Recovery of Natural Products from Deep Eutectic Solvents by

Subscriber access provided by UNIV OF SOUTHERN INDIANA

Article

Recovery of natural products from deep eutectic solvents by mimicking denaturation Haiyuan Tian, Jiaqin Wang, Yujie Li, Wentao Bi, and David D. Y. Chen ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b01012 • Publication Date (Web): 09 May 2019 Downloaded from http://pubs.acs.org on May 10, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22 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

ACS Sustainable Chemistry & Engineering

Recovery of natural products from deep eutectic solvents by mimicking denaturation Haiyuan Tian†, Jiaqin Wang†, Yujie Li†, Wentao Bi†*, David Da Yong Chen†‡* † Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Jiangsu

Key Laboratory of Biomedical Materials, College of Chemistry and Materials Science, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, China ‡ Department of Chemistry, University of British Columbia, Vancouver, BC, Canada

V6T 1Z1

*Corresponding

author. Tel.:+86 25 85891705; fax:+86 25 85891707

E-mail address: [email protected], [email protected] 1

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 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

Abstract A strategy was developed for recovering natural products from deep eutectic solvents based on denaturation of the solvents. Method similar to nucleic acid denaturation was used to disrupt the intermolecular forces in the deep eutectic solvents to reduce their ability to solubilize and interact with the flavonoids at recovery. MIL100 (Cr) was added to the system for sorptive separation and recovery of the target compounds from the denaturized deep eutectic solvents. The factors affecting denaturation of the deep eutectic solvents, and adsorption of the target compounds were systematically investigated and optimized. The recovery method was further combined with mechanochemical extraction to establish a convenient and efficient protocol for obtaining natural products from plants. Moreover, due to the conjugated structure of MIL-100 (Cr), the adsorbed natural products could be directly analyzed by atmosphere matrix assisted laser desorption ionization-mass spectrometry (AP/MALDI-MS) without elution. This study provided a strategy for improving the efficiency of separation and recovery of natural products using deep eutectic solvents, and also established a protocol for obtaining or analyzing natural products in a green, efficient, and convenient manner. Keywords: Denaturation; deep eutectic solvents; recovery method; extraction; analytical method

2


Page 2 of 22

Page 3 of 22 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


Introduction Conventional organic solvents are widely used to extract bioactive natural compounds from plant materials in the fields of biochemistry, chemical engineering, and analytical chemistry. However, the large amounts of organic solvents increases the environmental burden and leave solvent residues in extracts.1 Since the discovery of deep eutectic solvents (DES) by Abbott et al.,2 these solvents have received much attention and have emerged as a new type of green and sustainable solvent.3, 4 DESs are generally prepared by mixing two or more inexpensive and biodegradable components (i.e., a hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA)) to obtain a eutectic mixture.5 Owing to the properties of negligible volatility, biodegradability, low cost, and environmental friendliness, DESs have a well-deserved reputation as a green alternative to traditional solvents.6, 7 With the emerging use of DESs to extract plant materials, the low vapor pressure of DESs has been viewed as a shortcoming because it makes substance separation and purification difficult, and is thus a serious hindrance to be used for analysis and industrial applications.8 Moreover, the higher the power of DESs to extract and solubilize a compound, the more difficult it is to recover the solute because the partition co-efficient would favor partitioning of the solutes into the DES phase.9,

10

The recovery of target compounds from DES solutions has been

demonstrated by two main approaches, i.e., solid-liquid extraction and precipitation. Nevertheless, systematic research is still lacking and there is no clear-cut strategy for recovering the solutes. Herein, we propose a strategy in which DES solutions are denaturized to minimize the solubility of the target compounds in the DESs, and suitable sorbents are added to recover the target compounds. Denaturization of the DESs is the critical step in this strategy. By looking into the intermolecular forces of DESs, it seems that the hydrogen bonds in DES are similar to those in nucleic acids. In other words, base pairs linked by hydrogen bonds in nucleic acids can also be considered as DESs. Therefore, the methods used to denature nucleic acids may also be applicable to disrupting the hydrogen bonds in DESs.11, 12 Several methods, including the addition of antisolvents, and adjustment of the temperature, ionic strength, and pH 3



could thus be used to weaken the molecular interactions between DESs and the compounds of interests while increasing the adsorption capacity of the sorbents for the target compounds. To establish application protocols of DESs for purposes of analytical chemistry, or potential industrial separation and purification, only recovery method was not enough, suitable and efficient extraction method should be applied and combined. As is well known, high density and viscosity are important disadvantages of DESs compared to those of conventional solvents, where these features are hindrances to extraction.13 Increasing the temperature is an easy way to overcome these difficulties, thus microwave-assisted extraction (MAE)14 and heat reflux extraction (HRE)15 with solvent are widely used to extract target compounds from plants. However, thermally unstable compounds may degrade in the extraction process. Mechanochemical extraction (MCE)16, 17 can be used to enhance ambient temperature extraction and is used herein. The MCE method in this experiment is performed with high-speed movement of ceramic spheres in a matrix tube containing the sample, so that the external force acting in multiple directions can simultaneously act on the cells and break the cell wall to allow the natural products in the vacuole to be fully released.18 At the same time, cellulose or lignin in the cell wall dissolves into the DESs, which further promotes extraction of the natural products by the DESs. More importantly, the extraction process can be completed within one minute at room temperature.19 Overall, compared to traditional extraction methods, the DESs-MCE process greatly shortens the experiment time, and the extraction efficiency is also significantly improved. The combination of DES denaturation and MCE methods can effectively overcome the shortcomings of DESs and provide a convenient and fast protocol for obtaining natural products from plants. To systematically investigate the factors affecting the denaturization of DESs and adsorption of the target compounds on the sorbent, five flavonoids, including myricetin (MYR), quercetin (QUE), naringenin (NAR), kaempferol (KAE), and isorhamnetin (ISO),20,

21

with antioxidant and anti-

inflammatory properties, are used the extraction and recovery process.22, 23 A metal4


Page 4 of 22

Page 5 of 22 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


organic framework (MIL-100 (Cr))24 material was used as the sorbent because it can provide various interactions such as hydrophobic, coordination, and π-π interactions with flavonoids over a large surface area.25 The feasibility of the proposed protocol is tested by extracting, separating, and recovering three kinds of flavonoids (QUE, KAE, and ISO) from Flos Sophorae.26, 27 Moreover, due to the special conjugated structure of MIL-100 (Cr), the flavonoids adsorbed on this material can be directly analyzed using atmospheric pressure matrix-assisted laser desorption ionization-mass spectrometry (AP/MALDI-MS) in a few seconds without further treatment. The overarching aim of this study is to find ways to increase the efficiency of separation and recovery of target compounds in DESs, and to establish better protocol for the extraction, separation, and recovery of natural products from plants in a sustainable manner, and simplify the sample pre-treatment step for analysis of plant materials.

Experimental Preparation of Standard Solutions The mixed standard containing five flavonoids (MYR, QUE, NAR, KAE and ISO) shown in Fig.S1 were prepared at a concentration of 0.1 mg mL−1, and were stored at 4 °C until use. All working solutions were prepared by diluting the stock solutions with DESs and water. Synthesis of DESs The synthesis of DESs is accomplished by stirring the hydrogen bond donor (malic acid, citric acid, malonic acid, methyl urea, N, N-dimethylurea, urea, ethylene glycol, glycerol and 1,3-butanediol) and the hydrogen bond acceptor (choline chloride) at a certain molar ratio (1:1, 3:1) at 80.0 °C for half an hour. The prepared DESs in this study were listed in Table 1. Table 1 Abbreviations and components of DESs used in the experiments Abbreviation

HBA

HBD

Mole ratio

DESs-1

Malic acid

1:1,1:3

DESs-2

Citric acid

1:1,1:3

5



DESs-3

Page 6 of 22

Malonic acid

1:1,1:3

DESs-4

Choline

Methylurea

1:1,1:3

DESs-5

chloride

Urea

1:1,1:3

DESs-6

N,N-dimethylurea

1:1,1:3

DESs-7

1,3-butanediol

1:1,1:3

DESs-8

Ethylene glycol

1:1,1:3

DESs-9

Glycerol

1:1,1:3

Synthesis of MIL-100(Cr) MIL-100(Cr) was prepared in a Teflon-lined autoclave (100 mL) by mixing 0.5 g of CrO3, 1.05 g of trimesic acid, and 1.0 mL of a 5 M hydrofluoric acid in 24.0 mL of deionized water. After stirring for half an hour, the slurry was heated in Teflon-lined autoclave (100 mL) at 493 K for 96 h. Then, the product was cooled to room temperature and collected by filtration, washed with deionized water and ethanol, finally dried at room temperature. The detailed characterizations and explanations were listed in Supporting Information (Fig. S2). Multicomponent adsorption of flavonoids The sorbents (2.0 mg) were added into 1.0 mL of the DES solutions (20 μg mL-1 of flavonoids) with 10 %, 30 %, 50 %, 70 % and 90 % content of DESs at room temperature （ no precipitation occurs ） .The suspension was subjected to ultrasonication (20 min), vortex (2 min), and centrifugation (15 min). The concentrations of flavonoids in supernatants were determined, and the adsorbed amount calculated using the following equation:

𝑄𝑒 =

( 𝐶𝑜 ― 𝐶𝑒) 𝑉 𝑀

(1)

where 𝑄𝑒 is adsorbed amount of target compounds on sorbent (mg g−1), C0 and Ce are initial and equilibrium concentrations of the flavonoids (mg mL-1), M is the mass of sorbent (g), and V is the volume of solution (mL). To study the influence of pH during 6


Page 7 of 22 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


the adsorption, the pH of solution was adjust by 0.05 M acetic acid or triethylamine aqueous solution. Extract, recover and analyse flavonoids from Flos Sophorae As shown in Fig. 1, the dried Flos Sophorae was smashed by a pulveriser (FW100, Taisite Instrument Co., Ltd., China). The Flos Sophorae powder (200.0 mg) and DES solution (1.0 mL) (DES-8, HBA/HBD = 1:1, 60 %) were mixed in 2.0 mL Lysing Matrix D tubes (with 1.4 mm ceramic spheres, 1.1 g). MCE was completed in a short time by a tissue-and-cell homogenizer (FastPrep-24, MP Biomedicals LLC, USA). After MCE, the extract was centrifuged to obtain the solution containing the targeted compounds. The extract was then diluted to 10 % DES solution to break the hydrogen bonds in the DESs. Then MIL-100 (Cr) (24.0 mg) was added, and ultrasonication (1.0 min) (Scientz Ultrasonic Cleaner SB-3200DTN, Ningbo Co., Ltd., China) and vortex (5.0 min) (The US SCILOGEX Cyrus Czech MX-S Adjustable Mixer) were used for assisting adsorption at room temperature with adding 15 % (w/v) of NaCl. The sorbents were separated from the extract by centrifugation (TGL-16M desktop high speed refrigerated centrifuge, Xiangli Centrifuge Co., Ltd., China). The sorbents were then transferred into a tube and dried in oven, and the adsorbed target compounds were eluted with 0.4 mL of solvent with ultrasonication. The final eluent was separated from the sorbents by centrifugation. Finally, the eluent was analysed by UPLC (Thermo UltiMate 3000 Series). In addition, the target compounds adsorbed on MIL-100 (Cr) can be directly analyzed by AP/MALDI-MS without further treatment.

Fig. 1 Schemes of separation and recovery of flavonoids from DES solutions, and analysis by AP/MALDI-MS.

7



Operating conditions of UPLC and AP/MALDI-MS for analysis of Natural Products All liquid samples are analyzed using a UPLC system (Thermo UltiMate 3000 Series) with a WPS-3000TRS autosampler, a HPG- 3400RS pump, and a multiple wavelength diode array detector. A commercial C18 column (2.1 mm*100 mm, 1.9 μm) purchased from Thermo Fisher Scientific, Inc. (Shanghai, China) is used for this work. The mobile phase was acetonitrile/water/acetic acid = 70/30/0.1 (v/v/v) at 0.35 mL min-1. UV absorbance detection was performed at wavelengths of 254 and 268 nm. Instrument control and data processing were performed using Chromeleon 7. An AP/MALDI (ng) ion source (MassTech, Columbia, MD) was coupled with an Orbitrap Fusion Lumos (Thermo Fisher Scientific, San Jose, CA) mass spectrometer. The MS was operated with full-scan mode, automatic gain control (AGC) target set at 2e5, m/z range of 100-800, resolution of 120000, and maximum injection time of 100 ms. Laser energy was 30% and repetition rate was 3000 Hz. The target plate voltage was set at 3000 V. The mass spectrometer was controlled by Orbitrap Fusion Lumos 2.0 Tune (Thermo Fisher Scientific, USA). The ion transfer tube temperature was set at 300 °C. Finally, data is processed using Xcalibur software (Thermo Fisher Scientific, USA; the m/z of Quercetin is 301.24, Kaempferol is 285.24, Isorhamnetin is 315.26).

Results and discussion Denaturation of DESs for adsorption It is known that the viscosity, polarity, and hydrogen bond strength of various DESs are quite different. Therefore, the conditions for denaturing the DESs and adsorption of the target compounds by the sorbent must be systematically investigated and optimized. Effect of addition of antisolvents: Three types of DESs were tested in this research, namely acid-based, amine-based, and alcohol-based DESs (referring to the HBD type), in which the HBA was choline chloride. According to previous research, the addition of antisolvents (ethanol or water) is the most efficient and effective method of denaturing DESs. However, in compliance with Fig. 2, water and DESs formed homogeneous liquid phase when water and DESs were mixed, while ethanol cannot mix with acid-based or amine-based DESs forming 8


Page 8 of 22

Page 9 of 22 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


stratification. In other words, water can be used to break the hydrogen bonds of acidbased, amine-based, and alcohol-based DESs, while ethanol can only be used for alcohol-based DESs because ethanol is not sufficiently polar to efficiently break the hydrogen bonds in acid-based and amine-based DESs. This is because water can form hydrogen bonds with solute proton donors and acceptors, thus limits the formation of solute intermolecular or intramolecular hydrogen bonds. Consequently, hydrogen bonds between or within solute molecules dissolved in water are almost unfavorable relative to hydrogen bonds between water and the donors and acceptors for hydrogen bonds on those solutes.28 To better investigate the effect of antisolvents on nature of DESs, as shown in Fig.S3, different contents of DESs in water and ethanol were characterized by ultraviolet-visible (UV-Vis) spectroscopy. With the increase of water and ethanol content in DESs, the UV-Vis spectrum shifted and some characteristic peaks disappeared. This phenomena can be obviously observed in acid-based and amine-based DESs, indicating the change of structures and properties of DESs. Water with better performance was thus selected as the antisolvent to break the hydrogen bonds and denaturize the DESs.

Fig. 2 Solubility of different DESs in ethanol and water: (a) acid-based DESs in ethanol; (b) amine-based DESs in ethanol; (c) alcohol-based DESs in ethanol; (d) acid-based DESs in water; (e) amine-based DESs in water; (f) alcohol-based DESs in water.

In the case of the acid-based DESs, the amount of flavonoids adsorbed on MIL-100 (Cr) increased when water was added to the DES solutions. This was attributed to denaturation of the DESs via rupture of the hydrogen bonds in the DESs by adding water, leading to the solubility of the flavonoids in the DES solutions to decrease.28 Due to the hydrophobic, coordination, and π-π interactions between the flavonoids and MIL9



100 (Cr), the flavonoids were successfully adsorbed by the sorbent. The highest amount of flavonoids adsorbed on MIL-100 (Cr) was obtained when the content of water in the DES solutions was increased to 90 %. This is because water can function as a HBD instead of destroying the hydrogen bonds when the content of water is low. DES denaturation can only be achieved when the content of water is increased to a higher level. However, as shown in Fig. 3a-c, it was observed that the amount of flavonoids adsorbed in the acid-based DES solutions with a HBA:HBD molar ratio of 1:3 decreased slightly compared to that in the solvents with HBA: HBD molar ratios of 1:1, and among the acid-based DESs, optimal adsorption was observed in DES-3 with the most weakly acidic HBD. This may be because the ionized acid HBDs can inhibit ionization of the flavonoids, resulting in increased adsorption, but the acids would also prevent the interactions, especially the coordination interaction, between the flavonoids and MIL-100 (Cr).29 The results show that too much acidity has a negative effect on adsorption of the flavonoids. Fig.3d-f indicated that similar to the case of the acid-based DESs, the amount of flavonoids adsorbed on MIL-100 (Cr) in the amine-based DESs was optimal when the content of water in the DES solutions increased to 90%, and the adsorption in the amine-based DESs with a HBA: HBD molar ratio of 1:1 was superior to that in those with a HBA:HBD molar ratio of 1:3. Interestingly, however, when water was added to amine-based DESs, the hydrogen bonds between the HBDs and HBAs were interrupted, and ionization of the HBDs increased, thereby promoting the ionization and solubility of the flavonoids in the DES solutions, resulting in a slight decrease in the adsorption compared to that in the acid-based DESs. Therefore, the more amine HBDs, the higher the distribution coefficient of the flavonoids in the DES solutions. In Fig.3g-i, the adsorption performance in the alcohol-based DESs was better than that in the acid-based DESs and amine-based DESs because the HBDs in water did not ionize and thus influence ionization of the flavonoids and the interactions between the target compounds and sorbent. Unexpected trends were observed, wherein the amount of adsorbed flavonoids initially increased and then decreased when the content of water in the DESs reached 30−70 %, and finally increased to the highest adsorbed amount at 10


Page 10 of 22

Page 11 of 22 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


90 %. This trend was attributed to a decrease in the viscosity of the alcohol-based DESs when water was added to the DESs, which increases diffusion of the flavonoids in the DES solutions, resulting in higher adsorption. Further increasing the water content caused the solvent polarity to increase, thus enhancing the solubility of the flavonoids and decreasing adsorption. Finally, when the alcohol-based DESs were fully denaturized at 90 % water content, the flavonoids were maximally adsorbed on MIL100 (Cr). Little effect of the HBA: HBD ratio on adsorption was observed in 10 % DES solutions, confirming our previous explanation of the behavior of acid-based DESs and amine-based DESs. Moreover, the amount of flavonoids adsorbed in DES-7 and DES8 was higher than that in DES-9 due to the fewer hydroxide groups in the latter, which enhanced denaturization of the DESs. Based on thorough consideration, DES-8 (HBA/HBD=1/1, v/v) exhibited the best adsorption performance and was selected for further experiments.

11



Fig. 3 Adsorbed amounts of flavonoids on MIL-100(Cr) in different DESs: a-c acidbased (a) Choline chloride : Malic acid=1:1 (left) and 1:3 (right) ; (b) Choline chloride : Citric acid=1:1 (left) and 1:3 (right); (c) Choline chloride : Malonic acid=1:1 (left) and 1:3 (right)); d-f amine-based ((d) Choline chloride : Methylurea=1:1 (left) and 1:3 (right) ; (e) Choline chloride : Urea =1:1 (left) and 1:3 (right); (f) Choline chloride : N,N-dimethylurea=1:1 (left) and 1:3 (right)); g-I alcohol-based (g) Choline chloride : 1,3-butanediol =1:1 (left) and 1:3 (right) ; (h) Choline chloride : Ethylene glycol=1:1 (left) and 1:3 (right); (i) Choline chloride : Glycerol=1:1 (left) and 1:3 (right). Effect of pH: Because nucleic acids can be denatured by adjusting the pH, temperature, and salinity of the solution, these factors may also denature DESs. When acids or amines are added to the DES solution to adjust the pH from 1 to 11, they play a role similar to that of the acid or amine HBDs, as previously discussed. Compared with the 30, 50, 70, and 90% DES solutions, the amount of flavonoids adsorbed in the 10% DES solution was the highest (Fig. 4a and S4). With an increase in the water content of the DES solution, the adsorbed amount increased, which proves that the effect of adding water is stronger than that of changing pH. Among the five flavonoids, MYR was more strongly affected by the pH. This may be due to the fact that MYR contains more hydroxyl groups than the other flavonoids. Therefore, the existence of phenolic hydroxyl groups in the solution and the interactions between the phenolic hydroxyl groups and sorbents were greatly affected by the pH. In the solution of 10 % DES, adding acid and alkali will reduce the amount of flavonoids adsorbed on the sorbent. The adsorbed amount was highest at pH=5, which is the pH of 10 % DES solution without the addition of acid or base. Therefore, to obtain the maximum adsorption capacity, there is no need to adjust the pH value of the 10% DES solution. When the pH is decreased to 1, the peracidic environment may lead to structural damage of MIL-100 (Cr), resulting in a sharp decrease in the adsorption capacity.

12


Page 12 of 22

Page 13 of 22 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


Effect of temperature: Temperature is also an important factor affecting denaturization of the DESs and adsorption of the flavonoids on the sorbent. Typically, high temperature will weaken or destroy the hydrogen bonds of the DES and increase the rate of diffusion of the flavonoids, leading to an increase in the adsorption capacity of the sorbents for the flavonoids.30,

31

However, high temperature would weaken most of the interactions

between the flavonoids and MIL-100 (Cr), thus reducing the capacity for adsorption of the flavonoids on the sorbents. Therefore, choosing a suitable temperature is very important for improving the adsorption capacity. As can be seen from the Fig. 4b and S5, except for NAR, the maximum adsorption of most of the flavonoids was achieved at 20 ℃. The exception for NAR is mainly because NAR is more hydrophobic than the other flavonoids, which leads to more hydrophobic interaction between NAR and the sorbent with increasing temperature. Comprehensive consideration of the adsorbed amount of the five flavonoids shows that 20 ℃ was optimal under the conditions of this study. Effect of salinity: The effect of salinity on denaturation of the DES and adsorption of the flavonoid cannot be neglected. To investigate the effect of salinity, different concentrations of NaCl ranging from 0 to 25.0 % w/v were added. However, the solubility of NaCl in most DES solutions is low, thus only the 10, 30, and 50% DES solutions were studied. Generally, adding NaCl can increase the polarity of the solution and weaken the hydrogen bonding effect. In addition, chloride would also generate hydrogen bonds with HBD, further reducing its hydrogen bond interaction with HBA. Besides, the addition of NaCl can provide a salting-out effect,32 which can decrease the solubility of the target substances in the solutions and increase adsorption of the flavonoids on MIL100 (Cr). However, the only disadvantage is that too much salt will increase the viscosity of the solution and reduce the rate of diffusion of the target substance, and thus reduce the adsorption efficiency. It can be seen from the Fig. 4c and S6 that in the solutions with a high DES content, the effect of adding salt was not obvious, mainly because salt addition led to high viscosity and affected the adsorption efficiency. In the 10% DES solution, the effect of adding NaCl was more obvious. The optimum concentration of salt was 20%. However, considering the small increase compared with 13



15% (w/v) salt, 15% (w/v) salt was selected for subsequent research from the economic point of view.

Fig. 4 Effect of pH (temperature=20 ℃; salinity=0 %) (a), temperature (pH=5; salinity=0 %) (b), salinity (pH=5; temperature=20 oC) (c) on the adsorbed amounts of flavonoids on MIL-100 (Cr) (concentration of DES-8=10%). In short, increase of the adsorption capacity of flavonoids on MIL-100 (Cr) can be achieved by reducing the solubility of the target compounds in DESs and enhancing the interactions between the target compounds and sorbent. DESs can be denatured by the addition of water and adjustment of the pH, temperature, and salinity, leading to decrease of solubility. The main interactions between the target compounds and the sorbent were hydrophobic, coordination, and π-π interactions. Methods should be used to increase the interactions. However, the factors affecting the interactions were complex. For example, the addition of water and adjustment of the pH can affect the state of the target compounds and the strength of hydrogen bond and coordination bond. High temperature can reduce physical adsorption, decrease coordination, and π-π interactions, but enhance hydrophobic interaction. Salinity can enhance hydrophobic interaction but reduce diffusion rate. Therefore, it is necessary to optimize the conditions for different target compounds to achieve the maximum adsorption.

Extraction, separation, and recovery of flavonoids from Flos Sophorae To verify the proposed separation and recovery method based on DES denaturation, this method was combined with the MCE method to develop a protocol for obtaining natural products from plants. In the DESs-MCE process, the amount of water added to the DESs, sample/solvent ratio, vibration speed, and time all have influence on the 14


Page 14 of 22

Page 15 of 22 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


extraction efficiency. Therefore, these factors were systematically investigated and optimized.

Extraction of flavonoids from Flos Sophorae: The amount of water added to the DESs had a major influence on the viscosity and polarity, and thus affected the extraction efficiency of the target compounds. As shown in the Fig. S7a, the amount of flavonoids extracted increased with increasing addition of water, reaching the highest at 60 % DES-8 (HBA/HBD = 1/1, v/v). This is mainly due to the fact that 60 % DES can provide more suitable viscosity and polarity. The sample/solvent ratio is also an important factor affecting the extraction of flavonoids. In this experiment, eight ratios (0.01, 0.05, 0.1, 0.12, 0.15, 0.18, 0.2, 0.25 g mL−1) were tested. In Fig. S7b, the sample/solvent ratio had little effect on KAE and ISO, but had an appreciable impact on QUE. The extracted amount increased significantly from 0.01 to 0.2 g mL−1, then decreased at 0.25 g mL−1. Therefore, 0.2 g mL−1 was chosen for the following experiments. The MCE vibration speed and time also played important roles in the extraction. In general, a higher vibration speed and longer extraction time should result in better extraction efficiency. However, with increasing vibration speed and extraction time, the amount of flavonoids extracted initially increased and then decreased. This phenomenon was attributed to the principle of MCE. The natural products in cells can be released by the DESs-MCE method through rupture of the cell wall under the action of multiple external forces and dissolution of the cell wall by the DESs. Meanwhile, the solubility of impurities such as cellulose and lignin in the DES solution would increase at higher vibration speed and with longer extraction times, resulting in a decrease in the solubility of the target substance. Therefore, 5 m s−1 is a suitable vibration speed (Fig. S7c), and the best duration was 40 s (Fig. S7d). To verify the advantages of the DESs as an extractant, methanol, water, n-hexane, isopropyl alcohol, ethyl alcohol, acetone, and 60 % (HBA/HBD = 1/1, v/v) were compared under the same conditions. As expounded in Table S1, the highest extraction was achieved with 60 % DES-8, and the results proved that the DES was the best solvent for use with 15



MCE due to its ability to dissolve cellulose and lignin. In addition, ultrasound-assisted extraction (UAE) and HRE were used for extraction of the flavonoids from Flos Sophorae for comparison with MCE. In Table S2, the amount of flavonoids extracted by DESs-MCE in 40 s was several times higher than that extracted with UAE and HRE. A time of 1800 s was required for the traditional extraction methods to achieve the same effect as obtained with DESs-MCE, demonstrating that the DESs-MCE method is more eco-friendly and more efficient than the conventional methods.

Separation of flavonoids from DES extract: To recover the flavonoids from the DES extract, the DES was diluted with water (from 60 % to 10 %) with the addition of 15% of NaCl, as investigated previously, and MIL100 (Cr) was then added for adsorption of the target compounds. The amount of MIL100 (Cr) used for adsorption was a significant factor because too little sorbent leads to incomplete adsorption of the targets, whereas too much result in waste of resource. Thus, the sorbent/liquid ratio was varied from 2 to 30 mg mL−1 and the efficacy was evaluated based on the adsorption rate (the amount of target compounds adsorbed was divided by the total content of the target compounds in the extract). Fig. S8 shows that the adsorption rate tended to become constant with the use of a sorbent/liquid ratio of 24 mg mL−1. Therefore, 24 mg mL−1 was sufficient for adsorption of the flavonoids from the extract. The adsorption time, which consisted of the time for vortexing and ultrasonication, played an important role in the experiment. Ultrasound was used to disperse the sorbent, whereas vortexing was used to rapidly mix the sorbent and DES solution. As indicated in Fig. S9, ultrasonication for 1.0 min was enough as the ultrasonication time had little effect on adsorption. The vortex time was then optimized, indicating that the adsorption rate slowly increased with vortexing from 10 s to 300 s and then tended to become constant; thus, 300 s was chosen. Control tests were also made to better demonstrate the effect of ultrasonic and vortex time on the adsorption rate (Fig. S9). Similar trends were observed, but the adsorption rates were slightly lower than that treated by vortexing and ultrasonication together. Therefore, the optimal 16


Page 16 of 22

Page 17 of 22 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


adsorption time was 1.0 min of ultrasonication and 300 s of vortex.

Recovery of flavonoids: The last and most important step after adsorption of the flavonoids from the extract is to elute the adsorbed targets. It was essential to optimize the type of eluent, elution volume, and time to achieve the best performance. Seven solvents and their mixtures were compared in Fig. S10a. The highest recovery was obtained with methanol, possibly because the polarity of methanol is similar to that of the flavonoids. Ethanol and methanol have similar elution ability, so ethanol can also be used as an alternative elution solvent. The volume of methanol was then optimized as this may influence the elution efficiency. Increasing the eluent volume in the range of 200−600 μL resulted in an increase in the recovery (Fig. S10b). Upon further increasing the eluent volume, the recovery remained unchanged. Considering that increasing from 400 to 600 μL could not effectively improve the recovery, 400 μL was chosen as the most appropriate elution volume. When the elution time was optimized, it was found that the ultrasonication time had little effect on the elution; thus, 1.0 min was enough for elution (Fig. S11). In summary, under the optimal conditions, the recovery of QUE, KAE and ISO was 72.7 %, 89.2 % and 90.3 %, respectively. Because the QUE content in the Flos Sophorae is much higher than that of KAE and ISO, the choice of solid/liquid ratio at 24 mg/ml for economic consideration will result in a relatively low QUE recovery. Finally, the reuse of MIL-100 (Cr) was also investigated. As shown in Fig. S12, the efficiency has not changed much after five reuses, which proves that the material can be reused to reduce cost and waste.

Qualitative analysis of flavonoids by AP/MALDI-MS Generally, the flavonoids adsorbed on sorbent should be eluted with a solvent for UPLC or UPLC-MS analysis. However, elution and UPLC (or UPLC-MS) analysis still require the consumption of organic solvents. To avoid the use of organic solvents, ambient ionization mass spectrometry (AIMS) can be used to analyze the target 17



compounds if only for the purpose of analysis. Because of the conjugate structure of MIL-100 (Cr), it can absorb light in the ultraviolet region and transmit it to the targets adsorbed on it, thus ionizing the targets. AP/MALDI-MS, which requires an analytical time of only seconds, was used for analysis of the flavonoids adsorbed on MIL-100 (Cr) without consumption of any solvent. As shown in Fig. S13, three ions of the flavonoids with m/z values of 301.24, 286.24, and 316.26 were observed, and were assigned as QUE, KAE, and ISO ions, respectively. Moreover, only a very weak signal of choline chloride was detected, proving that adsorption of the DES on the sorbent was very weak. This work provides possible blueprint for the establishment of a new, fast, and efficient green analytical protocol based on the combination of DESs and materials, and further research is underway.

Conclusions DESs were successfully denatured by the addition of water and adjustment of the pH, temperature, and salinity, leading to improved adsorption of flavonoids on MIL-100 (Cr). It was clearly revealed in Fig.1 that the developed separation and recovery method was combined with MCE to establish a convenient and efficient protocol for obtaining natural products from plants. Moreover, by combination with AP/MALDI-MS, the established protocol can be used as a green, fast, and convenient analytical method. This research shows the efficient separation and recovery of target compounds from DESs and provides better understanding of the interactions between DESs. The conception of this study also establishes a link between nucleic acid and DESs, which may break the barriers of solvent molecules and biomolecules.

Supporting Information Chemicals, materials, and instruments used in this experiment; characterization of MIL100 (Cr); UV-Vis spectrograms of different contents of DESs in ethanol and water; investigation and optimization of factors affecting adsorption, extraction, separation 18


Page 18 of 22

Page 19 of 22 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


and recovery; qualitative analysis by AP/MALDI-MS; comparison of solvents and extraction methods.

Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant No. 21507062 and 21475061), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry ([2015]1098),Program of Natural Science Research of Jiangsu Higher Education Institutions of China (Grant No. 16KJB150022), and the Priority Academic Program Development of Jiangsu Higher Education Institutions, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials. References 1.

2.

3.

4.

5. 6.

7.

8.

Pena-Pereira, F.; Kloskowski, A.; Namieśnik, J. Perspectives on the Replacement of Harmful Organic Solvents in Analytical Methodologies: A Framework Toward the Implementation of a Generation of Eco‐Friendly Alternatives. Green Chem. 2015, 17, 3687-3705, DOI 10.1039/c5gc00611b. Abbott, A. P.; Capper, G.; Davies, D. L.; Rasheed R. K.; Tambyrajah, V. Novel solvent properties of choline chloride/urea mixtures. Chem. Comm. 2003, 7, 7071, DOI 10.1039/b210714g. Hammond, O. S.; Edler, K. J.; Bowron, D. T.; Torrente-Murciano, L. Deep eutectic-solvothermal synthesis of nanostructured ceria. Nature Comm. 2017, 8, 14150, DOI: 10.1038/ncomms14150. Kollau, L. J. B. M.; Vis, M.; van den Bruinhorst, A.; Esteves, A. C. C.; Tuinier, R. Quantification of the liquid window of deep eutectic solvents. Chem. Comm. 2018, 54, 13351-13354, DOI 10.1039/c8cc05815f Li, X.; Row, K. H. Development of deep eutectic solvents applied in extraction and separation. J. Sep. Sci. 2016, 39, 3505-3520, DOI 10.1002/jssc.201600633. Smith, E. L.; Abbott, A. P.; Ryder, K. S. Deep eutectic solvents (DESs) and their applications. Chem. Rev. 2014, 114, 11060-11082, DOI 10.1021/cr300162p. Duan, L.; Dou, L. L.; Guo, L.; Li, P.; Liu, E. H. Comprehensive evaluation of deep eutectic solvents in extraction of bioactive natural products. ACS Sustain. Chem. Eng. 2016, 4, 2405-2411, DOI 10.1021/acssuschemeng.6b00091. Shahbaz, K.; Mjalli, F. S.; Vakili-Nezhaad, G.; AlNashef, I. M.; Asadov, A.; Farid, M. M. Thermogravimetric measurement of deep eutectic solvents vapor pressure. J.Mol. Liq. 2016, 222, 61-66, DOI 10.1016/j.molliq.2016.06.106. 19



9.

10.

11. 12. 13.

14.

15.

16.

17.

18.

19.

20.

21.

Tang, X.; Zuo, M.; Li, Z.; Liu, H.; Xiong, C.; Zeng, X.; Sun, Y.; Hu, L.; Liu, S.; Lei, T.; Lin, L. Green Processing of Lignocellulosic Biomass and Its Derivatives in Deep Eutectic Solvents. ChemSusChem 2017, 10, 2696-2706, DOI 10.1002/cssc.201700457. Zhao, B.; Xu, P.; Yang, F.; Wu, H.; Zong, M.; Lou, W. Biocompatible deep eutectic solvents based on choline chloride: characterization and application to the extraction of rutin from sophora japonica. ACS Sustain. Chem. Eng. 2015, 3, 2746–2755, DOI 10.1021/acssuschemeng.5b00619. Wang, X.; Lim, H. J.; Son, A. Characterization of denaturation and renaturation of DNA for DNA hybridization. Environ. Health Toxicol. 2014, 29, e2014007. Marmur, J.; Ts'o, P.O.P.; Denaturation of deoxyribonucleic acid by formamide. Biochim. Biophys. Acta 1961, 51, 32-36, DOI 10.1016/0006-3002(61)91013-7. Ghaedi, H.; Ayoub, M.; Sufian, S.; Shariff, A. M.; Lal, B. The study on temperature dependence of viscosity and surface tension of several Phosphonium-based deep eutectic solvents. J. Mol. Liq. 2017, 241, 500-510, DOI 10.1016/j.molliq.2017.06.024. Chen, Z.; Wan, C. Ultrafast fractionation of lignocellulosic biomass by microwave-assisted deep eutectic solvent pretreatment. Bioresour. Technol. 2018, 250, 532-537, DOI 10.1016/j.biortech.2017.11.066. Vieira, V.; Prieto, M. A.; Barros, L.; Coutinho, J. A. P.; Ferreira, I. C. F. R.; Ferreira, O. Catechin-based extract optimization obtained from Arbutus unedo L. fruits using maceration/microwave/ultrasound extraction techniques. Ind. Crops. Prod. 2018, 115, 261-271, DOI 10.1016/j.indcrop.2016.10.050. Wang, M.; Wang, J.; Zhang, Y.; Xia, Q.; Bi, W.; Yang, X.; Chen, D. D. Y. Fast environment-friendly ball mill-assisted deep eutectic solvent-based extraction of natural products. J. Chromatogr. A 2016, 1443, 262-266, DOI 10.1016/j.chroma.2016.03.061. Wang, M.; Bi, W.; Huang X.; Chen, D. D. Y. Ball mill assisted rapid mechanochemical extraction method for natural products from plants. J. Chromatogr. A 2016, 1449, 8-16, DOI 10.1016/j.chroma.2016.04.044. Wang, M.; Wang, J.; Zhou, Y.; Zhang, M.; Xia, Q.; Bi, W.; Chen, D. D. Y. Ecofriendly mechanochemical extraction of bioactive compounds from plants with deep eutectic solvents. ACS Sustain. Chem. Eng., 2017, 5, 6297-6303, DOI 10.1021/acssuschemeng.7b01378. Wang, J.; Zhou, Y.; Wang, M.; Bi, W.; Li, H.; Chen, D. D. Y. High-throughput analysis for artemisinins with deep eutectic solvents mechanochemical extraction and direct analysis in real time mass spectrometry. Anal. Chem. 2018, 90, 3109-3117, DOI 10.1021/acs.analchem.7b04060. Wang, R.; Chang, Y.; Tan, Z.; Li, F. A novel combined process for extracting, separating and recovering flavonoids from flos sophorae immaturus. Sep. Purif. Technol. 2017, 172, 422-432, DOI 10.1016/j.seppur.2016.08.038. Xie, Z.; Lam, S.; Wu, J.; Yang, D.; Xu, X. Chemical fingerprint and simultaneous determination of flavonoids in Flos Sophorae Immaturus by 20


Page 20 of 22

Page 21 of 22 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


22.

23.

24.

25.

26.

27.

28. 29.

30.

31.

32.

HPLC-DAD and HPLC-DAD-ESI-MS/MS combined with chemometrics analysis. Anal. Methods 2014, 6, 4328-4335, DOI 10.1039/c4ay00289j. Wu, H.; Chen, M.; Fan, Y.; Elsebaei, F.; Zhu, Y. Determination of rutin and quercetin in Chinese herbal medicine by ionic liquid-based pressurized liquid extraction–liquid chromatography–chemiluminescence detection. Talanta 2012, 88, 222-229, DOI 10.1016/j.talanta.2011.10.036. Gullón, B.; T. Lú-Chau, T. A.; Moreira, M. T.; Lema, J. M.; Eibes, G. Rutin: A review on extraction, identification and purification methods, biological activities and approaches to enhance its bioavailability. Trends Food Sci. Tech. 2017, 67, 220-235, DOI 10.1016/j.tifs.2017.07.008. Palomino Cabello, C.; Gómez-Pozuelo, G.; Opanasenko, M.; Nachtigall, P.; Čejka, J. Metal–Organic Frameworks M‐MOF‐74 and M‐MIL‐100: comparison of textural, acidic, and catalytic properties. ChemPlusChem 2016, 81, 828-835, DOI 10.1002/cplu.201600168. Sun, D. T.; Gasilova, N.; Yang, S.; Oveisi, E.; Queen, W. L. Rapid, selective extraction of trace amounts of gold from complex water mixtures with a metal– organic framework (mof)/polymer composite. J. Am Chem. Soc. 2018, 140, 16697-16703, DOI 10.1021/jacs.8b09555. 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, DOI 10.1039/c4gc01556h. Mou, Q.; He, J.; Yin, R.; Yang, B.; Fu, M.; Fang, J.; Li, H. Response surface optimized infrared-assisted extraction and UHPLC determination of flavonoid types from Flos Sophorae. Molecules 2017, 22, E1000, DOI 10.3390/molecules22061000. Stahl, Neil.; Jencks, W. P. Hydrogen bonding between solutes in aqueous solution. J. Am. Chem. Soc. 1986, 108, 4196-4205, DOI: 10.1021/ja00274a058. Scordino, M.; Di Mauro, A.; Passerini, A.; Maccarone, E. Adsorption of flavonoids on resins: hesperidin. J. Agric. Food. Chem. 2003, 51(24), 69987004, DOI 10.1021/jf034496q. Zhang, Y.; Gao, B.; Xu, Z. Adsorption properties of polyvinyl-alcohol-grafted particles toward genistein driven by hydrogen-bond interaction. J. Phys. Chem. B 2013, 117(18), 5730-5736, DOI 10.1021/jp4004938. Lv, C.; Yang, J.; Liu, R.; Lu, Q.; Ding, Y.; Zhang, J.; Deng, J. A comparative study on the adsorption and desorption characteristics of flavonoids from honey by six resins. Food Chem. 2018, 268, 424-430, DOI 10.1016/j.foodchem.2018.06.100. Wang, J.; Zhu, J.; Si, L.; Du, Q.; Li, H.; Bi, W.; Chen, D. D. Y. High throughput screening of phenoxy carboxylic acids with dispersive solid phase extraction followed by direct analysis in real time mass spectrometry. Anal. Chim. Acta 2017, 996, 20-28, DOI 10.1016/j.aca.2017.10.007.

21



For TOC only

Synopsis: A strategy was developed for recovering natural products from deep eutectic solvents based on denaturation of the solvents.

22


Page 22 of 22

Recovery of Natural Products from Deep Eutectic Solvents by

Recommend Documents