolar solvents.52 Ethanol and methanol are relatively polar solvents, where methanol is more polar than ethanol.53 Despite their polarity of the hydroxyl group, these organic solvents have the ability to dissolve non-polar compounds because of the presence of their non-polar and hydrophobic ends. Their non-polar end allows them to dissolve polygodial, which is a relatively non-polar and hydrophobic compound.14 DESs that gave extraction yield between those of water and methanol most likely have higher polarity as compared to PEG-based and 1-dodecanol-based DESs (DES2 to DES4). This conclusion was also supported by the visual appearances of the used solvents after polygodial extraction 8


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(Figure 4). From Figure 4 it is also clear that most of the studied DESs had similar color (reddish) to that of water except for DES2, DES3 and DES4 which looked alike with methanol and ethanol mixtures (greenish). The reddish color of water and DES5 to DES14 was possibly due to the extraction of anthocyanins, water-soluble phenolic compounds that are responsible for the red coloration of horopito leaves.54 As can been seen from Figure 3, the alcohol-based and polyol-based DESs produced better polygodial extractability than the carboxylic acid-based DESs due to their lower polarity. In agreement with this, Dai et al. (2013) reported that the polarity of alcohol-based DESs was close to that of methanol, meanwhile carboxylic acid-based DESs have similar polarity to that of water.55 Bubalo et al. (2016) in their investigation on the extraction of anthocyanins from grape skin using ChCl:glycerol, ChCl:oxalic acid, ChCl:malic acid, ChCl:sorbose, and ChCl:proline:malic acid, have also reported that carboxylic acid-based DESs give the highest anthocyanin extraction yield.38 Similar to carboxylic acid-based DESs, the amino acid-based DESs did not exhibit better polygodial extractability than methanol. Duan et al. (2016) also reported that proline-based DESs were able to extract phenolic acid from Chinese herbal medicines with comparable results to that of methanol, which indicates that this type of DESs may be more suitable for polar bioactive compounds.39 Moreover, the addition of water to the amino acid-based DESs investigated in this work might also increase the polarity of the DESs. Based on the extraction results, it can be concluded that 1-dodecanol-based DES (DES4) provides markedly higher polygodial extractability due to its analogous polarity with polygodial. In addition, PEG-based DESs (DES2 and DES3) exhibited a lower extraction yield (8.86 and 7.72 mg/g dried leaf, respectively) compared to DES4 (9.89 mg/g dried leaf). This is because the PEGs are hydrophilic compounds, and DES2 and DES3 exhibit hydrophilic characteristic,56 thus impacting the polarity of the solvent. Moreover, the PEGbased DESs have higher viscosity compared to DES4. Since the viscosity of PEG300 is less than PEG600, it was not surprising to observe decreasing extraction yield as the molecular weight of PEG increased. The screening results confirmed that the newly introduced DES2 and DES4 offer better performances in extracting polygodial compared to the other prepared DESs. Hence, they were investigated further as alternatives to conventional solvents. In this respect, polygodial

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stability in the DESs, and the reusability of the DESs were examined and compared with ethanol as a conventional solvent. The stability of bioactive compounds in their carrier solvents is important, since it impacts their shelf life. Presently, polygodial-containing products are made by either adding the solvent-free extract in the formulation or mixing polygodial dissolved in ethanol with other ingredients in the product. These methods are not appropriate, because solvent removal is energy intensive and polygodial in ethanol has short shelf life due to degradation. It was reported that polygodial is relatively stable in ethanol at room temperature over a period of almost 4 days.57 However, longer shelf life is desired for commercialized product. Therefore, the stability of polygodial in the potential DESs was studied, in this work, and compared to ethanol by measuring the concentration of polygodial in the solvents for 10 days storage at room temperature without direct exposure to sunlight. The change of polygodial concentration with respect to initial concentration in DES2, DES4 and ethanol is shown in Figure 5. As can be seen from the figure, the degradation of polygodial was faster in ethanol, which was due to the susceptibility of the dialdehyde group of polygodial to degradation.57 It is a fact that most bioactive compounds are sensitive materials and they can degrade due to interaction with oxygen, moisture, light, elevated temperature, and microbial contamination.58 In this case, aldehydes in polygodial degrade in air via autoxidation process, and the presence of air during the storage of the solvent-extract mixtures will cause inevitable degradation. Even though the degradation of polygodial in DES2 and DES4 is significantly lower than ethanol, the hydrophilic characteristic of TBAC enabled the DESs to absorb moisture from air, which created undesirable effect to the ability of the DESs to retain polygodial. When it was combined with PEG300, the chemical interaction with air was still inevitable because of the hydrophilic nature of PEG300. On the other hand, DES4 was able to preserve polygodial better than DES2 because the hydrophobicity of 1-dodecanol limited the interaction between oxygen and water molecules in air with the DES structure. The observed results agreed with the report of Florindo et al. (2017), that DESs composed of hydrophobic HBA and HBD were more stable to interaction with water.56 The reusability of DES2 and DES4 for extracting polygodial from horopito powder was also investigated under the same conditions used for the DES extractability test (room temperature, 1 hour of stirring, 5%-w/v biomass, and stirring speed of 1000 rpm). Once one 10


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extraction cycle was done, the biomass was removed and the solvent was again loaded with the same percentage of fresh biomass. This procedure was repeated seven times. Figure 6 presents the reusability results for DES2, DES4 and ethanol for seven cycles of extraction. No significant decline in the extraction efficiency was noticed for DES2 and DES4 during 7 extraction cycles. In addition, they have comparable performance with ethanol after 5 cycles (1.88, 1.77, and 1.91 mg/mL solvent for ethanol, DES2, and DES4, respectively). Moreover, after 7 cycles, the final concentrations of polygodial in DES2 and DES4 were 6.65% and 7.35% higher than in ethanol. High reusability of DESs is important because it is related not only to the economical utilization of the solvents for extraction, but also to the capability of the solvent to retain more polygodial. As a result, the amount of DES needed to be incorporated in the formulation of polygodial-containing products can be reduced. In addition, the low volatility of DESs is expected to have more consistent reusability than that of ethanol which has high vapor pressure. At room temperature, ethanol evaporates easily and such property is not desirable for extraction because it affects the extraction efficiency and also the stability of the compound extracted, as demonstrated in the stability study. Besides polygodial stability in DES and solvent reusability, the applicability of the polygodial-containing extract for product formulation needs to be taken into account to select the most potential DES. Compared to PEG300 which is used mainly for coating and lubricant, 1-dodecanol has wider applications, especially in the formulation of emollient, cosmetics and pharmaceuticals.59 For these reasons, DES4 (TBAC: 1-dodecanol DES) was selected as the most promising DES for polygodial extraction. Prior to adding the extract in product formulation, TBAC has to be separated from the DES4polygodial mixture due to their unknown effects for human in topical applications, although it was reported that quaternary ammonium salts possess antimicrobial activity against several species of gram positive and gram negative bacteria.60-62 TBAC was also found to exhibit antiproliferative effects on human colon carcinoma cells of ionic liquids containing the compound.63 The separation of TBAC from the DES4-polygodial mixture can be done by the addition of antisolvent. Considering the hydrophilic nature of TBAC and hydrophobic nature of dodecanol, as well as the more hydrophobic properties of polygodial, there are two options in selecting the appropriate antisolvent in this case: (i) water, which is a very polar molecule and has the ability to attract TBAC from the DES mixture due to the hydrophilic nature of TBAC and (ii) organic solvents with low polarity, such as dichloromethane or ethyl acetate, 11



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which is miscible with the low-polarity 1-dodecanol/polygodial mixture but has very low miscibility with TBAC. Antisolvent with minimum impact to the quality of the dodecanol/polygodial mixture is more desirable. Based on our preliminary experiments, it was also found that usage of 1-dodecanol as a solvent, which is solid at 26 °C, for polygodial extraction made the biomass separation difficult at room temperature. The solidification of 1-dodecanol also caused inconsistent HPLC readings and blockage in the HPLC column. Moreover, the extraction yield of polygodial using 1-dodecanol was found to be lower than that of DES4. Optimization of Polygodial Extraction The optimization of polygodial extraction using DES4 was performed by Box-Behnken experimental Design (BBD). Response surface methodology (RSM), a valuable method in multivariate statistic techniques, was employed to simultaneously optimize levels of the investigated factors to attain the highest extraction yield in this study.64 Table 4 lists the coded and uncoded levels of the investigated independent variables. In this study, the conditions used for extractability study were set as the lower boundary to investigate whether the higher values of these factors could enhance the extraction yield. The results of the BBD experiments are presented in Table 5. The variables and response were processed to build a quadratic polynomial model for polygodial. By fitting the experimental data of the responses, the following mathematical regression model (Eq. 1) for polygodial extraction was established: Y = 7.9644 + 0.3204X1+ 1.7470 X2 - 0.9442 X3 - 0.0352 X1X2 + 0.0002 X1X3 + 0.0669 X2X3 2

2

2

– 0.0031X1 – 0.2788X2 + 0.0298X3

(1)

where Y is polygodial extracted (mg/g dried leaf), X1 is Temperature (°C), X2 is time (hour) and X3 is %biomass Table 6 shows the result of ANOVA for the established model, including the significance of the model terms which was determined by their p-values. The predictive model was significant due to the small p-value (probability of error value) which is less than 0.05. The small p-value of the model indicates that the model was sufficient to accurately represent the experimental data. Significant model terms (p < 0.05) were also identified, namely: X3, X1X2, X2X3, X12, X22, and X32. Although insignificant model terms can be removed from the model to improve the regression model and optimization results, they were kept to detect small 12


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changes in the results. The lack-of-fit testing produced a p-value > 0.05, which indicates that the model explains all data well in establishing a predictive mathematical model. It is interesting to note that X1X3 (interaction between temperature and %biomass) was found to be highly not significant. This was probably because increase in temperature only affected the diffusivity of polygodial into the solvent, but did not allow more polygodial to be released when more biomass was added to the DES. Therefore, more time was required by the compound to diffuse into the solvent. Although it is true that temperature increases the solubility of solute in a solvent, it is also possible that the effect of temperature in this research was possibly limited by the heat sensitive nature of polygodial. On the other hand, X1 (temperature) and X2 (time) as standalone variables did not have significant effect on the extraction yield. However, the quadratic effects of both variables (X12 and X22) were significant to the response of the model. The significance of the quadratic effects of X1 and X2 indicates that the extraction modeled in this research had a true optimum point, instead of a local optimum. It means that all polygodial in the sample used in the experiments was already extracted by the studied DES, which might be true, as the sample used was quite old and some of the polygodial in the sample has degraded, leaving only about half of the normal polygodial concentration in the sample. The quality of the model was evaluated in terms of the square of correlation coefficient (R2) and the lack-of-fit by the analysis of variance (ANOVA) at 95% confidence level, as shown in Table 7. The resulting R2 was 0.9829, which indicates a good agreement between the experimental data and predicted extraction yield. The value of predicted R-squared (0.9057) is in reasonable agreement with the adjusted R-squared, which means the model is good enough to predict responses for new observations. An adequate precision measure of 31.359 indicates that the signal to noise ratio is adequate (the minimum desired value is 4). Three-dimensional response plots of the response are illustrated in Figure 7. The statistical analysis described above and Figure 7 reveal the significance of temperature, time, and %biomass on the extraction yield of polygodial. Figure 7a shows that the extraction yield improved when low %-biomass was added to the solvent and the increase in temperature led to a higher extraction yield until approximately the middle of its range, then the amount of polygodial extracted became lower. The same effect of temperature was also shown by Figure 7b, where the extraction yield started to decrease at temperature higher than around 47.50 °C. As mentioned previously, although usually higher temperature is associated with 13



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higher extraction yield of bioactive compounds due to lowering of viscosity of the solvent, in this research the effect of temperature was limited by the susceptibility of polygodial to thermal degradation. Figure 7b and Figure 7c indicate that longer stirring time did not contribute positively to the extraction yield, because the low viscosity of the DES facilitated good diffusion of polygodial onto the solvent. Similarly, increasing %biomass did not increase the polygodial yield, which can be associated with the volume of minimum solvent required to dissolve all polygodial from the biomass. Overall, the maximum extraction yield occurred at less mixing time and %biomass, and slightly high temperature. Based on the developed model, Table 8 gives the optimum conditions that can give the maximum response within the defined range. The maximum extraction yield predicted from the model was 12.47 mg/g dried leaf, which can be obtained by setting the temperature, stirring time, and %biomass to 47.13 °C, 1.03 h, and 5.01%, respectively. To validate the model, triplicate verification experiments were conducted under these optimized parameters. The experimental values are also shown in Table 8. A reasonable accuracy of the model with an error of 0.95% was obtained, which indicates the suitability of the response model for optimization. Compared to extraction with ethanol at 25°C (Table 9), the extraction yield obtained by DES4 at optimized condition was better. The extraction using ethanol was not conducted at elevated temperature because of high vapor pressure of ethanol. Meanwhile, for DES4, slight temperature increase has a big impact on its viscosity, thus allowing better diffusion of polygodial into the designed solvent. According to this result, it can be concluded that a new DES based on TBAC as salt and 1-dodecanol as HBD for the extraction of polygodial from horopito leaves has been successfully prepared. However, this green extraction process still could be further improved by the application of other extraction methods, such as microwaveassisted extraction or ultrasound-assisted extraction to enable shorter extraction time and the use of higher solid/liquid ratio to reduce the amount of solvent.

CONCLUSIONS In this study, two new DESs (DES2 and DES4) were prepared and examined for extraction of polygodial from horopito leaves. It was demonstrated that the designed DESs showed close 14


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polygodial extractability to that of ethanol using a simple extraction method, with relative extraction efficiencies of 76.5% and 74.2% for TBAC:PEG-300 and TBAC:1-dodecanol, respectively. Compared to ethanol, DES2 and DES4 revealed significantly better stability in protection polygodial from degradation. 1-dodecanol-based DES (DES4) gave better results due to its lower polarity and viscosity than PEG300-based DES (DES2), therefore it was studied further for optimization. Response surface methodology gave 12.35 mg polygodial/g dried leaf at 47.13 °C, for 1.03 h, and %-biomass of 5.01%, DES4 as the optimum extraction conditions. The comparison of the optimum extraction yield between predicted and measured values was of a reasonable accuracy with an error of 0.95%. To sum up, 1-dodecanol-based DES has a strong potential as an alternative solvent to extract polygodial from horopito leaves with a simple and a cheap extraction method, and it may be applicable for extracting bioactive compounds with similar characteristics. ACKNOWLEDGEMENT The authors would like to thank Indonesia Endowment Fund for Education for the financial support and Forest Herbs Research Ltd. for providing the leaf sample. Furthermore, this research was carried out as part of the Food Industry Enabling Technologies programme funded by the New Zealand Ministry of Business, Innovation and Employment (contract MAUX1402).

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Table 1. Chemicals used in this study. Mass fraction purity 1,3-Propanediol 98% 1-Dodecanol ≥98% Acetic acid glacial >99.8% Choline Chloride ≥98% Ethanol >99.4% Ethylene glycol >99.8% Glycerol >99.5% Lactic acid 92% L-proline 99% Methanol >99.8% PEG-300 and PEG-600 99% Polygodial ≥97% Tetrabutylammonium chloride ≥97% Xylitol ≥99% Chemical

Source Sigma-Aldrich, St. Louis, MO, USA Merck KGaA, Darmstadt, Germany ROMIL Pure Chemistry, Waterbeach, Cambridge, UK Sigma-Aldrich, St. Louis, MO, USA ECP Labchem, Auckland, New Zealand ECP Labchem, Auckland, New Zealand Ajax Finechem, Auckland, New Zealand Sigma-Aldrich, St. Louis, MO, USA AK Scientific, Union City, CA, USA LOBA Chemie, Mumbai, India Merck Schuchardt OHG, Hohenbrunn, Germany Sigma-Aldrich, St. Louis, MO, USA Sigma-Aldrich, St. Louis, MO, USA Sigma-Aldrich, St. Louis, MO, USA

Table 2. Composition of the studied DESs and their abbreviations. Abbreviation DES1 DES2 DES3 DES4 DES5 DES6 DES7 DES8 DES9 DES10 DES11 DES12 DES13 DES14

HBA ChCl TBAC TBAC TBAC ChCl ChCl ChCl ChCl ChCl L-proline L-proline L-proline ChCl TBAC

HBD 1 1,3-propanediol PEG300 PEG600 1-dodecanol glycerol ethylene glycol xylitol lactic acid acetic acid 1,3-propanediol glycerol ethylene glycol L-proline L-proline

HBD 2 water water water water

Molar ratio 1:3 1:3 1:2 1:2 1:2 1:2 1:1:2 1:2 1:2 1:2:2 1:3 1:3 1:2:5 1:1:3

Table 3. Themophysical properties of the DES2 and DES4. DES

Molar ratio

Tm ( °C)

Td ( °C)

η @25 °C (cP)

DES2

1:3

-30.15

277.75

68.06

DES4

1:2

8.88

205.72

41.90

20


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Table 4. Independent variables and levels used for BBD experiments. Factor

Variable

X1 X2 X3

-1 25 1 5

Temperature ( °C) Time (h) % w/v biomass

Level 0 40 2 10

1 55 3 15

Table 5. Experimental data and obtained responses values with different combination of extraction temperature, stirring time, and %biomass. Run

X1: Temperature ( °C)

X2: time (h)

X3: %biomass

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

25 40 25 40 40 55 40 40 25 40 55 40 55 25 55

2 1 1 2 2 2 3 2 3 3 1 1 2 2 3

15 5 10 10 10 15 5 10 10 15 10 15 5 5 10

Response: extraction yield (mg PG/g dried leaves) 9.35 12.43 8.96 10.45 10.39 9.69 11.56 10.37 9.79 9.98 10.13 9.51 11.54 11.26 8.85

Table 6. ANOVA for the response surface quadratic model of polygodial extraction using DES4. Source

F Value

p-value Prob > F Remarks < 0.0001 significant 0.0765

Sum of Squares

df

Mean Square

Model X1-Temperature

14.68 0.089

9 1

1.63 0.089

90.49 4.96

X2-Time

0.092

1

0.092

5.11

X3-%biomass

8.51

1

8.51

472.39

< 0.0001 significant

X1X2

1.12

1

1.12

61.88

0.0005 significant

X1X3

7.142E-004

1

7.142E-004

0.040

0.8501

X2X3

0.45

1

0.45

24.82

0.0042 significant

X1

2

1.75

1

1.75

97.06

0.0002 significant

X2

2

0.29

1

0.29

15.92

0.0104 significant

X3

2

2.06

1

2.06

114.08

0.0001 significant

Residual

0.090

5

0.018

Lack of Fit

0.087

3

0.029

Pure Error

3.583E-003

2

1.792E-003

Cor Total

14.77

14

16.10

0.0733

0.0590 not significant

21



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Table 7. Analysis of the variance for the fitted quadratic polynomial model of polygodial extraction using DES4. Std. Dev. Mean

0.13425 10.28267

R2

0.9939 2

0.9829

2

0.9057

Adjusted R

C.V. %

1.305599

Predicted R

PRESS

1.392583

Adequate Precision 31.3594

Table 8. Experimental and predicted results of polygodial extraction using DES4 at optimum condition. Temperature ( °C)

time (h)

%-biomass

47.1

1.03

5.01

Polygodial yield (mg/g dried leaf) Predicted

Measured

12.47

12.35

Error (%) 0.95

Table 9. Comparison between extraction yield using ethanol at room temperature and DES4 at optimized condition. Solvent Ethanol DES4

Temperature ( °C) 25 47.1

time (h) 1 1.03

%-biomass 5 5.01

Polygodial yield* 10.58 ± 0.18 12.35 ± 0.05

*Mean ± standard deviation, in mg/g dried leaf

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Figure 1. Chemical structure of polygodial 100

80

Viscosity (cP)

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


60

40

20 DES2 DES4 0 10

20

30

40

50

60

70

80

90

Temperature (°C)

Figure 2. Viscosities of DES2 and DES4 at different temperature (Shear rate = 1-100 s1 ).

23


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Solvent


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Ethanol DES4 DES2 Methanol DES1 DES3 DES6 DES5 DES9 DES8 DES10 DES11 DES13 DES7 DES14 DES12 Water 0

2

4

6

8

10

12

14

Polygodial extracted (mg/g dried leaf)

Figure 3. Screening result of polygodial extractability in different DESs and conventional solvents.

Figure 4. Appearance of the used solvents after polygodial extraction: (a) DES1; (b) DES2; (c) DES3; (d) DES4; (e) DES5; (f) DES6; (g) DES7; (h) DES8; (i) DES9; (j)

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DES10; (k) DES11; (l) DES12; (m) DES13; (n) DES14; (o) water; (p) ethanol; (q) methanol.

100

Relative percent change

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


80

60

40 Ethanol DES2 DES4

20

0 0

1

2

3

4

5

6

7

8

9

10

11

Time (day) Figure 5. Percent change in polygodial concentration in DES2, DES4 and ethanol over 10 days.

25



3.0

Polygodial concentration in solvent (mg/mL)

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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Ethanol DES2 DES4

2.5

2.0

1.5

1.0

0.5

0.0 0

1

2

3

4

5

6

7

8

Number of extraction cycle

Figure 6. Total polygodial extracted in DES2, DES4 and ethanol for seven cycles of extraction.

26


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(b)

(a)

(c)

Figure 7. Response surface plots of polygodial extraction using DES4 presented in terms of the interaction between the studied factors: (a) extraction temperature and %biomass; (b) extraction temperature and time; (c) extraction time and %-biomass.

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(Horopito) Leaves Using Deep Eutectic Solvents - American Chemical

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