Green and efficient extraction of four bioactive flavonoids from Pollen Typhae by ultrasound-assisted deep eutectic solvents extraction

Accepted Manuscript Title: Green and efficient extraction of four bioactive flavonoids from Pollen Typhae by ultrasound-assisted deep eutectic solvents extraction Authors: Zhaorui Meng, Jing Zhao, Hongxia Duan, Yuanyuan Guan, Longshan Zhao PII: DOI: Reference:

S0731-7085(18)30567-3 https://doi.org/10.1016/j.jpba.2018.08.048 PBA 12179

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

7-3-2018 19-7-2018 24-8-2018

Please cite this article as: Meng Z, Jing Z, Hongxia D, Yuanyuan G, Longshan Z, Green and efficient extraction of four bioactive flavonoids from Pollen Typhae by ultrasoundassisted deep eutectic solvents extraction, Journal of Pharmaceutical and Biomedical Analysis (2018), https://doi.org/10.1016/j.jpba.2018.08.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Green and efficient extraction of four bioactive flavonoids from Pollen Typhae by ultrasound-assisted deep eutectic solvents extraction

Zhaorui Meng, Jing Zhao, Hongxia Duan, Yuanyuan Guan, Longshan Zhao*

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School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning Province, 110016, P. R. China *

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Correspondence: Longshan Zhao, School of Pharmacy, Shenyang Pharmaceutical University. 103 Wenhua Road in Shenhe District, Shenyang Liaoning Province, 110016, P. R. China E-mail address: [email protected]. Tel: +86 24 43520571. Fax: +86 24 43520571.

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This work described a novel method for the determination of four bioactive flavonoids in

(UAE-DES).

The efficiencies of synthetic DESs were thoroughly investigated and optimized in this study.

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Pollen Typhae using ultrasound-assisted extraction based on deep eutectic solvents

DESs exhibited higher extraction efficiency comparing with conventional solvents.

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DESs were firstly applied for extraction of Pollen Typhae combined with acid hydrolysis

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Abstract

Recently, deep eutectic solvents (DESs) have been recognized as a novel class of

sustainable solvents to replace common organic solvents. In this study, a highly and efficient extraction technique for determination of four bioactive flavonoids from Pollen Typhae using a combination of ultrasound-assisted extraction and natural deep eutectic solvents (NADESs) was developed. A series of DESs containing various hydrogen bond acceptors combined with 1

different hydrogen bond donors were synthesized and screened for high extraction efficiencies based on the flavonoids extraction yields. The extraction conditions including composition of DES, water content in DES, liquid-solid ratio, and extraction time were statistically optimized by single-factor experiment. As a result, DES composed of choline chloride and 1,2-propanediol (ChPri) at 1:4 molar ratio, 30% of aqueous solution, 50:1 mg·mL-1 for solid-liquid ratio, and 35 min for extraction time were selected as the most

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effective process for extraction of flavonoids in Pollen Typhae. Under the optimal conditions, the target compounds recoveries were in the range of 86.87%-98.89%. Meanwhile, DESs

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showed greater extraction efficiency for extraction of quercetin, naringenin, kaempferol and

isorhamnetin from Pollen Typhae comparing with conventional solvents such as methanol and 75% of aqueous ethanol. Comparing DESs to the conventional organic solvents, in

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addition to their reduced environmental impacts, they proved to provide higher extraction

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efficiency for flavonoids, and therefore have a great potential as possible alternatives to those

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organic solvents in extraction of Chinese herbal medicines.

1. Introduction

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Key Words: Deep eutectic solvents; Green extraction; Flavonoids; Pollen Typhae

Solvent extraction remains one of the most widely used sample preparation techniques

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for chromatographic analysis. Several types of solvents including ionic liquids (ILs) have been suggested as “green” solutions to replace volatile organic solvents. In recent years,

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much attention has been paid to ILs as sustainable alternatives to hazardous organic solvents [1-4]. However, most ILs have the disadvantages of high price and toxicity [5,6]. To overcome the limitations of ILs, a novel class of sustainable solvents with similar properties

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to ILs have emerged [7-9], Abbott et al. presented a new solvent system, termed “deep eutectic solvents (DESs)” which are formed by mixing natural and renewable starting materials with high melting points [10]. DESs are mixtures prepared by combining two or more components from hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) with a lower melting point than that of its individual components mainly due to the generation of inter molecular hydrogen bonds [11-14]. Meanwhile, DESs have attracted more 2

attention as a rapidly emerging new green solvent that replaces traditional solvents or even ILs owing to their unique advantages of biodegradability, non-toxicity, and low costs for synthesis [10-12]. They have been widely used in catalysis, organic synthesis, electrochemical materials and solvents of extraction because of the green property and variety of DESs [14-18]. In previous studies, DESs have been successfully applied for efficient

[20], anthocyanins [21], glaucarubinone [22] and catechins [23,24] .

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extraction of different types of bioactive compounds including quercetin [19], ginsenosides

Pollen Typhae, the dried pollen of Cattail plants, is commonly used in Chinese medicine

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in the treatment of stranguria, hematuria, dysmenorrhea, and metrorrhagia [25,26]. Pharmacological studies have shown that isorhamnetin-3-O-neohesperidin, isorhamnetin,

quercetin and other flavonoids are the main active constituents in Pollen Typhae which

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possess antioxidant, antiinflammatory, antigenotoxic, and antiprotozoal activities [27,28]. The

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flavonoid constituents in Pollen Typhae, which cover a wide range of polarities, contribute to

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its pharmacological activities and therapeutic efficacy. A series of methods have been reported for the efficient extraction of bioactive flavonoids from Pollen Typhae [26-29].

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Because the solubility of flavonoids in water is generally low, various organic solvents

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including ethanol, methanol, acetone, and ethyl acetate have been commonly used as extraction solvents. The selection of analytical methods for sample pre-treatment is also crucial since the method of extraction not only affects the extraction efficiency, but also

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determines accuracy of developed method [30]. These commonly used extraction methods mainly include immersion extraction (ME), soxhlet extraction (SE) [31], heat reflux

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extraction (HRE), pressurized liquid extraction (PLE), ultrasound-assisted extraction (UAE) [32-37] and microwave assisted extraction (MAE) [38-41]. However, some of these reported methods are time consuming, labor intensive and require a large number of organic solvents

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[21].

In present study, we aimed to develop a simple and green method for extraction of four

bioactive flavonoids including quercetin, kaempferol, isorhamnetin and naringenin in Pollen Typhae by using DESs as extraction solvent. Simultaneously, we also established a simple and rapid method of acid hydrolysis, through the acid hydrolysis method, the flavonoid glycosides which were not easy to be quantified in the extracts could convert to their 3

corresponding aglycons forms [42,43] so as to accurately reflect the extraction efficiency of each extraction solvent. The ultrasound assisted-deep eutectic solvent extraction (UAE-DES) method was applied to extract the analytes, and the high performance liquid chromatography (HPLC) method was developed for their sensitive and selective quantification. The efficiencies of DESs were thoroughly investigated and optimized in this study. Finally, the Pollen Typhae extraction yields of the optimized method were compared with those of

based on these DESs have been reported for extraction of Pollen Typhae.

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2. Experimental

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conventional extraction methods. To the best of our knowledge, no extraction procedures

2.1 Materials

Pollen Typhae was purchased from the local medicine market in Liaoning Province,

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China. The samples were dried to constant weight and stored in the shade to use. Choline

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chloride (>98.0%), 1,4-butanediol (>98.0%), 1,2-propanediol (>98.0%), glucose (>98.0%),

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glycerol (>98.0%), L-proline (>99%), lactic acid (>98.0%) and ethylene glycol (>99.5%) were purchased from Shanghai Aladdin Chemistry Co., Ltd (Shanghai, China). Analytical

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standard quercetin (≥98.5%, E1712092), kaempferol (≥98.0%, B1704064), naringenin

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(≥98.0%, K1502023) and isorhamnetin (≥98.0%, 161017) were purchased from Sigma-Aldrich (Shanghai, China). The structures of the reference standards are shown in Fig. 1. Ultra-pure water used in this experiment was obtained from Wahaha Group Co., Ltd.

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(Hangzhou, China). Chromatographic grade acetonitrile, methanol and phosphoric acid were purchased from Shandong Yuwang Pharmaceutical Co., Ltd. (Shandong, China). Ultrasonic

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irradiation (KQ-5200) was purchased from Kunshan Ultrasonic Instrument Co., Ltd. (Jiangsu, China).

2.2 Preparation of DESs

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In this study, eight kinds of DESs were synthesized using the heating method as

previously described [11]. The produced DESs included choline chloride-1,4-butanediol (ChBu), choline chloride-glucose (ChGlu), choline chloride-glycerol (ChGly), choline chloride-1,4-butanediol-glycerol

(ChBuGly),

L-proline-glycerol

chloride-lactic acid (ChLac), choline chloride-ethylene

(ProGly),

choline

glycol (ChEtgly), choline

chloride-1,2-propanediol (ChPri). Accurately weighed components at proper molar ratios 4

were mixed in a glass vial and stirred at 60~80℃ until a clear liquid was obtained. The list of prepared DESs is displayed in Table 1. 2.3 Properties of the DESs The physicochemical properties of DESs are usually attributed to the compositions and the structures of DESs, the related properties such as conductivity, density, and viscosity have been reported in few literatures [10-13]. In the present work, we summarized some of the

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reported properties of DESs related to our experiment (Table S2) and investigated FT-IR

spectra of pure choline chloride, 1,2-propanediol and prepared ChPri（1:3, 1:4, 1:5) to

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confirm the formation of hydrogen bonds.

2.4 Flavonoids extraction from Pollen Typhae by UAE-DES and hydrolysis of the extracts For initial screening of the extraction solvent and method, accurately weighed 100 mg of

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Pollen Typhae powder was added to 2 mL of extraction solvent (DES or traditional solvents)

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in a 10 mL centrifuge tube. The mixture was briefly vortexed for 5 min and extracted by an

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ultrasonic irradiation for 35 min, followed by centrifugation at 4200 rpm for 25 min. 1 mL of supernatant was transferred and mixed with 1 mL of 7 M aqueous hydrochloric acid solution.

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The mixture was heated and stirred at 60℃ for 20 min, in order to convert the flavonoid

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glycosides in the extract to their corresponding aglycons, acid hydrolysis approach was applied.

2.5 HPLC-UV analysis for quantification of flavonoids in Pollen Typhae

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High perforfance liquid chromatography coupled to ultraviolet detection (HPLC-UV) was performed using an Agilent 1100 Series HPLC system (Agilent, San Jose, CA, USA)

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equipped with a diode array detector (DAD). Chromatographic separation was carried out on a Diamonsil C18 reversed-phase column (250 mm × 4.6 mm, 5 μm). The flow rate was 1.0 mL·min-1 and the column temperature was set at 35℃. The mobile phase consists of 0.05%

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(v/v) phosphoric acid aqueous water (A) and acetonitrile (B). Gradient elution was performed as follows: 0–12 min, 15–23% B; 12–15 min, 23–40% B; 15–20 min, 40–30% B; 20–30 min, 30–20% B; 30–32 min, 20–15% B, and they were filtered through a 0.45 μm membrane filter before use. The detection wavelength of quercetin, kaempferol, isorhamnetin were set at 370 nm and naringenin was verified at wavelength of 290 nm. The injection volume was 20 μL, all the samples were passed through a 0.45 μm membrane filter prior to injection. 5

2.6 LC-MS analysis for qualitative analysis Waters Acquity UPLC system (Waters, Milford, MA, USA) coupled to triple-quadrupole tandem spectrometer was performed for qualitative analysis to confirm the presence of aglycone. Flavonoid standards and extracts of Pollen Typhae in methanol or DESs were chromatographed on an Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 µm) from Waters (Milford, MA, USA). Mobile phase consists of aqueous formic acid (0.1%, v/v) (A)

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and acetonitrile (B). The flow rate was 0.3 mL·min-1. Aglycone were detected in multiple reaction monitoring (MRM) mode. The direct infusion of each compound at 100 μg·L-1 was

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performed to optimize instrument parameters and the optimal MRM conditions are shown in

Table S2. Data was acquired using electrospray positive ionization (ESI+). Capillary voltage was set as 3.00 kV, while desolvation gas flow was optimized at 1000 L·h -1. Desolvation

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temperature and source temperature were maintained at 300 ℃ and 150 ℃, respectively.

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2.7 Optimization of extraction conditions

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For screening of extraction solvent, various types of DESs were compared based on the extraction yields of major flavonoids in Pollen Typhae. Then several extraction parameters of

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target compounds were optimized using single factor experiment design. The extraction parameters including molar ratios of ChPri (1:2, 1:3, 1:4, 1:5, and 1:6), water content in

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ChPri (0%, 30%, 50%, and 70%), solid-liquid ratio (50:1, 100:1, 150:1, and 200:1 mg·mL-1), and extraction time (15, 25, 35, 45, and 60 min) were optimized in order to maximize the

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extraction efficiency.

2.8 Statistical Analysis

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All statistical analysis was performed using GraphPad Prism software (version 6.00, La

Jolla California, USA). The test results are based on the average of three parallel determinations, expressed as mean ± standard deviation (SD). The significance of difference

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was conducted by multiple comparisons test and two-way analysis of variance (ANOVA) of the peak areas of flavones aglycones, and p values ≤ 0.05 were considered as significant difference. 2.9 Recovery of extracted flavonoids from DES The recovery was determined by adding a known amount of standard material to the Pollen Typhae powder. It was estimated at three concentration levels (n = 3 for each 6

concentration). The sample spiked with the standard substance was prepared under the optimized conditions according to section 2.4 and was analyzed based on section 2.5. 3. Results and Discussion 3.1 Characterization of DES Most DESs can be prepared with various ratios at room temperature or at a certain temperature to form a transparent and uniform liquid. The prepared DESs have a lower

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freezing point than their individual constituents, which is attributed to the reduction of the coulomb forces of DESs with the large volume and asymmetric charge distribution of

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molecular ions [18]. It has been proved that the densities of mostly prepared DESs are higher

than that of water [11]. DES of ChCl/1,2-propanediol (1:4) has a higher density of 1.062 (30 ℃）than 1,2-propanediol (1.037, 30 ℃). Viscosity is also one of the most important

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characteristics and greatly affected by water content and temperature, it restricts the

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application of DESs due to their higher viscosities. The viscosity of ChCl/1,2-propanediol is

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83 cP (30 ℃), which is higher than the pure 1,2-propanediol (61 cP, 25 ℃). FT-IR spectra of pure choline chloride, 1,2-propanediol and prepared ChPri（1:3, 1:4, 1:5)

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were investigated to confirm the formation of hydrogen bond between choline chloride and 1,2-propanediol. The FT-IR spectra results are presented in Fig. 2. In the FT-IR spectra the

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characteristic peaks presented at 3228 cm−1 and 1085 cm−1 belong to O-H and C-N vibration of pure choline chloride displayed in Fig. 2(A), respectively. The O-H and C-H vibration of

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pure 1,2-propanediol are positioned at 3384 cm-1, 2970 cm-1 and 2932 cm-1 in Fig. 2(B). By comparison among the various spectra, O-H stretching vibration absorption peak of

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1,2-propanediol is a wide peak, which may be due to the formation of hydrogen bonds in the molecule. Both characteristic peaks of choline chloride and 1,2-propanediol are observed in prepared ChPri spectrums [44]. The stretching vibration absorption peaks of methyl group

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(vCH3) in choline chloride and 1,2-propanediol shift from 1483 cm-1 and 1377 cm-1 to 1402 cm-1, 1407 cm-1, 1402 cm-1 in prepared ChPri (1:3, 1:4, 1:5), respectively. The O-H vibration of ChPri (1:3, 1:4, 1:5) are positioned at 3423 cm-1 in Fig. 2(C), 3408 cm-1 in Fig. 2(D), and 3419 cm-1 in Fig. 2(E), respectively. The shift of hydroxyl group and characteristic group indicates the formation of hydrogen bonds in ChPri [37]. The absorption peak of hydroxyl group in ChPri (1:4) is broader than that in ChPri (1:3) and ChPri (1:5), which indicates that 7

the hydrogen bonds in the ChPri (1:4) are more than those in the ChPri (1:3) and ChPri (1:5) [45-46]. 3.2 Selection of initial extraction conditions According to the previous reports on Pollen Typhae extraction, heat reflux extraction was the most commonly used method, which was complicated and need a large amount of herbs and solvents [25, 27-29]. In this study, UAE was selected in the initial screening

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procedure because it is generally simple, fast, and effective, which can be applied regardless of the solvent type.

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Based on the preliminary experiment, we found there was large amount of glycosides content in Pollen Typhae with similar polarity. Flavonoid levels may be measured in their original form. However, the various forms of the conjugates lead to too many peaks that

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significantly vary in retention time, which makes chromatographic separation very

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challenging using a conventional LC system. As a result, flavonoids are usually converted to

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the corresponding aglycons by acid hydrolysis, which results in only a few major aglycons and simple chromatographic patterns. Therefore, it is possible to determine the amount of

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aglycons to reflect the extraction efficiency [34]. In order to make the extract completely

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acidified, heating and stirring method was selected in current method. Reference materials, unhydrolyzed extracts and acid hydrolyzed extracts from Pollen Typhae were analyzed using UPLC-MS to identify aglycone. Based on their retention time, accurate masses of molecular

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and fragment ions in comparison to literature, it was concluded that extracts are converted into target aglycone after acid hydrolysis (Fig. S2, Fig. S3). During the experiment, we found

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that when the extract was fully converted to aglycon form, the sample solution turned dark red in color and the heating time varied depending on the extraction efficiency. 3.3 Optimization of extraction conditions

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3.3.1 Screening of DESs for enhanced flavonoid extraction Different types of DESs have great influence on its physical properties, including density,

viscosity, conductivity, refractive index and other physical properties. Since the polarity of bioactive natural products varies greatly, maximized extractability can be achieved by adjusting the polarity of the extraction solvent [20]. Based on previous reports and our preliminary experiences, a number of HBAs and HBDs were combined at various ratios. As a 8

result, 8 different types of stable DESs at room temperature include acid-based, alcohol-based and sugar-based DESs were selected and tested for flavonoids extraction (Table 1). The extraction ability depends on whether there is an easy interaction between the components of DESs and the extracted material (eg, hydrogen bonding, π-π). As can be seen in the Fig. 3, each DES has different extraction efficiency for the target compounds. For high polar analytes, ChPri (1:4) was found to be the most effective DES for extraction flavonoids from

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Pollen Typhae. 3.3.2 Effect of water content on extraction efficiency

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Based on a series of preliminary experiments, it was found that the extraction solvent, water content in DES and extraction time have a greater impact on the extraction efficiency,

especially the water content [47]. The viscosity of DES is tens or even hundreds of times

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greater than the viscosity of water, which makes it difficult to fully mix with Pollen Typhae

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powder. As shown in Fig. 4(A), extraction efficiency increased as water content in ChPri

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ranged from 0 to 30% (p < 0.05) since the addition of suitable water could effectively reduce the viscosity and could contribute to perform better effects on polar compounds. However,

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extraction efficiency decreased when the water content increased above 30% because an

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excessive concentration of water would result in the loss of existing hydrogen bonds, and consequently, the disappearance of the special structure of DES, and simultaneously reduce the solubility of the flavonoids in DESs, which cause a negative influence on the interactions

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between DES and target bioactive flavonoids. In previous study, it has been demonstrated that there is a strong interaction between the protons on the hydroxyl groups from choline

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chloride, 1,2-propanediol and water, implying that hydrogen bonds are formed between these hydroxyl groups, which suggests that water might also participate in the supermolecular structure of DES [11]. Above all, a water content of 30% of DES was selected for subsequent

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experiments.

3.3.3 Effect of salt to HBD ratio The composition ratio of ChCl/HBD will affect the polarity of DESs, thus affecting the extraction ability. DESs are not easily formed and not stable when the proportion of HBDs is too high, and therefore, it is important to adjust the molar ratio of the components. Generally speaking, for most of DESs, the higher the temperature and hydrogen bond donor content, the 9

smaller the viscosity of DESs. In addition, the presence of additional hydroxyl groups allows the formation of more hydrogen bonds and thus increases the stability of the liquid [11]. The molar ratio of ChPri from 1:2 to 1:6 was investigated and the results showed in (Fig. 4(B)). The amounts of the target flavonoids compounds significantly increased as the molar ratio increased from 1:2 to 1:4 (p < 0.05) due to a decrease in viscosity and surface tension. Meanwhile, proper increased ratio of 1,2-propanediol in DES also improve its diffusion and

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mass transfer, thus enhance the extraction yield. However, the quantity of extracted flavonoids decreased with the reduced concentration of ChCl when the molar ratio reached at

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1: 5, this is because exceeding ratio of 1,2-propanediol led to decrease in the basicity and

interaction between the target flavonoids compounds and chloride anion. Therefore, the molar ratio of 1:4 was selected for the following investigation.

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3.3.4 Effect of sonication time

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The effect of ultrasonic time ranging from 15 to 60 min on the extraction yield was

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investigated. It can be observed in Fig. 4(C) that the extraction yields of the target compounds obviously increased as increasing of the ultrasonic time before reached 35 min (p

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< 0.05), and then the extraction yields were not significantly improved with a further

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increasing of ultrasonic time, which indicates that there is a dynamic balance between extracts and extraction time with the extension of the ultrasound time. Thus, taking into account the extraction efficiency, 35 min was selected for the extraction of the target

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compounds in further experiments.

3.3.5 Effect of solid to solvent ratio

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The solid to solvent ratio will affect the diffusion of solute into the solvent, in order to

maximize extraction efficiency and reduce solvent waste, we studied the effect of solid to solvent ratios (50:1, 100:1, 150:1, and 200:1 mg·mL-1). It was found that maximum extract

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yields of target flavones occurred at the ratio of 50:1 mg·mL-1 (Fig. 4(D)), if a higher extraction yield was needed, less amount of solid material would be added. At solid to solvent ratio more than 50:1 mg·mL-1, sample and solvent were not sufficiently mixed because of the more solid particles existed in the solution and might adsorb more solvent and resulting in the lower extraction yields. Considering the extraction recoveries of four target flavones and the solvent consumption, a solid to solvent ratio of 50:1 mg·mL-1 was eventually adopted as the 10

optimal solid to solvent ratio for the extraction. 3.3.6 Evaluation of extraction efficiency of the optimized conditions in comparison to conventional solvents In this study, conventional solvents and DES of ChPri (1:4) used for the extraction of flavonoids from Pollen Typhae were compared. The traditional solvents mainly included ethanol, water, methanol and 75% ethanol. The same volume of extraction solvents was

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added to sample under the optimized conditions, compared to the traditional solvents, the

extraction of flavonoids by DES of ChPri (1:4) showed excellent advantages. As shown in the

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Fig. 5, 75% ethanol and ChPri (1:4) showed higher extraction efficiency in extraction of quercetin, isorhamnetin and kaempferol than that of ethanol, water and methanol.

Furthermore, the extraction efficiency of ChPri (1:4) was significantly higher than that using

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75% ethanol for extraction of isorhamnetin (p<0.05). Methanol showed the highest

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naringenin extraction efficiency compared to other solvents while solvent of water

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demonstrated the worst extraction efficiency for naringenin, meanwhile, 75% ethanol and ChPri (1:4) exhibited similar extraction efficiency in extraction of naringenin. In addition to

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the use of DES as the extraction solvent could increase the solubility of active constituents,

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DES also possessed outstanding advantages such as environmental friendly, low cost. Therefore, considering all the factors, ChPri (1:4) was selected as the final extraction solvent due to its higher extraction efficiency and sustainable property.

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3.4 Method validation

The determination of four target analytes in Pollen Typhae by HPLC was validated in

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this section. The linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy were examined for the developed method. Fig. S1 showed the chromatograms of the four analytes standard mixture (Fig. S1(a)) and extracts by UAE-DES (Fig. S1(b)). The

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results indicated that all the target compounds can be well separated in both the standard mixture and Pollen Typhae extracts. 3.4.1 Linearity, limit of detection (LOD) and limit of quantification (LOQ) Linearity of the method was evaluated via a six points calibration curves for each analyte with the range of 2.4-64 μg·mL-1 for quercetin, 0.40-8.0 μg·mL-1 for naringenin, 4.0-80 μg·mL-1 for kaempferol, and 16-96 μg·mL-1 for isorhamnetin. The correlation 11

coefficients (r2) of the four calibration curves are greater than 0.9991. The LOD and LOQ were estimated for the concentration of each analyte that corresponding signal-to-noise ratios (S/N) of 3 and 10, respectively. The LODs and LOQs of the method varied from 0.05-0.14 μg·mL-1 and 0.17-0.48 μg·mL-1, respectively, indicating the analytical method possessed excellent sensitivity. The results are summarized in Table 2. 3.4.2 Precision and accuracy

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The precision of the method was determined by analyzing the same Pollen Typhae sample in triplicate by six times. Relative standard deviations (RSDs) for the retention time

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(tR) and peak area (Pa) were analyzed to assess the precision. The results of the precision test

are presented in Table 3. The deviations for tR ranged from 0.07 to 0.14% and Pa ranged from 1.11 to 2.48%. Accuracy (recovery) was determined by adding three concentration levels of

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four target standards to the raw materials. The materials mixed with standards were prepared

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according to Section 2.4 and the samples were analyzed by HPLC according to Section 2.5.

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For each concentration level, three replicate experiments were performed. The recovery was calculated with the following formula: recovery (%) = (amount found - original

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amount)/amount spiked × 100. Consequently, it could be observed from the Table 3 that the

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average recoveries of four targets varied between 86.87% and 98.89% with deviation less than 4.38%. The results demonstrated the higher accuracy of the developed method for the quantification of four flavonoid aglycones in Pollen Typhae.

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3.5 Analysis of samples

The developed method was successfully applied to the determination of four flavonoid

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aglycones in Pollen Typhae. Three parallel experiments were performed under optimal parameters, as shown in Table 4, the average concentrations of quercetin, naringenin, kaempferol and isorhamnetin were 0.383, 0.048, 0.391 and 3.149 μg·mg-1, respectively. The

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results showed that the content of isorhamnetin was the highest and the content of naringenin was the lowest. These significant differences may be due to the glycosides of isorhamnetin was the major active component, flavonol glycosides converted to its corresponding aglycon through acid hydrolysis. 4. Conclusion In the present study, a green and efficient extraction method using DESs was established 12

for the extraction of flavonoids from Pollen Typhae. DESs showed greater extraction efficiency for extraction of quercetin, naringenin, kaempferol and isorhamnetin from Pollen Typhae comparing with conventional solvents such as methanol and 75% of aqueous ethanol. ChPri (1:4) with 30% water content showed the highest extraction efficiency among all prepared DESs. This study suggests the excellent properties of DESs demonstrate their

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promising and potential as green solvents in extraction of Chinese herbal medicines. Acknowledgment

This work was supported by the National Natural Science Foundation of China

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(No. 81503029) and Young and Middle-aged Backbone Personnel Training Program of Shenyang Pharmaceutical University (ZQN2016011). References

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[1] A. Berthod, M.J. Ruiz-Ángel, S. Carda-Broch, Ionic liquids in separation techniques, J.

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Chromatogr. A 1184 (2008) 6-18.

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[2] D.R. Prabhu, D.R. Raut, M.S. Murali, P.K. Mohapatra, Extraction of plutonium(IV) by

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diglycolamide extractants in room temperature ionic liquids. Radiochim. Acta. (2017). [3] B. Pospiech, W. Kujawski, Ionic liquids as selective extractants and ion carriers of heavy

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metal ions from aqueous solutions utilized in extraction and membrane separation, Rev. Chem. Eng. 31 (2015) 171-191.

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[4] C.F. Poole, S.K. Poole, Extraction of organic compounds with room temperature ionic liquids, J. Chromatogr. A. 1217 (2010) 2268-2286.

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[5] T.P. Pham, C.W. Cho, Y.S. Yun, Structural effects of ionic liquids on microalgal growth inhibition and microbial degradation, Environ. Sci. Pollut. Res. Int. 23 (2016) 4294-4300. [6] P. Stepnowski, A.C. Składanowski, A. Ludwiczak, E. Laczyńska, Evaluating the

A

cytotoxicity of ionic liquids using human cell line HeLa, Hum. Exp. Toxicol. 23 (2004) 513-517. [7] D.Z. Troter, Z.B. Todorović, D.R. Đokić-Stojanović, O.S. Stamenković, V.B. Veljković, Application of ionic liquids and deep eutectic solvents in biodiesel production: A review, Renewable and Sustainable Energy Reviews. 61 (2016) 473-500. [8] Y. Dai, J. van Spronsen, G.J. Witkamp, R. Verpoorte, Y.H. Choi, Ionic liquids and deep 13

eutectic solvents in natural products research: mixtures of solids as extraction solvents, J. Nat. Prod. 76 (2013) 2162-2173. [9] Y.R. Lee, K.H. Row, Comparison of ionic liquids and deep eutectic solvents as additives for the ultrasonic extraction of astaxanthin from marine plants, J. Ind. Eng. Chem. 39 (2016) 87-92. [10] A.P. Abbot, G. Capper, D.L. Davies, R.K. Rasheed, V. Tambyrajah, Novel solvent

IP T

properties of choline chloride/urea mixtures, Chem. Commun. 9 (2002) 70-71.

[11] Y.T. Dai, J. Spronsen, G.J. Witkamp, R. Verpoorte, Y.H. Choi, Natural deep eutectic

SC R

solvents as new potential media for green technology, Anal. Chim. Acta. 766 (2013) 61-68.

[12] R. Craveiro, I. Aroso, V. Flammia, T. Carvalho, M.T. Viciosa, M. Dionísio, S. Barreiros, R.L. Reis, A.R.C. Duarte, A. Paiva, Properties and thermal behavior of natural deep eutectic

U

solvents, J. Mol. Liq. 215 (2016) 534-540.

N

[13] M. Espino, M. de los Ángeles Fernández, F.J.V. Gomez, M.F. Silva, Natural designer

A

solvents for greening analytical chemistry, TrAC. Trends Anal. Chem. 76 (2016) 126-136. [14] S. Khandelwal, Y.K. Tailor, M. Kumar, Deep eutectic solvents (DESs) as eco-friendly

M

and sustainable solvent/catalyst systems in organic transformations, J. Mol. Liq. 215 (2016)

ED

345-386.

[15] X. Li, K.H. Row, Development of deep eutectic solvents applied in extraction and separation, J. Sep. Sci. 39 (2016) 3505-3520.

PT

[16] K.M. Jeong, J. Zhao, Y. Jin, S.R. Heo, S.Y. Han, E. Yoo da, J. Lee, Highly efficient extraction of anthocyanins from grape skin using deep eutectic solvents as green and tunable

CC E

media, Arch. Pharmacal Res. 38 (2015) 2143-2152. [17] B. Zhuang, L.L. Dou, P. Li, E.H. Liu, Deep eutectic solvents as green media for extraction of flavonoid glycosides and aglycones from Platycladi Cacumen, J. Pharm.

A

Biomed. Anal. 134 (2017) 214-219. [18] B. Tang, H. Zhang, K.H. Row, Application of deep eutectic solvents in the extraction and separation of target compounds from various samples, J. Sep. Sci. 38 (2015) 1053-1064. [19] B. Tang, H.E. Park, K.H. Row, Simultaneous extraction of flavonoids from chamaecyparis obtusa using deep eutectic solvents as additives of conventional extractions solvents, J. Chromatogr. Sci. 53 (2015) 836. 14

[20] K.M. Jeong, M.S. Lee, M.W. Nam, J. Zhao, Y. Jin, D.K. Lee, S.W. Kwon, J.H. Jeong, J. Lee, Tailoring and recycling of deep eutectic solvents as sustainable and efficient extraction media, J. Chromatogr. A 1424 (2015) 10-17. [21] Y. Dai, E. Rozema, R. Verpoorte, Y.H. Choi, Application of natural deep eutectic solvents to the extraction of anthocyanins from Catharanthus roseus with high extractability and stability replacing conventional organic solvents, J. Chromatogr. A 1434 (2016) 50-56.

IP T

[22] F. Kholiya, N. Bhatt, M.R. Rathod, R. Meena, K. Prasad, Fundamental studies on the

feasibility of deep eutectic solvents for the selective partition of glaucarubinone present in the

SC R

roots of Simarouba glauca, J. Sep. Sci. 38(2015) 3170-3175.

[23] K.M. Jeong, J. Ko, J. Zhao, Y. Jin, E.Y. Da, S.Y. Han, J. Lee, Multi-functioning deep eutectic solvents as extraction and storage media for bioactive natural products that are

U

readily applicable to cosmetic products, J. Cleaner Prod. 151 (2017) 87-95.

N

[24] K.H. Row, Evaluation of alcohol-based deep eutectic solvent in extraction and

A

determination of flavonoids with response surface methodology optimization, J. Chromatogr. A 1285(2013) 22-30.

M

[25] F. Qin, H.X. Sun, Immunosuppressive activity of Pollen Typhae ethanol extract on the

ED

immune responses in mice, J. Ethnopharmacology. 102 (2005) 424-429. [26] L. Han, X. Liu, N. Yang, J. Li, B. Cai, S. Cheng, Simultaneous chromatographic fingerprinting and quantitative analysis of flavonoids in Pollen Typhae by high-performance

PT

capillary electrophoresis, Acta Pharm. Sinica B. 2 (2012) 602-609. [27] D.X. Fu, X.W. Cheng, W. Wei, J.K. Chen, T.S. Zhou, Determination of Typhaneoside

CC E

and Isorhamnetin-3-O-neohesperidoside in Total Flavonoid Extracts of Pollen typhae by HPLC, Chin. J. Pharm. 35 (2004) 360-361. [28] B. Liu, Y. Lu, HPLC determination of two flavonoids in pollen Typhae (puhuang) and its

A

different processed products, Chin. J. Pharm. Anal. 23 (1998) 402-404. [29] S.H. Zhang, Y.H. Yang, P. Cai, Q. Liu, G.X. Cai, Effect of microwave-assisted extraction on flavone constituents of Pollen Tyhae micropowder, Chin. Traditional Patent Med. (2004). [30] S. Bajkacz, J. Adamek, Evaluation of new natural deep eutectic solvents for the extraction of isoflavones from soy products, Talanta. 168 (2017) 329. [31] S.B. Hawthorne, C.B. Grabanski, E. Martin, D.J. Miller, C.F. Poole, Comparisons of 15

Soxhlet extraction, pressurized liquid extraction, supercritical fluid extraction and subcritical water extraction for environmental solids: recovery, selectivity and effects on sample matrix, J. Chromatogr. A. 892 (2000) 421-433. [32] M.W. Nam, J. Zhao, M.S. Lee, J.H. Jeong, J. Lee, Enhanced extraction of bioactive natural products using tailor-made deep eutectic solvents: application to flavonoid extraction from Flos sophorae, Green Chem. 17 (2015) 1718-1727.

IP T

[33] C. Bakirtzi, K. Triantafyllidou, D.P. Makris, Novel lactic acid-based natural deep eutectic solvents: Efficiency in the ultrasound-assisted extraction of antioxidant polyphenols

SC R

from common native Greek medicinal plants, J. Appl. Res. Med. Aromat. Plants. 3 (2016) 120-127.

[34] E. Yilmaz, M. Soylak, Ultrasound assisted-deep eutectic solvent based on emulsification

U

liquid phase microextraction combined with microsample injection flame atomic absorption

N

spectrometry for valence speciation of chromium(III/VI) in environmental samples, Talanta.

A

160 (2016) 680-685.

[35] E. Yilmaz, M. Soylak, Ultrasound assisted-deep eutectic solvent extraction of iron from

M

sheep, bovine and chicken liver samples, Talanta. 136 (2015) 170-173. [36] T. Bosiljkov, F. Dujmić, M. Cvjetko Bubalo, J. Hribar, R. Vidrih, M. Brnčić, E. Zlatic, I.

ED

Radojčić Redovniković, S. Jokić, Natural deep eutectic solvents and ultrasound-assisted extraction: Green approaches for extraction of wine lees anthocyanins, Food Bioprod.

PT

Process. 102 (2017) 195-203.

[37] T. Khezeli, A. Daneshfar, R. Sahraei, A green ultrasonic-assisted liquid–liquid

CC E

microextraction based on deep eutectic solvent for the HPLC-UV determination of ferulic, caffeic and cinnamic acid from olive, almond, sesame and cinnamon oil, Talanta. 150 (2016) 577-585.

A

[38] Q. Cui, X. Peng, X.-H. Yao, Z.-F. Wei, M. Luo, W. Wang, C.-J. Zhao, Y.-J. Fu, Y.-G. Zu, Deep eutectic solvent-based microwave-assisted extraction of genistin, genistein and apigenin from pigeon pea roots, Sep. Purif. Technol. 150 (2015) 63-72. [39] H.-F. Zhang, X. Zhang, X.-H. Yang, N.-X. Qiu, Y. Wang, Z.-Z. Wang, Microwave assisted extraction of flavonoids from cultivated Epimedium sagittatum: Extraction yield and mechanism, antioxidant activity and chemical composition, Ind. Crops and Prod. 50 (2013) 16

857-865. [40] Z. Karami, Z. Emam-Djomeh, H.A. Mirzaee, M. Khomeiri, A.S. Mahoonak, E. Aydani, Optimization of microwave assisted extraction (MAE) and soxhlet extraction of phenolic compound from licorice root, J. Food Sci. Technol. 52 (2015) 3242-3253. [41] D. Wang, B.B. Zhang, X.X. Qu, F. Gao, M.Y. Yuan, Microwave-assisted extraction of polysaccharides from Yupingfeng powder and their antioxidant activity, Phcog. Mag. 11

IP T

(2015) 546-554.

[42] L.X. Wang, L.Y. Niu, Y.N. Pan, W. Q, X.Q. Liu, Content Determination of Total Favonol

SC R

Glycosideds of Typhae Pollen Extract by UPLC, Mod. Chin. Med. 16 (2014) 755-758.

[43] H.M. Wen, Y.Y. Wang, G.P. Peng, W. Li, Determination of Flavones in Pollen t yphae by HPLC, Res.

Pract. Chi. Med. 20 (2006) 41-44.

U

[44] Khezeli T, Daneshfar A, Sahraei R. Emulsification liquid-liquid microextraction based

N

on deep eutectic solvent: An extraction method for the determination of benzene, toluene,

A

ethylbenzene and seven polycyclic aromatic hydrocarbons from water samples. J Chromatogr A. 2015;1425:25-33.

M

[45] R. Xin, S. Qi, C. Zeng, F.I. Khan, B. Yang, Y. Wang, A functional natural deep eutectic

ED

solvent based on trehalose: Structural and physicochemical properties, Food Chem. 217 (2017) 560-567.

[46] M.K. AlOmar, M.A. Alsaadi, M. Hayyan, S. Akib, M.A. Hashim, Functionalization of

PT

CNTs surface with phosphonuim based deep eutectic solvents for arsenic removal from water, Appl. Surf. Sci. 389 (2016) 216-226.

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[47] X. Peng, M.H. Duan, X.H. Yao, Y.H. Zhang, C.J. Zhao, Y.G. Zu, Y.J. Fu, Green extraction of five target phenolic acids from Lonicerae japonicae Flos with deep eutectic

A

solvent, Sep. Purif. Technol. 157 (2016) 249-257.

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Figure captions:

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Fig. 1. Chemical structures of the four reference standards.

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Fig. 2. IR spectra of choline chloride (A); 1,2 propanediol (B); choline chloride-1,2

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propanediol (1:3) (C); choline chloride-1,2 propanediol (1:4) (D); choline chloride-1,2

A

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ED

M

A

propanediol (1:5) (E).

18

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Fig. 3. Comparison extraction efficiency of different types of DESs.

Fig. 4. Effect of water content on extraction efficiency (A); Effect of salt to HBD ratio (B);

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A

N

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Effect of ultrasonic time (C); Effect of solvent to solid ratio (D).

A

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Fig. 5. Flavonoid extraction yields of prepared DESs and conventional solvents.

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Table

Table 1 List of DESs synthesized in this study Component 1

Component 2

Component 3

Mole ratio

ChBu

Choline

1,4-butanediol

/

1:4

ChGlu

Choline chloride

Glucose

/

ChGly

Choline chloride

Glycerol

/

ChBuGly

Choline chloride

1,4-butanediol

Glycerol

ProGly

L-proline chloride

Glycerol

/

4:11

ChLac

Choline

Lactic acid

/

1:4

ChEtgly

Choline chloride

Ethylene glycol

/

1:4

ChPri

Choline chloride

1,2-propanediol

/

1:4

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Abbreviation

1:4 1:4

N

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1:2:2

M

A

chloride

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Table 2 Linearity, limit of detection (LOD) and limit of quantification (LOQ) of the method

r2

Linearity range (μg·mL-1)

LOD (μg·mL-1)

LOQ (μg·mL-1)

y=102.3x-66.6

0.9993

2.0~64

0.05

0.17

y=68.8x-3.3

0.9995

0.40~8.0

0.10

0.32

Kaempferol

y=91.6x-28.3

0.9999

4.0~80

0.12

0.40

Isorhamnetin

y=1402.8x-180.7

0.9994

16~96

0.14

0.48

Quercetin

A

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Naringenin

Calibration curve

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Analyte

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Table 3 Precision and recovery

1.78

0.07

0.386

Naringenin

2.48

0.09

0.048

Kaempferol

1.11

0.12

0.396

Isorhamnetin

1.11

0.14

3.145

RSD (%)

86.87

4.38

98.34

3.09

97.64

3.55

98.89

1.27

M

A

N

Quercetin

0.308 0.393 0.585 0.036 0.048 0.058 0.309 0.391 0.470 2.509 3.136 3.763

Recovery (%)

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tR (%)

Spiked (μg·mg-1)

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Pa (%)

Original (μg·mg-1)

U

Precision（%RSD)

Analyte

Analytes

1

Batch 2

3

Average (μg·mg-1)

0.372

0.390

0.387

0.383

0.047

0.049

0.047

0.048

0.385

0.394

0.395

0.391

3.148

3.157

3.142

3.149

A

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Quercetin (μg·mg-1) Naringenin (μg·mg-1) Kaempferol (μg·mg-1) Isorhamnetin (μg·mg-1)

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Table 4 Analysis Pollen Typhae samples

21