Subscriber access provided by ORTA DOGU TEKNIK UNIVERSITESI KUTUPHANESI

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

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 free 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 accessible to all
readers and 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.

ACS Sustainable Chemistry & Engineering 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 33 ACS Sustainable Chemistry & Engineering

1
2
3 Comprehensive evaluation of deep eutectic solvents in extraction of
4
5
6 bioactive natural products
7
8
9
10
Li Duan1, Li-Li Dou1, Long Guo, Ping Li*, E-Hu Liu*
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26 State Key Laboratory of Natural Medicines, China Pharmaceutical University, No. 24
27
28 Tongjia Lane, Nanjing, P. R. China
29
30
31
32 1
33 These authors contributed equally to this work.
34
*
35 Corresponding authors.
36
*
37 E-Hu Liu, E-mail: liuehu2011@163.com. Tel. / fax: +86 25 83271379.
38
39 *
Ping Li, E-mail: liping2004@126.com. Tel. / fax: +86 25 83271379
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59 1
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 2 of 33

1
2
3
4 Abstract
5
6 Deep eutectic solvents (DESs) are emerging as green and sustainable solvents for
7
8
9 efficient extraction of bioactive compounds or drugs. This work aimed to
10
11 comprehensively evaluate the potential and effectiveness of DESs for extraction of
12
13
14 different types of natural compounds from biomass. Five Chinese herbal medicines
15
16 including Berberidis Radix, Epimedii Folium, Notoginseng Radix et Rhizoma, Rhei
17
18
19 Rhizoma et Radix, and Salviae Miltiorrhizae Radix et Rhizoma were selected to
20
21 assess the efficiency of DESs on extraction of alkaloids, flavonoids, saponins,
22
23
24 anthraquinones, and phenolic acids, respectively. Totally 43 types of choline chloride-,
25
26 betaine- and L-proline-based DESs with different polarity, viscosity, composition, and
27
28
29 solubilization abilities were tailored to test their extraction efficiency, and the
30
31 operation conditions were statistically optimized using response surface methodology
32
33
34 to produce the most efficient process. In this work, DES solvents were firstly
35
36 introduced to extract alkaloids and anthraquinones. The results indicated that most
37
38
39 prepared DESs proved to be efficient solvents for extraction of alkaloids, but lower
40
41
extractability for anthraquinones. The extraction capacity of DES may be correlated
42
43
44 with their physical-chemical properties, including H-bonding interactions, polarity,
45
46
viscosity and pH. This study demonstrated that DESs were suitable green extraction
47
48
49 solvents for selectively and efficiently extracting bioactive compounds from
50
51
biomaterials.
52
53
54 ：Deep eutectic solvents; green extraction; natural products; alkaloids;
Keywords：
55
56
57
laevulinic acid
58
59 2
60
ACS Paragon Plus Environment
Page 3 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 INTRODUCTION
5
6 The development of green solvents with low toxicity and cost is a key issue for
7
8
9 the pharmaceutical industry.1,2 Nowadays, conventional organic solvents e.g.,
10
11 methanol, alcohols, chloroform, and ethyl acetate are widely used for extraction of
12
13
14 bioactive natural compounds from plant material.3,4 However, the consumption of
15
16 large amounts of these volatile and hazardous organic solvents may contribute to
17
18
19 environmental pollution and leave unacceptable solvent residues in extracts.
20
21 Since being introduced by Abbott et al. in 2003,5 deep eutectic solvents (DESs)
22
23
24 are rapidly emerging as a new type of green and sustainable solvent. DESs are
25
26 prepared by simply mixing two or more naturally-occurring, inexpensive, and
27
28
29 biodegradable components together to obtain a eutectic mixture.5 The availability, low
30
31 cost, biodegradability and environmental friendliness of the components makes the
32
33
34 DESs versatile alternatives to conventional organic solvents.6-9
35
36 Due to their excellent properties, DESs have been widely used to generate
37
38
39 nanoscale materials, extract DNA and desulfurize the products of fuels.10-13 Recently,
40
41
DESs were applied for extraction and separation of bioactive phenolic compounds,
42
43
44 saponins or flavonoids from plant materials.14-19 Nonetheless, the number of reports
45
46
on the application of DESs to the extraction of natural products is limited, and the
47
48
49 efficiency of DESs on extraction of other types of active natural compounds (e.g.,
50
51
alkaloids, anthraquinones) still remains unknown. To the best of our knowledge, there
52
53
54 is still no report on comprehensive evaluation of the potential and effectiveness of
55
56
57
DESs as green solvents for extraction of different bioactive compounds from natural
58
59 3
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 4 of 33

1
2
3
4 sources.
5
6 To extend the applications of DESs in extraction of bioactive natural-occurring
7
8
9 compounds, in this study, five Chinese herbal medicines (HMs) including Berberidis
10
11 Radix (BR), Epimedii Folium (EF), Notoginseng Radix et Rhizoma (NRR), Rhei
12
13
14 Rhizoma et Radix (RRR), and Salviae Miltiorrhizae Radix et Rhizoma (SMR) were
15
16 selected to comprehensively evaluate the efficiency of DESs on extraction of
17
18
19 alkaloids, flavonoids, saponins, anthraquinones, and phenolic acids, respectively. A
20
21 series of choline chloride (ChCl)-, betaine (Bet)- and L-proline (Pro)-based DESs with
22
23
24 different mixing ratios were firstly tailored, and the operation conditions were
25
26 statistically optimized using response surface methodology (RSM) to produce the
27
28
29 most efficient process. The interactions between the DESs and natural products, as
30
31 well as the optimal conditions for extraction were analyzed by high-performance
32
33
34 liquid chromatography (HPLC).
35
36
37
38
39 EXPERIMENTAL
40
41
42
43
44 Materials and reagents.
45
46
EF, NRR, RRR, SMR and BR were purchased from a local TCM market
47
48
49 (Nanjing, China). Reference compounds (Fig. 1) including aloe-emodin, rhein,
50
51
emodin, chrysophanol, physcion, ginsenoside RG1, ginsenoside RB1,
52
53
54 notoginsenoside R1, rosmarinic acid, lithospermic acid, salvianolic acid B,
55
56
57
jatrorrhizine hydrochloride (Jat), berberine hydrochloride (Ber), palmatine
58
59 4
60
ACS Paragon Plus Environment
Page 5 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 hydrochloride (Pal) and icariin were purchased from Chengdu Must Bio-technology
5
6 Co., Ltd. (Chengdu, China). Compounds for DES preparation including ChCl, Bet,
7
8
9 Pro, urea, 1-methylurea, N,N’-dimethylurea, acetamide, glycerol, xylitol, D-sorbitol,
10
11 ethylene glycol, sucrose, D-glucose, maltose, citric acid, malonate, laevulinic acid,
12
13
14 oxalic acid, DL-malic acid and lactic acid were obtained from Aladdin Reagent
15
16 Company (Shanghai, China). HPLC-grade acetonitrile was purchased from TEDIA
17
18
19 (Tedia Company, Inc., USA). Deionized water used in experiments was purified by a
20
21 Milli-Q system (Millipore, Milford, MA, USA). All other reagents and chemicals
22
23
24 used were of analytical grade
25
26
27
28
29 Preparation of DESs.
30
31 The syntheses of natural DESs were conducted as described previously.20 Briefly,
32
33
34 DESs were obtained by simply mixing the hydrogen bond acceptors (HBAs) and
35
36 hydrogen bond donors (HBD) together at a proper molar ratio with magnetic agitation
37
38
39 at 80.0 ºC until a clear and homogeneous liquid formed, and a known amount of water
40
41
was added when necessary.
42
43
44
45
46
Extraction procedure.
47
48
49 For initial screening of the extraction solvent, ultrasound-assisted extraction
50
51
(UAE) was performed in a 2 mL microtube with 25 mg of plant material powder and
52
53
54 1.0 mL of DES-water (3:1) solvent. The mixture was ultrasounded (KQ5200B, 200W,
55
56
57
40KHz, KunShan, China) at 50 ºC for 30 min and then centrifuged at 16200 G for 10
58
59 5
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 6 of 33

1
2
3
4 min. The suspension was diluted ten times with water or methanol prior to HPLC
5
6 analysis. Each extraction was performed by triplicate.
7
8
9
10
11 HPLC-UV analysis for quantification of extracted bioactive components.
12
13
14 HPLC-UV analysis was performed using a Shimadzu HPLC system equipped
15
16 with a pump (LC-20AT), an auto-sampler (SIL-20A), a diode array detector
17
18
19 (SPD-M20A) and an automatic column temperature control oven (CTO-20A).
20
21 Separation was performed on a Thermo BDS Hypersil C18 column (100 mm × 4.6
22
23
24 mm, 2.4 µm). For analysis of EF, NRR, RRR, and SMR, the mobile phase consisted
25
26 of water containing 0.1% formic acid (eluent A) and acetonitrile (eluent B). For
27
28
29 analysis of BR, the mobile phase consisted of 0.02 mol/L potassium phosphate
30
31 monobasic (eluent A) and acetonitrile (eluent B). (1) HPLC analysis of alkaloids in
32
33
34 BR extract. The gradient program was as follows: 0-5 min, 25%-32% B; 5-10 min, 32%
35
36 B; 10-11 min, 32%-25% B; 11-15 min, 25% B. The flow rate was kept constant at 0.8
37
38
39 mL/min, and the effluents were monitored at 265 nm. (2) HPLC analysis of phenolic
40
41
acids in SMR extract. The gradient program was set at: 0-7 min, 18%-25% B; 7-9 min,
42
43
44 25% B; 9-11 min, 25%-95% B; 11-13 min, 95% B. The flow rate was kept constant at
45
46
0.9 mL/min, and the wavelength was set at 286 nm; (3) HPLC analysis of
47
48
49 anthraquinones in RRR extract. The gradient program was as follows: 0-15 min,
50
51
35%-65% B; 15-25 min, 65%-100% B. The flow rate was kept constant at 0.9
52
53
54 mL/min, and the effluents were monitored at 254 nm; (4) HPLC analysis of saponins
55
56
57
in NRR extract. The gradient program was set at: 0-13 min, 18%-28% B; 13-17 min,
58
59 6
60
ACS Paragon Plus Environment
Page 7 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 28%-33% B; 17-24 min, 33%-50% B; 24-25 min, 50%-54% B. The flow rate was
5
6 kept constant at 0.9 mL/min, and the wavelength was set at 203 nm; (5) HPLC
7
8
9 analysis of flavonoids in EF extract. The gradient program was as follows: 0-10 min,
10
11 25% B; 10-15 min, 25%-90% B. The flow rate was kept constant at 0.7 mL/min, and
12
13
14 the effluents were monitored at 265 nm.
15
16
17
18
19 Experimental design and statistical analysis.
20
21 RSM was performed using the Design-Expert Ver. 8.0.6 (Stat-Ease Inc.,
22
23
24 Minneapolis, MN, USA). After determining the range of extraction variables on the
25
26 basis of preliminary single-factor test, Box–Behnken design (BBD) was used to find
27
28
29 the optimal values for four independent variables: water content (%) in the selected
30
31 DES (A), temperature (B), ultrasonic irradiation time (C) and solid (sample
32
33
34 powder)-liquid (extractant) ratio (D) at three levels (-1, 0, 1). Whole experiments
35
36 were composed of 29 experimental points that included five replicates of the centre
37
38
39 points. The extraction amounts were taken as the response of the design experiments.
40
41
Regression analysis was performed according to the experimental data. Subsequently,
42
43
44 three additional confirmation experiments were conducted to confirm the validity of
45
46
the statistical experimental strategies.
47
48
49
50
51
52
RESULTS AND DISCUSSION
53
54
55
56
57
Preparation of DES.
58
59 7
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 8 of 33

1
2
3
4 For DES preparation, a number of HBAs and HBDs from renewable,
5
6 inexpensive, and readily accessible resources have been tested as potential DES
7
8
9 components. In this work, four groups of HBDs (amides, carboxylic acid, alcohols
10
11 and sugars) were used in combination with three types of HBAs (ChCl, Bet, Pro) to
12
13
14 produce DESs. A total of 51 combinations of DESs were initially tested and 43 of
15
16 them were eventually found to be stable as a clear, viscous liquid without
17
18
19 precipitation over the course of time (Table 1).
20
21 During the preparation process, we found that the chemical structures of HBAs played
22
23
24 an important role in the formation and stability of DESs. Natural DESs could be
25
26 successfully formed by heating ChCl or Bet with ethylene glycol, in contrast, Pro was
27
28
29 proved incapable of producing a stable liquid when heated with ethylene glycol. Also,
30
31 it was easier to synthesize a clear and less viscous DES without any precipitation
32
33
34 when HBDs were in liquid form (e.g., lactic acid, glycerol, levulinic acid, and
35
36 ethylene glycol). Moreover, a known amount of water was necessary to be added in
37
38
39 some combinations to form a clear liquid, for example, when betaine was mixed and
40
41
heated with lactic acid, it was difficult to prepare a clear DES without adding water.
42
43
44 We also discovered that different ratios of HBAs and HBDs may affect the synthesis
45
46
and stability of DES. For instance, when levulinic acid were heated with ChCl in the
47
48
49 mole ratios of 1 : 3, 1 : 2, 3 : 2, 2 : 1, and 3 : 1, a clear liquid can be only prepared
50
51
with 3 : 2, 2 : 1, or 3 : 1 mole ratio.
52
53
54
55
56
57
Comprehensive evaluation of DES in extraction of bioactive natural products.
58
59 8
60
ACS Paragon Plus Environment
Page 9 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 Discovery of natural bioactive products meets numerous challenges, and sample
5
6 extraction is the first step. In this work, a total of 43 DESs with different polarity,
7
8
9 viscosity, composition, and solubilization abilities were selected to test their
10
11 extraction efficiency for five different types of bioactive natural-occurring compounds
12
13
14 (flavonoid, alkaloid, anthraquinone, saponin, and phenolic acid) from HMs. The
15
16 potential and effectiveness of DESs as green solvents for extraction was
17
18
19 comprehensively evaluated. Although certain DESs containing liquid-form HBDs
20
21 (e.g., ChCl-La) were relatively fluidic, most of the DESs prepared were still viscous.
22
23
24 Compared with conventional solvents, the high viscosity of most DES at room
25
26 temperature restricted their application due to a slow mass transfer. The addition of
27
28
29 water to DES can have significant benefits in decreasing viscosity. In this paper,
30
31 extraction conditions were adjusted to reduce the viscosity by heating and adding
32
33
34 certain amount of water. The extraction temperature was set at 50 ºC and 75% DES
35
36 solution in water (v/v) was employed in the initial screening. In this study, methanol
37
38
39 was used as the control extraction solvents.
40
41
42
43
44 Extraction of alkaloid
45
46
Ber, Jat and Pal are three natural bioactive nitrogen-containing compounds
47
48
49 belonging to the protoberberine hydrochloride group of isoquinoline alkaloids, and
50
51
they are the major bioactive compounds of the medicinal herb Berberidis Radix. The
52
53
54 compound Ber has shown potent biological activity against fungal and bacterial/viral
55
56
57
infections21 and Jat has been demonstrated to possess antimelanoma effects.22 To
58
59 9
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 10 of 33

1
2
3
4 extend the applications of DESs and reduce the environmental pollution by organic
5
6 solvents, Ber, Jat and Pal were extracted from BR by the prepared DESs and
7
8
9 determined by HPLC.
10
11 Different DESs were tested under the same condition using the same protocol
12
13
14 (solid to solvent ratio: 25 mg/mL, extraction time: 30 min). The HPLC fingerprints of
15
16 DES extracts showed that the three high peaks were respectively identified as Jat, Pal
17
18
19 and Ber by comparison with reference compounds (Fig. 2a). As shown in Fig. 3a,
20
21 significant differences in the extractability of alkaloids with the tested DESs were
22
23
24 reflected by HPLC profiles and the calculated content of Jat, Pal and Ber in BR.
25
26 Clearly, the extraction capacity of the tested DESs varied greatly depending on
27
28
29 the type of HBD in DES. Sugars- and alcohols- based DESs exhibited relatively low
30
31 extraction capacity, capable of extracting only about 75% alkaloids compared with
32
33
34 methanol, while amides-based DESs showed similar yields compared with methanol
35
36 for Jat, Pal and Ber extraction. Surprisingly, the extraction efficiencies of carboxylic
37
38
39 acid- based DESs were significantly higher than that of other DESs, as well as
40
41
methanol. Among the carboxylic acid- based DES, La-based DES, such as ChCl-La,
42
43
44 Bet-La and Pro-La, produced the highest extractability of alkaloids from natural
45
46
sources. The differences observed between DESs and MeOH for alkaloids may be due
47
48
49 to the lack of extractability of partially ionized compounds by MeOH when compared
50
51
with DESs, where electrostatic interactions could significantly contribute to their
52
53
54 extraction. In conclusion, the La-based DESs, relatively inexpensive and
55
56
57
biodegradable, were found to be the best DESs for the extraction of alkaloids.
58
59 10
60
ACS Paragon Plus Environment
Page 11 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4
5
6 Extraction of phenolic acid
7
8
9 SMR is one of the most frequently used HMs and has been widely used for the
10
11 treatment of cardiovascular and cerebrovascular disease in clinic.23 The phenolic acids
12
13
14 (e,g., salvianolic acid B, rosmarinic acid and lithospermic acid) are the main
15
16 beneficial compounds responsible for the pharmacological activities and therapeutic
17
18
19 efficacy of SMR.24
20
21 In order to find the best extraction solvents for rosmarinic acid, lithospermic acid
22
23
24 and salvianolic acid B from SMR, the prepared DESs which had different properties
25
26 like polarity, solubility, diffusion, and viscosity were examined in this study. The total
27
28
29 contents of these three phenolic acids detected by HPLC were employed to assess the
30
31 extraction efficiency of DESs (Fig. 2b).
32
33
34 The results indicated that extraction yields for phenolic acid were relatively less
35
36 affected by solvent type of DESs. As shown in Fig. 3b, a majority of DESs exhibited
37
38
39 higher yields of phenolic acid compared with methanol. The main reason could be
40
41
superior dissolving capacity of the DES for the polar compounds. Among the three
42
43
44 HBAs, the extraction capacity of ChCl-based DESs was markedly higher than that of
45
46
Bet- and Pro-based DESs. Also, amides-based DES showed the highest efficiency
47
48
49 compared with other three types of HBDs. Overall, considering the toxicity of
50
51
methanol, DES was a suitable green solvent for extraction of phenolic acid from
52
53
54 medicinal herbs.
55
56
57
58
59 11
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 12 of 33

1
2
3
4 Extraction of anthraquinone
5
6 RRR, a well-known medicinal herb wildly used in oriental preparations, has been
7
8
9 recognized for centuries for its multiple pharmacological actions including laxative,
10
11 antibacterial, hemostatic and antispasmodic activities.25 The major active components
12
13
14 of the herb are five anthraquinones, namely aloe-emodin, rhein, emodin, chrysophanol
15
16 and physcion.
17
18
19 To evaluate the extraction efficiency of DESs for anthraquinone, RRR was taken
20
21 as a case in our work. The total contents of five anthraquinones including aloe-emodin,
22
23
24 rhein, emodin, chrysophanol and physcion in RRR were quantified by HPLC method
25
26 (Fig. 2c). Unfortunately, the results showed that most DESs showed the lower
27
28
29 extraction yields than methanol, and only four synthesized DESs (ChCl-La, ChCl-Ox,
30
31 Bet-La and Pro-La), produced similar extraction efficiency for anthraquinone
32
33
34 compared with methanol (Fig. 3c). We speculate that the low extractability of DES
35
36 solvents may be attributed to the low polarity of anthaquinones.
37
38
39
40
41
Extraction of saponin
42
43
44 NRR, is a highly valuable and important Chinese medicinal herb mainly
45
46
cultivated and processed in Yunnan Province, China. It has been widely used as a
47
48
49 tonic and hemostatic agent for the treatment of cardiovascular diseases, inflammation,
50
51
body pains, trauma, and internal or external bleeding caused by injury.26,27 The
52
53
54 dammarane-type saponins, including ginsenosides and notoginsenosides, are the main
55
56
57
bioactive components contributing to the pharmacological activities of NRR.
58
59 12
60
ACS Paragon Plus Environment
Page 13 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 In order to get insight into the DES excellence in the extraction of saponins from
5
6 NRR, a comparative study was carried out to illustrate the extraction efficiency of
7
8
9 methanol, and DESs for Ginsenoside RG1, ginsenoside RB1 and notoginsenoside R1
10
11 (Fig. 2d). As displayed in Fig. 3d, the extraction efficiencies of DESs varied
12
13
14 extremely depending on the type of HBD. Several carboxylic acid-based DESs, such
15
16 as ChCl-Ca, ChCl-Mal, ChCl-Ox and ChCl-Maa could hardly extract the saponins
17
18
19 from NRR. Contrastly, amide-based DESs exhibited higher yields of saponin than
20
21 other DESs. Based on the data, twelve DESs demonstrated comparable extraction
22
23
24 yields with methanol, particularly ChCl-Du and Pro-Mu extracted higher amounts of
25
26 saponins with significant difference. All in all, certain DESs could be used as the
27
28
29 excellent solvents for saponin extraction.
30
31
32
33
34 Extraction of flavonoid
35
36 The medicinal herb EF has been historically used in combination with other
37
38
39 herbs to treat skeletal diseases in traditional Chinese medicines. Its main active
40
41
components are flavonoids (e.g., icariine), which exhibit multiple biological activities
42
43
44 including promotion of sexual function, protection of the nervous system, and
45
46
prevention of cardiovascular diseases.28,29
47
48
49 To compare the flavonoid extraction efficiency of DESs and conventional
50
51
organic solvents, the extraction yields of icariine, which was the main bioactive
52
53
54 flavonoid in EE, were detected and determined by HPLC (Fig. 2e). The results
55
56
57
indicated that the extraction efficiency was influenced by the types of DES solvents,
58
59 13
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 14 of 33

1
2
3
4 and different types of DES resulted in different extraction yields. The flavonoid
5
6 icariine was highly extractable in Pro-based DESs, while sugar-based and
7
8
9 alcohol-based DESs demonstrated lower extractability for icariine (Fig. 3e). Our data
10
11 showed that 14 types of DES, such as Pro-Mu, Pro-Mal, Pro-La, Bet-Maa and
12
13
14 Chcl-Du, exhibited higher yields of icariine compared with methanol. In sum, the
15
16 excellent properties of DESs highlight their potential as promising green solvents for
17
18
19 the extraction of flavonoid.
20
21
22
23
24 Generally, the extraction capacity of DES for bioactive natural products is correlated
25
26 with their physical-chemical properties, including H-bonding interactions, polarity,
27
28
29 viscosity and pH. The high extractability of phenolic acids with DES may be
30
31 attributed to H-bonding interactions between DES molecules and phenolic
32
33
34 compounds. The polarity of DESs is an important factor affecting their extraction
35
36 efficiency. Since the synthesized DESs have relatively higher polarity, they
37
38
39 demonstrate lower extraction yields for low-polar natural compounds, e.g.,
40
41
anthraquinones in the present study. Compared with conventional solvents, the high
42
43
44 viscosity of DESs restricts their application as extraction solvents. Our results
45
46
demonstrate that the less viscous La-based DESs show higher efficiency for
47
48
49 natural-occurring compounds than other DESs. Moreover, the extractability of DESs
50
51
is affected by acidity and basicity of natural compounds and DESs. Our data indicate
52
53
54 that extraction yields of carboxylic acids-based DES for alkaloids are 1.5 times higher
55
56
57
than other DESs, most likely due to the acid-base neutralization reaction.
58
59 14
60
ACS Paragon Plus Environment
Page 15 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4
5
6 Optimization of the operational conditions for alkaloids extraction using RSM.
7
8
9
10
11 Optimization of extraction conditions
12
13
14 The above extraction investigations showcased the tuneability of DESs as
15
16 designer solvents to selectively extract bioactive compounds from biomass. Next, the
17
18
19 effect of key parameters on DES extraction was investigated in detail using BR as a
20
21 practical example. The variables affecting the extraction capacity, including the
22
23
24 concentration of water (%) in extraction solvent, temperature, extraction time and
25
26 solid-liquid ratio, were optimized in order to obtain the optimum conditions of
27
28
29 extraction. RSM, a valuable statistical technique to determine the optimal values of
30
31 the independent variables and to enable the user to effectively investigate the effects
32
33
34 of multiple factors and their interactions, was employed to produce the highest
35
36 extraction efficiency in this study.30
37
38
39 In this section, UAE was selected as the extraction method. As described above,
40
41
ChCl-La produced the highest extractability of alkaloids from BR. In initial screening,
42
43
44 the eutectic mixtures of ChCl-La were firstly prepared at mole ratios of 2 : 3, 1 : 2 and
45
46
1 : 3 to extract alkaloids from BR, and the results demonstrated that ChCl-La of 1 : 2
47
48
49 ratio showed relatively higher extraction ability with no significant difference.
50
51
Subsequent preliminary experiments for UAE condition optimization revealed
52
53
54 that the extraction temperature owned a positive relationship with extraction yields
55
56
57
and the extraction efficiency was also enhanced with the decrease of solid-to-liquid
58
59 15
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 16 of 33

1
2
3
4 ratio. Therefore, the following four independent variables were chosen for
5
6 RSM-based optimization: water addition (%) in the selected DES (10%-50%),
7
8
9 temperature (40-60 ºC), extraction time (10-30 min) and solid-to-liquid ratio (20-60
10
11 mg/mL). Table 2 lists the coded and uncoded levels of the independent variables.
12
13
14 Extraction yields of Jat, Pal and Ber in BR were investigated as the response function
15
16 of the BBD method. The experimental orders and levels of coded and uncoded
17
18
19 variables are summarized in Supporting Information.
20
21 The variables and response were processed to build a quadratic multiple
22
23
24 regression model for alkaloids. The model quality was evaluated in terms of the
25
26 square of correlation coefficient (R2) and the lack-of-fit by the analysis of variance
27
28
29 (ANOVA) at the 95% confidence level (Supporting Information). The resulting R2
30
31 value was 0.9277, indicating that the experimental data were in relatively good
32
33
34 agreement with predicted extraction yields. Besides, the lack-of-fit, which evaluates
35
36 the failure of the model to represent the data in the experimental domain points, was
37
38
39 insignificant for the response with p-value of 0.124 (>0.05). This implied that the
40
41
model equation was adequate. The model was expressed as second order polynominal
42
43
44 quadratic equations for the extraction yield (Y) and coded factors (A, B, C and D) as
45
46
follows:
47
48
49 Y = 2.93 + 0.081A + 0.031B + 0.039C - 0.12D - 0.0043AB - 0.075 AC + 0.0045AD - 0.02BC - 0.015BD
50 2 2 2 2
-0.022CD - 0.16 A + 0.0024B - 0.016C + 0.053D
51
52
53
54
55 Statistical analysis and 3D response plots (Fig. 4) illustrate the significant variables
56
57
58 affecting extraction yields of the alkaloids and the interaction effects between the
59 16
60
ACS Paragon Plus Environment
Page 17 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 variables. In the model, the concentration of water (%) in extraction solvent (variable
5
6 A) and the solid-to-liquid ratio (variable D) led to significantly different extraction
7
8
9 yields. Generally, the extraction efficiency for alkaloids increased with lower
10
11 solid-to-liquid ratio. The amount of extracted increased with increasing percentage of
12
13
14 water until the water concentration reached 35%, but an excessively high
15
16 concentration of water decreased the yields of alkaloids, most likely due to the
17
18
19 decreased interaction between DES and alkaloids with increasing polarity of the
20
21 mixture. On the other hand, extraction time (variable C) had a mild positive effect on
22
23
24 extraction efficiency of alkaloids (p = 0.0245) and temperature (variable B) showed an
25
26 insignificant effect (p = 0.0689).
27
28
29
30
31 Validation of the optimized conditions
32
33
34 Triplicate experiments were carried out under the optimized conditions and mean
35
36 values of experimental results were compared with the predicted values. The alkaloids
37
38
39 extraction model yielded optimal conditions with A = 34% v/v, B = 60 ºC, C = 21 min,
40
41
D = 20 mg/mL. Under these optimum conditions, the predicted extraction yield was
42
43
44 3.09 ± 0.06%. The experimental values were close to the predicted value (3.11 ±
45
46
0.08%) obtained using RSM. The results obtained through confirmation experiments
47
48
49 indicated the suitability of the developed quadratic models and it may be noted that
50
51
these optimal values are valid within the specified range of process parameters
52
53
54 (Supporting Information).
55
56
57
58
59 17
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 18 of 33

1
2
3
4 CONCLUSIONS
5
6 In the present study, a green DES extraction method was described for
7
8
9 comprehensive extraction of different types of bioactive natural compounds from
10
11 HMs. The tailor-made DESs proved to be efficient solvents for the extraction of
12
13
14 alkaloids, phenolic acids, flavonoids and saponins, but demonstrated lower
15
16 extractability for anthraquinones. Subsequent optimization for alkaloids extraction
17
18
19 using RSM indicated that the concentration of water in DES-water mixture and
20
21 solid-to-liquid ratio were key parameters affecting extraction yields. This work
22
23
24 provided practical examples showcasing the tuneability of DESs that selectively and
25
26 efficiently extract bioactive compounds from biomaterials. Moreover, DES solvents
27
28
29 were firstly introduced to extract alkaloids, and ChCl-La was shown to be
30
31 significantly more efficient than previous methods requiring the consumption of
32
33
34 organic solvents. This study holds promise for further utilization of DESs in
35
36 pharmaceutical, biochemical, and food industries for the extraction of bioactive
37
38
39 natural products.
40
41
42
43
44 ACKNOWLEDGEMENTS
45
46
This work was supported by the National Natural Science Foundation of China (No.
47
48
49 81473343), Foundation for Innovative Research Groups of the National Natural
50
51
Science Foundation of China (No. 81421005), Program for New Century Excellent
52
53
54 Talents in University (No. NECT-13-1034), Natural Science Foundation of Jiangsu
55
56
57
Province (No. BK20130652) and a Project Funded by the Priority Academic Program
58
59 18
60
ACS Paragon Plus Environment
Page 19 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 Development of Jiangsu Higher Education Institutions.
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 19
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 20 of 33

1
2
3
4 REFERENCES
5
6 (1) Paiva, A.; Craveiro, R.; Aroso, I.; Martins, M.; Reis, R. L.; Duarte, A. R. C.
7
8
9 Natural Deep Eutectic Solvents-Solvents for the 21st Century. ACS Sustainable
10
11 Chem. Eng. 2014, 2, 1063-1071.
12
13
14 (2) Chemat, F.; Vian, M. A.; Cravotto, G. Green Extraction of Natural Products:
15
16 Concept and Principles. Int. J. Mol. Sci. 2012, 13, 8615-8627.
17
18
19 (3) Duan, L.; Guo, L.; Liu, K.; Liu, E. H.; Li, P. Characterization and classification of
20
21 seven Citrus herbs by liquid chromatography–quadrupole time-of-flight mass
22
23
24 spectrometry and genetic algorithm optimized support vector machines. J.
25
26 Chromatogr. A, 2014, 1339, 118-127.
27
28
29 (4) W. Bi, M. Tian and K. H. Row, Evaluation of alcohol-based deep eutectic solvent
30
31 in extraction and determination of flavonoids with response surface methodology
32
33
34 optimization. J. Chromatogr. A 1285 (2013) 22-30.
35
36 (5) Abbott, A. P.; Capper, G.; Davies, D. L.; Rasheed, R. K.; Tambyrajah, V. Novel
37
38
39 solvent properties of choline chloride/urea mixtures. Chem. Commun. 2003, 1,
40
41
70-71.
42
43
44 (6) Dai, Y.; Witkamp, G. J.; Verpoorte, R.; Choi, Y. H. Tailoring properties of natural
45
46
deep eutectic solvents with water to facilitate their applications. Food Chem.
47
48
49 2015, 187, 14-19.
50
51
(7) Gu, T.; Zhang, M.; Tan; T.; Chen, J.; Li, Z.; Zhang, Q.; Qiu, H. Deep eutectic
52
53
54 solvents as novel extraction media for phenolic compounds from model oil. Chem.
55
56
57
Commun. 2014, 50, 11749-11752.
58
59 20
60
ACS Paragon Plus Environment
Page 21 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 (8) Jeong, K. M.; Zhao, J.; Jin, Y.; Heo, S. R.; Han, S. Y.; Yoo, D. E.; Lee, J. Highly
5
6 efficient extraction of anthocyanins from grape skin using deep eutectic solvents
7
8
9 as green and tunable media. Arch. Pharmacal Res. 2015, 38, 2143-2152.
10
11 (9) Yilmaz, E.; Soylak, M. Ultrasound assisted-deep eutectic solvent extraction of
12
13
14 iron from sheep, bovine and chicken liver samples. Talanta 2015, 136, 170-173.
15
16 (10) Wagle, D. V.; Zhao, H.; Baker, G. A. Deep eutectic solvents: sustainable media
17
18
19 for nanoscale and functional materials. Acc. Chem. Res. 2014, 47, 2299-2308.
20
21 (11) Zhao, H. DNA stability in ionic liquids and deep eutectic solvents, J. Appl. Chem.
22
23
24 Biotechnol. 2015, 90, 19-25.
25
26 (12) Li, C.; Li, D.; Zou, S.; Li, Z.; Yin, J.; Wang, A.; Cui, Y.; Yao, Z.; Zhao, Q.
27
28
29 Extraction desulfurization process of fuels with ammonium-based deep eutectic
30
31 solvents. Green Chem. 2013, 15, 2793-2799.
32
33
34 (13) Phadtare, S. B.; Shankarling, G. S. Halogenation reactions in biodegradable
35
36 solvent: Efficient bromination of substituted 1-aminoanthra-9,10-quinone in deep
37
38
39 eutectic solvent (choline chloride : urea). Green Chem. 2010, 12, 458-462.
40
41
(14) Dai, Y.; Witkamp, G. J.; Verpoorte, R.; Choi, Y. H. Natural deep eutectic solvents
42
43
44 as a new extraction media for phenolic metabolites in Carthamus tinctorius L.
45
46
Anal. Chem. 2013, 85, 6272-6278.
47
48
49 (15) Nam, M. W.; Zhao, J.; Lee, M. S.; Jeong, J. H.; Lee, J. Enhanced extraction of
50
51
bioactive natural products using tailor-made deep eutectic solvents: Application
52
53
54 to flavonoid extraction from Flos sophorae. Green Chem. 2015, 17, 1718-1727.
55
56
57
(16) Xia, B.; Yan, D.; Bai, Y.; Xie, J.; Cao, Y.; Liao, D.; Lin, L. Determination of
58
59 21
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 22 of 33

1
2
3
4 phenolic acids in Prunella vulgaris L.: a safe and green extraction method using
5
6 alcohol-based deep eutectic solvents. Anal. Methods 2015, 7, 9354-9364.
7
8
9 (17) Wei, Z. F.; Wang, X. Q.; Peng, X.; Wang, W.; Zhao, C. J.; Zu, Y. G.; Fu, Y. J. Fast
10
11 and green extraction and separation of main bioactive flavonoids from Radix
12
13
14 Scutellariae. Ind. Crops Prod. 2015, 63, 175-181.
15
16 (18) Li, J.; Han, Z.; Zou, Y.; Yu, B. Efficient extraction of major catechins in Camellia
17
18
19 sinensis leaves using green choline chloride-based deep eutectic solvents. RSC
20
21 Adv. 2015, 5, 93937-93944.
22
23
24 (19) Zhao, B. Y.; Xu, P.; Yang, F. X.; Wu, H.; Zong, M. H.; Lou, W. Y. Biocompatible
25
26 Deep Eutectic Solvents Based on Choline Chloride: Characterization and
27
28
29 Application to the Extraction of Rutin fromSophora japonica. ACS Sustainable
30
31 Chem Eng. 2015, 3, 2746-2755.
32
33
34 (20) Dai, Y.; van Spronsen, J.; Witkamp, G. J.; Verpoorte, R.; Choi, Y. H. Natural deep
35
36 eutectic solvents as new potential media for green technology. Anal. Chim. Acta.
37
38
39 2013, 766, 61-68.
40
41
(21) Song, S.; Qiu, M.; Chu, Y.; Chen, D.; Wang, X.; Su, A.; Wu, Z. Downregulation
42
43
44 of Cellular c-Jun N-Terminal Protein Kinase and NF-κB Activation by Berberine
45
46
May Result in Inhibition of Herpes Simplex Virus Replication. Antimicrob.
47
48
49 Agents Chemother. 2014, 58, 5068-5078.
50
51
(22) Liu, R.; Cao, Z.; Pan, Y.; Zhang, G.; Yang, P.; Guo, P.; Zhou, Q. Jatrorrhizine
52
53
54 hydrochloride inhibits the proliferation and neovascularization of C8161
55
56
57
metastatic melanoma cells. Anti-Cancer Drugs 2013, 24, 667-676.
58
59 22
60
ACS Paragon Plus Environment
Page 23 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 (23) Zhou, L.; Zuo, Z.; Chow, M. S. Danshen: an overview of its chemistry,
5
6 pharmacology, pharmacokinetics, and clinical use. J. Clin. Pharmacol. 2005, 45,
7
8
9 1345-1359.
10
11 (24) Liu, P.; Yang, H.; Long, F.; Hao, H.P.; Xu, X.; Liu, Y.; Shi, X.W.; Zhang, D.D.
12
13
14 Zheng, H. C.; Wen, Q. Y.; Li, W. W.; Ji, H.; Jiang, X.J.; Zhang, B.L.; Qi, L.W.; Li,
15
16 P. Bioactive Equivalence of Combinatorial Components Identified in Screening
17
18
19 of an Herbal Medicine. Pharm. Res. 2014, 31, 1788-1800.
20
21 (25) Azelmat, J.; Larente, J. F.; Grenier, D. The anthraquinone rhein exhibits
22
23
24 synergistic antibacterial activity in association with metronidazole or natural
25
26 compounds and attenuates virulence gene expression in Porphyromonas
27
28
29 gingivalis. Arch. Oral Biol. 2015, 60, 342-346.
30
31 (26) Wang, M.; Zhang, X. J.; Liu, F.; Hu, Y.; He, C.; Li, P.; Su, H.; Wan, J. B.
32
33
34 Saponins isolated from the leaves of Panax notoginseng protect against alcoholic
35
36 liver injury via inhibiting ethanol-induced oxidative stress and gut-derived
37
38
39 endotoxin-mediated inflammation. J. Funct. Foods 2015, 19, 214-224.
40
41
(27) Lee, C. H.; Kim, J. H. The anthraquinone rhein exhibits synergistic antibacterial
42
43
44 activity in association with metronidazole or natural compounds and attenuates
45
46
virulence gene expression in Porphyromonas gingivalis. J. Ginseng Res. 2014, 38,
47
48
49 161-166.
50
51
(28) Makarova, M. N.; Pozharitskaya, O. N.; Shikov, A. N.; Tesakova, S. V.; Makarov,
52
53
54 V. G.; Tikhonov, V. P. Effect of lipid-based suspension of Epimedium koreanum
55
56
57
Nakai extract on sexual behavior in rats. J. Ethnopharmacol. 2007, 114, 412-416.
58
59 23
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 24 of 33

1
2
3
4 (29) Xu, H. B. Huang, Z. Q. Icariin enhances endothelial nitric-oxide synthase
5
6 expression on human endothelial cells in vitro. Vasc. Pharmacol. 2007, 47, 18-24.
7
8
9 (30) Cheng, X. L.; Qi, L. W.; Wang, Q.; Liu, X. G.; Boubertakh, B.; Wan, J. Y.; Liu, E.
10
11 H.; Li, P. Highly efficient sample preparation and quantification of constituents
12
13
14 from traditional Chinese herbal medicines using matrix solid-phase dispersion
15
16 extraction and UPLC-MS/MS. Analyst 2013, 138, 2279-2288.
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 24
60
ACS Paragon Plus Environment
Page 25 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 Figure captions
5
6 Fig. 1. Chemical structures of different type of natural compounds. (1) Aloe-emodin;
7
8
9 (2) Rhein; (3) Emodin; (4) Physcion; (5) chrysophanol; (6) ginsenoside Rg1; (7)
10
11 notoginsenoside R1; (8) ginsenoside Rb1; (9) lithospermic acid; (10) rosmarinic acid;
12
13
14 (11) salvianolic acid; B (12) jatrorrhizine hydrochloride; (13) palmatine hydrochloride;
15
16 (14) berberine hydrochloride; (15) icariin.
17
18
19 Fig. 2. Representative HPLC chromatogram of (a) BR, (b) SMR, (c) RRR, (d) NRR
20
21 and (e) EF. (a1, jatrorrhizine hydrochloride; a2, palmatine hydrochloride; a3,
22
23
24 berberine hydrochloride; b1, rosmarinic acid; b2, lithospermic acid; b3, salvianolic
25
26 acid B; c1, aloe-emodin; c2, rhein; c3, emodin; c4, chrysophanol; c5, physcion; d1,
27
28
29 ginsenoside RG1; d2, ginsenoside RB1; d3, notoginsenoside R1; e1, icariine)
30
31 Fig. 3. Extraction yields (mg of extracted compounds per 100 g of HM powder) of
32
33
34 DESs for (a) alkaloid, (b) phenolic acid, (c) anthraquinone, (d) saponin, (e) flavonoid.
35
36 Numbers on horizontal axis are in accordance with the numbers in Table 1 and M
37
38
39 represented MeOH. ChCl-based DESs were indicated by green bars, Bet-based DESs
40
41
by orange bars and Pro-based DES by blue bars. Extraction efficiencies of DESs
42
43
44 compared with MeOH were indicated with * (p<0.05, mean value > MeOH) and #
45
46
(p>0.05). Error bars indicate the SD (n=3).
47
48
49 Fig. 4 Response surface plots of the model for extraction of alkaloids in BR.
50
51
52
53
54
55
56
57
58
59 25
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 26 of 33

1
2
3
4 Tables
5
6 Table 1 List of DESs initially tested for extraction.
7 No. Abbreviation HBA HBD Mole ratio
8 1 ChCl-Glu D-Glucose 1 : 1 (2)
9 2 ChCl-Ma Maltose 2 : 1 (4)
10 3 ChCl-Suc Sucrose 4 : 1 (3)
11 4 ChCl-Xyl Xylitol 5 : 2 (5)
12
5 ChCl-Sor D-Sorbitol 3 : 1 (3)
13
6 ChCl-Eg Ethylene Glycol 1:2
14
15 7 ChCl-Gly Glycerol 1:2
16 8 ChCl-Ca Citric Acid 2:1
Choline
17 9 ChCl-La Laevulinic Acid 1:2
Chloride
18 10 ChCl-Ox Oxalic Acid 1:1
19 11 ChCl-Lac Lactic Acid 1:1
20
12 ChCl-Maa DL-Malic Acid 1:1
21
13 ChCl-Mal Malonate 1 : 1 (1)
22
23 14 ChCl-Ur Urea 1:2
24 15 ChCl-Mu 1-Methylurea 1 : 1 (1)
25 16 ChCl-Du N,N'-Dimethylurea 1 : 1 (2)
26 17 ChCl-Am Acetamide 1 : 1 (1)
27 18 Bet-Glu D-Glucose 1 : 1 (1)
28 19 Bet-Ma Maltose 5 : 2 (6)
29
20 Bet-Suc Sucrose 2 : 1 (7)
30
31 21 Bet-Xyl Xylitol 1 : 1 (1)

32 22 Bet-Sor D-Sorbitol 1 : 1.2 (3)

33 23 Bet-Eg Ethylene Glycol 1 : 2 (1)
34 24 Bet-Gly Betaine Glycerol 1:2
35 25 Bet-Ca Citric Acid 2 : 1 (6)
36 26 Bet-La Laevulinic Acid 1:2
37
27 Bet-Lac Lactic Acid 1 : 1 (1)
38
39 28 Bet-Maa DL-Malic Acid 1 : 1 (1)

40 29 Bet-Ur Urea 1 : 1 (2)

41 30 Bet-Mu 1-Methylurea 1 : 1 (2)
42 31 Pro-Glu D-Glucose 1 : 1 (5)
43 32 Pro-Suc Sucrose 2 : 1 (10)
44 33 Pro-Sor D-Sorbitol 1 : 2 (4)
45
34 Pro-Gly Glycerol 2:5
46
35 Pro-Ca Citric Acid 1 : 1 (2)
47
48 36 Pro-La Laevulinic Acid 1:2
49 37 Pro-Ox L-Proline Oxalic Acid 1:1
50 38 Pro-Lac Lactic Acid 1:1
51 39 Pro-Maa DL-Malic Acid 1 : 1 (1)
52 40 Pro-Mal Malonate 1 : 1 (2)
53
41 Pro-Ur Urea 1 : 1 (3)
54
42 Pro-Mu 1-Methylurea 1 : 1 (2)
55
56 43 Pro-Am Acetamide 1 : 1 (2)
57
58
59 26
60
ACS Paragon Plus Environment
Page 27 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 Table 2 independent variables and levels used for BBD
5
Level
6 No. Variables
-1 0 1
7
8 A Water content (%) 10 30 50
9 B Temperature (ºC) 40 50 60
10 C Time (min) 10 20 30
11 D Solid/Liquid ratio (mg / mL) 20 40 60
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 27
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 28 of 33

1
2
3
4 Figure graphics
5
6 Fig. 1
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 28
60
ACS Paragon Plus Environment
Page 29 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 Fig. 2
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 29
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 30 of 33

1
2
3
4 Fig. 3
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 30
60
ACS Paragon Plus Environment
Page 31 of 33 ACS Sustainable Chemistry & Engineering

1
2
3
4 Fig. 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 31
60
ACS Paragon Plus Environment
 ACS Sustainable Chemistry & Engineering Page 32 of 33

1
2
3
4 Table of Contents graphic
5
6
7
8
9 Comprehensive evaluation of deep eutectic solvents in extraction of bioactive
10
11 natural products
12
13
14 Li Duan, Li-Li Dou, Long Guo, Ping Li, E-Hu Liu
15
16
17
18
19 Synopsis: The potential and effectiveness of DESs for extraction of different types of
20
21 natural compounds from biomass was evaluated.
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 32
60
ACS Paragon Plus Environment
Page 33 of 33 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 81x47mm (300 x 300 DPI)
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 Paragon Plus Environment