Comprehensive Evaluation of Deep Eutectic Solvents in Extraction of

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Comprehensive evaluation of deep eutectic solvents in extraction of bioactive natural products Li Duan, Li-Li Dou, Long Guo, Ping Li, and E-Hu Liu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00091 • Publication Date (Web): 18 Feb 2016 Downloaded from http://pubs.acs.org on February 22, 2016

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ACS Sustainable Chemistry & Engineering

Comprehensive evaluation of deep eutectic solvents in extraction of bioactive natural products

Li Duan1, Li-Li Dou1, Long Guo, Ping Li*, E-Hu Liu*

State Key Laboratory of Natural Medicines, China Pharmaceutical University, No. 24 Tongjia Lane, Nanjing, P. R. China

1

These authors contributed equally to this work.

*

Corresponding authors.

*

E-Hu Liu, E-mail: [email protected]. Tel. / fax: +86 25 83271379.

*

Ping Li, E-mail: [email protected]. Tel. / fax: +86 25 83271379

1

ACS Paragon Plus Environment


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Abstract Deep eutectic solvents (DESs) are emerging as green and sustainable solvents for efficient extraction of bioactive compounds or drugs. This work aimed to comprehensively evaluate the potential and effectiveness of DESs for extraction of different types of natural compounds from biomass. Five Chinese herbal medicines including Berberidis Radix, Epimedii Folium, Notoginseng Radix et Rhizoma, Rhei Rhizoma et Radix, and Salviae Miltiorrhizae Radix et Rhizoma were selected to assess the efficiency of DESs on extraction of alkaloids, flavonoids, saponins, anthraquinones, and phenolic acids, respectively. Totally 43 types of choline chloride-, betaine- and L-proline-based DESs with different polarity, viscosity, composition, and solubilization abilities were tailored to test their extraction efficiency, and the operation conditions were statistically optimized using response surface methodology to produce the most efficient process. In this work, DES solvents were firstly introduced to extract alkaloids and anthraquinones. The results indicated that most prepared DESs proved to be efficient solvents for extraction of alkaloids, but lower extractability for anthraquinones. The extraction capacity of DES may be correlated with their physical-chemical properties, including H-bonding interactions, polarity, viscosity and pH. This study demonstrated that DESs were suitable green extraction solvents for selectively and efficiently extracting bioactive compounds from biomaterials. Keywords：：Deep eutectic solvents; green extraction; natural products; alkaloids; laevulinic acid 2


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INTRODUCTION The development of green solvents with low toxicity and cost is a key issue for the pharmaceutical industry.1,2 Nowadays, conventional organic solvents e.g., methanol, alcohols, chloroform, and ethyl acetate are widely used for extraction of bioactive natural compounds from plant material.3,4 However, the consumption of large amounts of these volatile and hazardous organic solvents may contribute to environmental pollution and leave unacceptable solvent residues in extracts. Since being introduced by Abbott et al. in 2003,5 deep eutectic solvents (DESs) are rapidly emerging as a new type of green and sustainable solvent. DESs are prepared by simply mixing two or more naturally-occurring, inexpensive, and biodegradable components together to obtain a eutectic mixture.5 The availability, low cost, biodegradability and environmental friendliness of the components makes the DESs versatile alternatives to conventional organic solvents.6-9 Due to their excellent properties, DESs have been widely used to generate nanoscale materials, extract DNA and desulfurize the products of fuels.10-13 Recently, DESs were applied for extraction and separation of bioactive phenolic compounds, saponins or flavonoids from plant materials.14-19 Nonetheless, the number of reports on the application of DESs to the extraction of natural products is limited, and the efficiency of DESs on extraction of other types of active natural compounds (e.g., alkaloids, anthraquinones) still remains unknown. To the best of our knowledge, there is still no report on comprehensive evaluation of the potential and effectiveness of DESs as green solvents for extraction of different bioactive compounds from natural 3



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sources. To extend the applications of DESs in extraction of bioactive natural-occurring compounds, in this study, five Chinese herbal medicines (HMs) including Berberidis Radix (BR), Epimedii Folium (EF), Notoginseng Radix et Rhizoma (NRR), Rhei Rhizoma et Radix (RRR), and Salviae Miltiorrhizae Radix et Rhizoma (SMR) were selected to comprehensively evaluate the efficiency of DESs on extraction of alkaloids, flavonoids, saponins, anthraquinones, and phenolic acids, respectively. A series of choline chloride (ChCl)-, betaine (Bet)- and L-proline (Pro)-based DESs with different mixing ratios were firstly tailored, and the operation conditions were statistically optimized using response surface methodology (RSM) to produce the most efficient process. The interactions between the DESs and natural products, as well as the optimal conditions for extraction were analyzed by high-performance liquid chromatography (HPLC).

EXPERIMENTAL

Materials and reagents. EF, NRR, RRR, SMR and BR were purchased from a local TCM market (Nanjing, China). Reference compounds (Fig. 1) including aloe-emodin, rhein, emodin,

chrysophanol,

physcion,

ginsenoside

RG1,

ginsenoside

RB1,

notoginsenoside R1, rosmarinic acid, lithospermic acid, salvianolic acid B, jatrorrhizine

hydrochloride

(Jat),

berberine

hydrochloride

4


(Ber),

palmatine

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hydrochloride (Pal) and icariin were purchased from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). Compounds for DES preparation including ChCl, Bet, Pro, urea, 1-methylurea, N,N’-dimethylurea, acetamide, glycerol, xylitol, D-sorbitol, ethylene glycol, sucrose, D-glucose, maltose, citric acid, malonate, laevulinic acid, oxalic acid, DL-malic acid and lactic acid were obtained from Aladdin Reagent Company (Shanghai, China). HPLC-grade acetonitrile was purchased from TEDIA (Tedia Company, Inc., USA). Deionized water used in experiments was purified by a Milli-Q system (Millipore, Milford, MA, USA). All other reagents and chemicals used were of analytical grade

Preparation of DESs. The syntheses of natural DESs were conducted as described previously.20 Briefly, DESs were obtained by simply mixing the hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBD) together at a proper molar ratio with magnetic agitation at 80.0 ºC until a clear and homogeneous liquid formed, and a known amount of water was added when necessary.

Extraction procedure. For initial screening of the extraction solvent, ultrasound-assisted extraction (UAE) was performed in a 2 mL microtube with 25 mg of plant material powder and 1.0 mL of DES-water (3:1) solvent. The mixture was ultrasounded (KQ5200B, 200W, 40KHz, KunShan, China) at 50 ºC for 30 min and then centrifuged at 16200 G for 10 5



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min. The suspension was diluted ten times with water or methanol prior to HPLC analysis. Each extraction was performed by triplicate.

HPLC-UV analysis for quantification of extracted bioactive components. HPLC-UV analysis was performed using a Shimadzu HPLC system equipped with a pump (LC-20AT), an auto-sampler (SIL-20A), a diode array detector (SPD-M20A) and an automatic column temperature control oven (CTO-20A). Separation was performed on a Thermo BDS Hypersil C18 column (100 mm × 4.6 mm, 2.4 µm). For analysis of EF, NRR, RRR, and SMR, the mobile phase consisted of water containing 0.1% formic acid (eluent A) and acetonitrile (eluent B). For analysis of BR, the mobile phase consisted of 0.02 mol/L potassium phosphate monobasic (eluent A) and acetonitrile (eluent B). (1) HPLC analysis of alkaloids in BR extract. The gradient program was as follows: 0-5 min, 25%-32% B; 5-10 min, 32% B; 10-11 min, 32%-25% B; 11-15 min, 25% B. The flow rate was kept constant at 0.8 mL/min, and the effluents were monitored at 265 nm. (2) HPLC analysis of phenolic acids in SMR extract. The gradient program was set at: 0-7 min, 18%-25% B; 7-9 min, 25% B; 9-11 min, 25%-95% B; 11-13 min, 95% B. The flow rate was kept constant at 0.9 mL/min, and the wavelength was set at 286 nm; (3) HPLC analysis of anthraquinones in RRR extract. The gradient program was as follows: 0-15 min, 35%-65% B; 15-25 min, 65%-100% B. The flow rate was kept constant at 0.9 mL/min, and the effluents were monitored at 254 nm; (4) HPLC analysis of saponins in NRR extract. The gradient program was set at: 0-13 min, 18%-28% B; 13-17 min, 6


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28%-33% B; 17-24 min, 33%-50% B; 24-25 min, 50%-54% B. The flow rate was kept constant at 0.9 mL/min, and the wavelength was set at 203 nm; (5) HPLC analysis of flavonoids in EF extract. The gradient program was as follows: 0-10 min, 25% B; 10-15 min, 25%-90% B. The flow rate was kept constant at 0.7 mL/min, and the effluents were monitored at 265 nm.

Experimental design and statistical analysis. RSM was performed using the Design-Expert Ver. 8.0.6 (Stat-Ease Inc., Minneapolis, MN, USA). After determining the range of extraction variables on the basis of preliminary single-factor test, Box–Behnken design (BBD) was used to find the optimal values for four independent variables: water content (%) in the selected DES (A), temperature (B), ultrasonic irradiation time (C) and solid (sample powder)-liquid (extractant) ratio (D) at three levels (-1, 0, 1). Whole experiments were composed of 29 experimental points that included five replicates of the centre points. The extraction amounts were taken as the response of the design experiments. Regression analysis was performed according to the experimental data. Subsequently, three additional confirmation experiments were conducted to confirm the validity of the statistical experimental strategies.

RESULTS AND DISCUSSION

Preparation of DES. 7



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For DES preparation, a number of HBAs and HBDs from renewable, inexpensive, and readily accessible resources have been tested as potential DES components. In this work, four groups of HBDs (amides, carboxylic acid, alcohols and sugars) were used in combination with three types of HBAs (ChCl, Bet, Pro) to produce DESs. A total of 51 combinations of DESs were initially tested and 43 of them were eventually found to be stable as a clear, viscous liquid without precipitation over the course of time (Table 1). During the preparation process, we found that the chemical structures of HBAs played an important role in the formation and stability of DESs. Natural DESs could be successfully formed by heating ChCl or Bet with ethylene glycol, in contrast, Pro was proved incapable of producing a stable liquid when heated with ethylene glycol. Also, it was easier to synthesize a clear and less viscous DES without any precipitation when HBDs were in liquid form (e.g., lactic acid, glycerol, levulinic acid, and ethylene glycol). Moreover, a known amount of water was necessary to be added in some combinations to form a clear liquid, for example, when betaine was mixed and heated with lactic acid, it was difficult to prepare a clear DES without adding water. We also discovered that different ratios of HBAs and HBDs may affect the synthesis and stability of DES. For instance, when levulinic acid were heated with ChCl in the mole ratios of 1 : 3, 1 : 2, 3 : 2, 2 : 1, and 3 : 1, a clear liquid can be only prepared with 3 : 2, 2 : 1, or 3 : 1 mole ratio.

Comprehensive evaluation of DES in extraction of bioactive natural products. 8


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Discovery of natural bioactive products meets numerous challenges, and sample extraction is the first step. In this work, a total of 43 DESs with different polarity, viscosity, composition, and solubilization abilities were selected to test their extraction efficiency for five different types of bioactive natural-occurring compounds (flavonoid, alkaloid, anthraquinone, saponin, and phenolic acid) from HMs. The potential and effectiveness of DESs as green solvents for extraction was comprehensively evaluated. Although certain DESs containing liquid-form HBDs (e.g., ChCl-La) were relatively fluidic, most of the DESs prepared were still viscous. Compared with conventional solvents, the high viscosity of most DES at room temperature restricted their application due to a slow mass transfer. The addition of water to DES can have significant benefits in decreasing viscosity. In this paper, extraction conditions were adjusted to reduce the viscosity by heating and adding certain amount of water. The extraction temperature was set at 50 ºC and 75% DES solution in water (v/v) was employed in the initial screening. In this study, methanol was used as the control extraction solvents.

Extraction of alkaloid Ber, Jat and Pal are three natural bioactive nitrogen-containing compounds belonging to the protoberberine hydrochloride group of isoquinoline alkaloids, and they are the major bioactive compounds of the medicinal herb Berberidis Radix. The compound Ber has shown potent biological activity against fungal and bacterial/viral infections21 and Jat has been demonstrated to possess antimelanoma effects.22 To 9



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extend the applications of DESs and reduce the environmental pollution by organic solvents, Ber, Jat and Pal were extracted from BR by the prepared DESs and determined by HPLC. Different DESs were tested under the same condition using the same protocol (solid to solvent ratio: 25 mg/mL, extraction time: 30 min). The HPLC fingerprints of DES extracts showed that the three high peaks were respectively identified as Jat, Pal and Ber by comparison with reference compounds (Fig. 2a). As shown in Fig. 3a, significant differences in the extractability of alkaloids with the tested DESs were reflected by HPLC profiles and the calculated content of Jat, Pal and Ber in BR. Clearly, the extraction capacity of the tested DESs varied greatly depending on the type of HBD in DES. Sugars- and alcohols- based DESs exhibited relatively low extraction capacity, capable of extracting only about 75% alkaloids compared with methanol, while amides-based DESs showed similar yields compared with methanol for Jat, Pal and Ber extraction. Surprisingly, the extraction efficiencies of carboxylic acid- based DESs were significantly higher than that of other DESs, as well as methanol. Among the carboxylic acid- based DES, La-based DES, such as ChCl-La, Bet-La and Pro-La, produced the highest extractability of alkaloids from natural sources. The differences observed between DESs and MeOH for alkaloids may be due to the lack of extractability of partially ionized compounds by MeOH when compared with DESs, where electrostatic interactions could significantly contribute to their extraction. In conclusion, the La-based DESs, relatively inexpensive and biodegradable, were found to be the best DESs for the extraction of alkaloids. 10


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Extraction of phenolic acid SMR is one of the most frequently used HMs and has been widely used for the treatment of cardiovascular and cerebrovascular disease in clinic.23 The phenolic acids (e,g., salvianolic acid B, rosmarinic acid and lithospermic acid) are the main beneficial compounds responsible for the pharmacological activities and therapeutic efficacy of SMR.24 In order to find the best extraction solvents for rosmarinic acid, lithospermic acid and salvianolic acid B from SMR, the prepared DESs which had different properties like polarity, solubility, diffusion, and viscosity were examined in this study. The total contents of these three phenolic acids detected by HPLC were employed to assess the extraction efficiency of DESs (Fig. 2b). The results indicated that extraction yields for phenolic acid were relatively less affected by solvent type of DESs. As shown in Fig. 3b, a majority of DESs exhibited higher yields of phenolic acid compared with methanol. The main reason could be superior dissolving capacity of the DES for the polar compounds. Among the three HBAs, the extraction capacity of ChCl-based DESs was markedly higher than that of Bet- and Pro-based DESs. Also, amides-based DES showed the highest efficiency compared with other three types of HBDs. Overall, considering the toxicity of methanol, DES was a suitable green solvent for extraction of phenolic acid from medicinal herbs.

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Extraction of anthraquinone RRR, a well-known medicinal herb wildly used in oriental preparations, has been recognized for centuries for its multiple pharmacological actions including laxative, antibacterial, hemostatic and antispasmodic activities.25 The major active components of the herb are five anthraquinones, namely aloe-emodin, rhein, emodin, chrysophanol and physcion. To evaluate the extraction efficiency of DESs for anthraquinone, RRR was taken as a case in our work. The total contents of five anthraquinones including aloe-emodin, rhein, emodin, chrysophanol and physcion in RRR were quantified by HPLC method (Fig. 2c). Unfortunately, the results showed that most DESs showed the lower extraction yields than methanol, and only four synthesized DESs (ChCl-La, ChCl-Ox, Bet-La and Pro-La), produced similar extraction efficiency for anthraquinone compared with methanol (Fig. 3c). We speculate that the low extractability of DES solvents may be attributed to the low polarity of anthaquinones.

Extraction of saponin NRR, is a highly valuable and important Chinese medicinal herb mainly cultivated and processed in Yunnan Province, China. It has been widely used as a tonic and hemostatic agent for the treatment of cardiovascular diseases, inflammation, body pains, trauma, and internal or external bleeding caused by injury.26,27 The dammarane-type saponins, including ginsenosides and notoginsenosides, are the main bioactive components contributing to the pharmacological activities of NRR. 12


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In order to get insight into the DES excellence in the extraction of saponins from NRR, a comparative study was carried out to illustrate the extraction efficiency of methanol, and DESs for Ginsenoside RG1, ginsenoside RB1 and notoginsenoside R1 (Fig. 2d). As displayed in Fig. 3d, the extraction efficiencies of DESs varied extremely depending on the type of HBD. Several carboxylic acid-based DESs, such as ChCl-Ca, ChCl-Mal, ChCl-Ox and ChCl-Maa could hardly extract the saponins from NRR. Contrastly, amide-based DESs exhibited higher yields of saponin than other DESs. Based on the data, twelve DESs demonstrated comparable extraction yields with methanol, particularly ChCl-Du and Pro-Mu extracted higher amounts of saponins with significant difference. All in all, certain DESs could be used as the excellent solvents for saponin extraction.

Extraction of flavonoid The medicinal herb EF has been historically used in combination with other herbs to treat skeletal diseases in traditional Chinese medicines. Its main active components are flavonoids (e.g., icariine), which exhibit multiple biological activities including promotion of sexual function, protection of the nervous system, and prevention of cardiovascular diseases.28,29 To compare the flavonoid extraction efficiency of DESs and conventional organic solvents, the extraction yields of icariine, which was the main bioactive flavonoid in EE, were detected and determined by HPLC (Fig. 2e). The results indicated that the extraction efficiency was influenced by the types of DES solvents, 13



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and different types of DES resulted in different extraction yields. The flavonoid icariine was highly extractable in Pro-based DESs, while sugar-based and alcohol-based DESs demonstrated lower extractability for icariine (Fig. 3e). Our data showed that 14 types of DES, such as Pro-Mu, Pro-Mal, Pro-La, Bet-Maa and Chcl-Du, exhibited higher yields of icariine compared with methanol. In sum, the excellent properties of DESs highlight their potential as promising green solvents for the extraction of flavonoid.

Generally, the extraction capacity of DES for bioactive natural products is correlated with their physical-chemical properties, including H-bonding interactions, polarity, viscosity and pH. The high extractability of phenolic acids with DES may be attributed to H-bonding interactions between DES molecules and phenolic compounds. The polarity of DESs is an important factor affecting their extraction efficiency. Since the synthesized DESs have relatively higher polarity, they demonstrate lower extraction yields for low-polar natural compounds, e.g., anthraquinones in the present study. Compared with conventional solvents, the high viscosity of DESs restricts their application as extraction solvents. Our results demonstrate that the less viscous La-based DESs show higher efficiency for natural-occurring compounds than other DESs. Moreover, the extractability of DESs is affected by acidity and basicity of natural compounds and DESs. Our data indicate that extraction yields of carboxylic acids-based DES for alkaloids are 1.5 times higher than other DESs, most likely due to the acid-base neutralization reaction. 14


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Optimization of the operational conditions for alkaloids extraction using RSM.

Optimization of extraction conditions The above extraction investigations showcased the tuneability of DESs as designer solvents to selectively extract bioactive compounds from biomass. Next, the effect of key parameters on DES extraction was investigated in detail using BR as a practical example. The variables affecting the extraction capacity, including the concentration of water (%) in extraction solvent, temperature, extraction time and solid-liquid ratio, were optimized in order to obtain the optimum conditions of extraction. RSM, a valuable statistical technique to determine the optimal values of the independent variables and to enable the user to effectively investigate the effects of multiple factors and their interactions, was employed to produce the highest extraction efficiency in this study.30 In this section, UAE was selected as the extraction method. As described above, ChCl-La produced the highest extractability of alkaloids from BR. In initial screening, the eutectic mixtures of ChCl-La were firstly prepared at mole ratios of 2 : 3, 1 : 2 and 1 : 3 to extract alkaloids from BR, and the results demonstrated that ChCl-La of 1 : 2 ratio showed relatively higher extraction ability with no significant difference. Subsequent preliminary experiments for UAE condition optimization revealed that the extraction temperature owned a positive relationship with extraction yields and the extraction efficiency was also enhanced with the decrease of solid-to-liquid 15



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ratio. Therefore, the following four independent variables were chosen for RSM-based optimization: water addition (%) in the selected DES (10%-50%), temperature (40-60 ºC), extraction time (10-30 min) and solid-to-liquid ratio (20-60 mg/mL). Table 2 lists the coded and uncoded levels of the independent variables. Extraction yields of Jat, Pal and Ber in BR were investigated as the response function of the BBD method. The experimental orders and levels of coded and uncoded variables are summarized in Supporting Information. The variables and response were processed to build a quadratic multiple regression model for alkaloids. The model quality was evaluated in terms of the square of correlation coefficient (R2) and the lack-of-fit by the analysis of variance (ANOVA) at the 95% confidence level (Supporting Information). The resulting R2 value was 0.9277, indicating that the experimental data were in relatively good agreement with predicted extraction yields. Besides, the lack-of-fit, which evaluates the failure of the model to represent the data in the experimental domain points, was insignificant for the response with p-value of 0.124 (>0.05). This implied that the model equation was adequate. The model was expressed as second order polynominal quadratic equations for the extraction yield (Y) and coded factors (A, B, C and D) as follows: Y = 2.93 + 0.081A + 0.031B + 0.039C - 0.12D - 0.0043AB - 0.075 AC + 0.0045AD - 0.02BC - 0.015BD 2 2 2 2 -0.022CD - 0.16 A + 0.0024B - 0.016C + 0.053D

Statistical analysis and 3D response plots (Fig. 4) illustrate the significant variables affecting extraction yields of the alkaloids and the interaction effects between the 16


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variables. In the model, the concentration of water (%) in extraction solvent (variable A) and the solid-to-liquid ratio (variable D) led to significantly different extraction yields. Generally, the extraction efficiency for alkaloids increased with lower solid-to-liquid ratio. The amount of extracted increased with increasing percentage of water until the water concentration reached 35%, but an excessively high concentration of water decreased the yields of alkaloids, most likely due to the decreased interaction between DES and alkaloids with increasing polarity of the mixture. On the other hand, extraction time (variable C) had a mild positive effect on extraction efficiency of alkaloids (p = 0.0245) and temperature (variable B) showed an insignificant effect (p = 0.0689).

Validation of the optimized conditions Triplicate experiments were carried out under the optimized conditions and mean values of experimental results were compared with the predicted values. The alkaloids extraction model yielded optimal conditions with A = 34% v/v, B = 60 ºC, C = 21 min, D = 20 mg/mL. Under these optimum conditions, the predicted extraction yield was 3.09 ± 0.06%. The experimental values were close to the predicted value (3.11 ± 0.08%) obtained using RSM. The results obtained through confirmation experiments indicated the suitability of the developed quadratic models and it may be noted that these optimal values are valid within the specified range of process parameters (Supporting Information).

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CONCLUSIONS In the present study, a green DES extraction method was described for comprehensive extraction of different types of bioactive natural compounds from HMs. The tailor-made DESs proved to be efficient solvents for the extraction of alkaloids, phenolic acids, flavonoids and saponins, but demonstrated lower extractability for anthraquinones. Subsequent optimization for alkaloids extraction using RSM indicated that the concentration of water in DES-water mixture and solid-to-liquid ratio were key parameters affecting extraction yields. This work provided practical examples showcasing the tuneability of DESs that selectively and efficiently extract bioactive compounds from biomaterials. Moreover, DES solvents were firstly introduced to extract alkaloids, and ChCl-La was shown to be significantly more efficient than previous methods requiring the consumption of organic solvents. This study holds promise for further utilization of DESs in pharmaceutical, biochemical, and food industries for the extraction of bioactive natural products.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No. 81473343), Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 81421005), Program for New Century Excellent Talents in University (No. NECT-13-1034), Natural Science Foundation of Jiangsu Province (No. BK20130652) and a Project Funded by the Priority Academic Program 18


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Development of Jiangsu Higher Education Institutions.

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REFERENCES (1) Paiva, A.; Craveiro, R.; Aroso, I.; Martins, M.; Reis, R. L.; Duarte, A. R. C. Natural Deep Eutectic Solvents-Solvents for the 21st Century. ACS Sustainable Chem. Eng. 2014, 2, 1063-1071. (2) Chemat, F.; Vian, M. A.; Cravotto, G. Green Extraction of Natural Products: Concept and Principles. Int. J. Mol. Sci. 2012, 13, 8615-8627. (3) Duan, L.; Guo, L.; Liu, K.; Liu, E. H.; Li, P. Characterization and classification of seven Citrus herbs by liquid chromatography–quadrupole time-of-flight mass spectrometry and genetic algorithm optimized support vector machines. J. Chromatogr. A, 2014, 1339, 118-127. (4) W. Bi, M. Tian and K. H. Row, Evaluation of alcohol-based deep eutectic solvent in extraction and determination of flavonoids with response surface methodology optimization. J. Chromatogr. A 1285 (2013) 22-30. (5) Abbott, A. P.; Capper, G.; Davies, D. L.; Rasheed, R. K.; Tambyrajah, V. Novel solvent properties of choline chloride/urea mixtures. Chem. Commun. 2003, 1, 70-71. (6) Dai, Y.; Witkamp, G. J.; Verpoorte, R.; Choi, Y. H. Tailoring properties of natural deep eutectic solvents with water to facilitate their applications. Food Chem. 2015, 187, 14-19. (7) Gu, T.; Zhang, M.; Tan; T.; Chen, J.; Li, Z.; Zhang, Q.; Qiu, H. Deep eutectic solvents as novel extraction media for phenolic compounds from model oil. Chem. Commun. 2014, 50, 11749-11752. 20


Page 20 of 33

Page 21 of 33

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(8) Jeong, K. M.; Zhao, J.; Jin, Y.; Heo, S. R.; Han, S. Y.; Yoo, D. E.; Lee, J. Highly efficient extraction of anthocyanins from grape skin using deep eutectic solvents as green and tunable media. Arch. Pharmacal Res. 2015, 38, 2143-2152. (9) Yilmaz, E.; Soylak, M. Ultrasound assisted-deep eutectic solvent extraction of iron from sheep, bovine and chicken liver samples. Talanta 2015, 136, 170-173. (10) Wagle, D. V.; Zhao, H.; Baker, G. A. Deep eutectic solvents: sustainable media for nanoscale and functional materials. Acc. Chem. Res. 2014, 47, 2299-2308. (11) Zhao, H. DNA stability in ionic liquids and deep eutectic solvents, J. Appl. Chem. Biotechnol. 2015, 90, 19-25. (12) Li, C.; Li, D.; Zou, S.; Li, Z.; Yin, J.; Wang, A.; Cui, Y.; Yao, Z.; Zhao, Q. Extraction desulfurization process of fuels with ammonium-based deep eutectic solvents. Green Chem. 2013, 15, 2793-2799. (13) Phadtare, S. B.; Shankarling, G. S. Halogenation reactions in biodegradable solvent: Efficient bromination of substituted 1-aminoanthra-9,10-quinone in deep eutectic solvent (choline chloride : urea). Green Chem. 2010, 12, 458-462. (14) Dai, Y.; Witkamp, G. J.; Verpoorte, R.; Choi, Y. H. Natural deep eutectic solvents as a new extraction media for phenolic metabolites in Carthamus tinctorius L. Anal. Chem. 2013, 85, 6272-6278. (15) Nam, M. W.; Zhao, J.; Lee, M. S.; Jeong, J. H.; Lee, J. Enhanced extraction of bioactive natural products using tailor-made deep eutectic solvents: Application to flavonoid extraction from Flos sophorae. Green Chem. 2015, 17, 1718-1727. (16) Xia, B.; Yan, D.; Bai, Y.; Xie, J.; Cao, Y.; Liao, D.; Lin, L. Determination of 21



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phenolic acids in Prunella vulgaris L.: a safe and green extraction method using alcohol-based deep eutectic solvents. Anal. Methods 2015, 7, 9354-9364. (17) Wei, Z. F.; Wang, X. Q.; Peng, X.; Wang, W.; Zhao, C. J.; Zu, Y. G.; Fu, Y. J. Fast and green extraction and separation of main bioactive flavonoids from Radix Scutellariae. Ind. Crops Prod. 2015, 63, 175-181. (18) Li, J.; Han, Z.; Zou, Y.; Yu, B. Efficient extraction of major catechins in Camellia sinensis leaves using green choline chloride-based deep eutectic solvents. RSC Adv. 2015, 5, 93937-93944. (19) Zhao, B. Y.; Xu, P.; Yang, F. X.; Wu, H.; Zong, M. H.; Lou, W. Y. Biocompatible Deep Eutectic Solvents Based on Choline Chloride: Characterization and Application to the Extraction of Rutin fromSophora japonica. ACS Sustainable Chem Eng. 2015, 3, 2746-2755. (20) Dai, Y.; van Spronsen, J.; Witkamp, G. J.; Verpoorte, R.; Choi, Y. H. Natural deep eutectic solvents as new potential media for green technology. Anal. Chim. Acta. 2013, 766, 61-68. (21) Song, S.; Qiu, M.; Chu, Y.; Chen, D.; Wang, X.; Su, A.; Wu, Z. Downregulation of Cellular c-Jun N-Terminal Protein Kinase and NF-κB Activation by Berberine May Result in Inhibition of Herpes Simplex Virus Replication. Antimicrob. Agents Chemother. 2014, 58, 5068-5078. (22) Liu, R.; Cao, Z.; Pan, Y.; Zhang, G.; Yang, P.; Guo, P.; Zhou, Q. Jatrorrhizine hydrochloride inhibits the proliferation and neovascularization of C8161 metastatic melanoma cells. Anti-Cancer Drugs 2013, 24, 667-676. 22


Page 22 of 33

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(23) Zhou, L.; Zuo, Z.; Chow, M. S. Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J. Clin. Pharmacol. 2005, 45, 1345-1359. (24) Liu, P.; Yang, H.; Long, F.; Hao, H.P.; Xu, X.; Liu, Y.; Shi, X.W.; Zhang, D.D. Zheng, H. C.; Wen, Q. Y.; Li, W. W.; Ji, H.; Jiang, X.J.; Zhang, B.L.; Qi, L.W.; Li, P. Bioactive Equivalence of Combinatorial Components Identified in Screening of an Herbal Medicine. Pharm. Res. 2014, 31, 1788-1800. (25) Azelmat, J.; Larente, J. F.; Grenier, D. The anthraquinone rhein exhibits synergistic antibacterial activity in association with metronidazole or natural compounds and attenuates virulence gene expression in Porphyromonas gingivalis. Arch. Oral Biol. 2015, 60, 342-346. (26) Wang, M.; Zhang, X. J.; Liu, F.; Hu, Y.; He, C.; Li, P.; Su, H.; Wan, J. B. Saponins isolated from the leaves of Panax notoginseng protect against alcoholic liver injury via inhibiting ethanol-induced oxidative stress and gut-derived endotoxin-mediated inflammation. J. Funct. Foods 2015, 19, 214-224. (27) Lee, C. H.; Kim, J. H. The anthraquinone rhein exhibits synergistic antibacterial activity in association with metronidazole or natural compounds and attenuates virulence gene expression in Porphyromonas gingivalis. J. Ginseng Res. 2014, 38, 161-166. (28) Makarova, M. N.; Pozharitskaya, O. N.; Shikov, A. N.; Tesakova, S. V.; Makarov, V. G.; Tikhonov, V. P. Effect of lipid-based suspension of Epimedium koreanum Nakai extract on sexual behavior in rats. J. Ethnopharmacol. 2007, 114, 412-416. 23



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(29) Xu, H. B. Huang, Z. Q. Icariin enhances endothelial nitric-oxide synthase expression on human endothelial cells in vitro. Vasc. Pharmacol. 2007, 47, 18-24. (30) Cheng, X. L.; Qi, L. W.; Wang, Q.; Liu, X. G.; Boubertakh, B.; Wan, J. Y.; Liu, E. H.; Li, P. Highly efficient sample preparation and quantification of constituents from traditional Chinese herbal medicines using matrix solid-phase dispersion extraction and UPLC-MS/MS. Analyst 2013, 138, 2279-2288.

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Figure captions Fig. 1. Chemical structures of different type of natural compounds. (1) Aloe-emodin; (2) Rhein; (3) Emodin; (4) Physcion; (5) chrysophanol; (6) ginsenoside Rg1; (7) notoginsenoside R1; (8) ginsenoside Rb1; (9) lithospermic acid; (10) rosmarinic acid; (11) salvianolic acid; B (12) jatrorrhizine hydrochloride; (13) palmatine hydrochloride; (14) berberine hydrochloride; (15) icariin. Fig. 2. Representative HPLC chromatogram of (a) BR, (b) SMR, (c) RRR, (d) NRR and (e) EF. (a1, jatrorrhizine hydrochloride; a2, palmatine hydrochloride; a3, berberine hydrochloride; b1, rosmarinic acid; b2, lithospermic acid; b3, salvianolic acid B; c1, aloe-emodin; c2, rhein; c3, emodin; c4, chrysophanol; c5, physcion; d1, ginsenoside RG1; d2, ginsenoside RB1; d3, notoginsenoside R1; e1, icariine) Fig. 3. Extraction yields (mg of extracted compounds per 100 g of HM powder) of DESs for (a) alkaloid, (b) phenolic acid, (c) anthraquinone, (d) saponin, (e) flavonoid. Numbers on horizontal axis are in accordance with the numbers in Table 1 and M represented MeOH. ChCl-based DESs were indicated by green bars, Bet-based DESs by orange bars and Pro-based DES by blue bars. Extraction efficiencies of DESs compared with MeOH were indicated with * (p MeOH) and # (p>0.05). Error bars indicate the SD (n=3). Fig. 4 Response surface plots of the model for extraction of alkaloids in BR.

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Tables Table 1 List of DESs initially tested for extraction. No.

Abbreviation

1

ChCl-Glu

HBA

D-Glucose

1 : 1 (2)

2

ChCl-Ma

Maltose

2 : 1 (4)

3

ChCl-Suc

Sucrose

4 : 1 (3)

4

ChCl-Xyl

Xylitol

5 : 2 (5)

5

ChCl-Sor

D-Sorbitol

3 : 1 (3)

6

ChCl-Eg

Ethylene Glycol

1:2

7

ChCl-Gly

Glycerol

1:2

8

ChCl-Ca

Citric Acid

2:1

9

ChCl-La

Laevulinic Acid

1:2

10

ChCl-Ox

Oxalic Acid

1:1

11

ChCl-Lac

Lactic Acid

1:1

12

ChCl-Maa

DL-Malic Acid

1:1

13

ChCl-Mal

Malonate

1 : 1 (1)

14

ChCl-Ur

Urea

1:2

15

ChCl-Mu

1-Methylurea

1 : 1 (1)

16

ChCl-Du

N,N'-Dimethylurea

1 : 1 (2)

17

ChCl-Am

Acetamide

1 : 1 (1) 1 : 1 (1)

Choline Chloride

HBD

Mole ratio

18

Bet-Glu

D-Glucose

19

Bet-Ma

Maltose

5 : 2 (6)

20

Bet-Suc

Sucrose

2 : 1 (7)

21

Bet-Xyl

Xylitol

1 : 1 (1)

22

Bet-Sor

D-Sorbitol

1 : 1.2 (3)

23

Bet-Eg

Ethylene Glycol

1 : 2 (1)

24

Bet-Gly

Glycerol

1:2

25

Bet-Ca

Citric Acid

2 : 1 (6)

26

Bet-La

Laevulinic Acid

1:2

Betaine

27

Bet-Lac

Lactic Acid

1 : 1 (1)

28

Bet-Maa

DL-Malic Acid

1 : 1 (1)

29

Bet-Ur

Urea

1 : 1 (2)

30

Bet-Mu

1-Methylurea

1 : 1 (2)

31

Pro-Glu

D-Glucose

1 : 1 (5)

32

Pro-Suc

Sucrose

2 : 1 (10)

33

Pro-Sor

D-Sorbitol

1 : 2 (4)

34

Pro-Gly

Glycerol

2:5

35

Pro-Ca

Citric Acid

1 : 1 (2)

Laevulinic Acid

1:2

Oxalic Acid

1:1

36

Pro-La

37

Pro-Ox

38

Pro-Lac

Lactic Acid

1:1

39

Pro-Maa

DL-Malic Acid

1 : 1 (1)

40

Pro-Mal

Malonate

1 : 1 (2)

41

Pro-Ur

Urea

1 : 1 (3)

42

Pro-Mu

1-Methylurea

1 : 1 (2)

43

Pro-Am

Acetamide

1 : 1 (2)

L-Proline

26


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Table 2 independent variables and levels used for BBD Level 0

No.

Variables

A

Water content (%)

10

30

50

B

Temperature (ºC)

40

50

60

-1

1

C

Time (min)

10

20

30

D

Solid/Liquid ratio (mg / mL)

20

40

60

27



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Figure graphics Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Table of Contents graphic

Comprehensive evaluation of deep eutectic solvents in extraction of bioactive natural products Li Duan, Li-Li Dou, Long Guo, Ping Li, E-Hu Liu

Synopsis: The potential and effectiveness of DESs for extraction of different types of natural compounds from biomass was evaluated.

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81x47mm (300 x 300 DPI)


Comprehensive Evaluation of Deep Eutectic Solvents in Extraction of

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