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Article Cite This: J. Agric. Food Chem. 2018, 66, 1008−1014

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An Efficient Strategy Based on Liquid−Liquid Extraction with ThreePhase Solvent System and High Speed Counter-Current Chromatography for Rapid Enrichment and Separation of Epimers of Minor Bufadienolide from Toad Meat Denglang Zou,† Xuelin Zhu,† Fan Zhang,† Yurong Du,‡ Jianbin Ma,*,‡ and Renwang Jiang*,† †

Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China ‡ Key Laboratory of Medicinal Plant and Animal Resources of Tibet Plateau, School of Life and Geography Science, Qinghai Normal University, Xining 810000, P. R. China S Supporting Information *

ABSTRACT: This study presents an efficient strategy based on liquid−liquid extraction with three-phase solvent system and high speed counter-current chromatography for rapid enrichment and separation of epimers of minor bufadienolide from toad meat. The reflux extraction conditions were optimized by response surface methodology first, and a novel three-phase solvent system composed of n-hexane/methyl acetate/acetonitrile/water (3:6:5:5, v/v) was developed for liquid−liquid extraction of the crude extract. This integrative extraction process could enrich minor bufadienolide from complex matrix efficiently and minimize the loss of minor targets induced by repeated extraction with different kinds of organic solvents occurring in the classical liquid two-phase extraction. As a result, four epimers of minor bufadienolide were greatly enriched in the middle phase and total content of these epimers of minor bufadienolide was increased from 3.25% to 46.23%. Then, the enriched four epimers were separated by HSCCC with a two-phase solvent system composed of chloroform/methanol/water (4:2:2, v/v) successfully. Furthermore, we tested Na+,K+-ATPase (NKA) inhibitory effect of the four epimers. 3β-Isomers of bufadienolide showed stronger (>8-fold) inhibitory activity than 3α-isomers. The characterization of minor bufadienolide in toad meat and their significant difference of inhibitory effect on NKA would promote the further quantitative analysis and safety evaluation of toad meat as a food source. KEYWORDS: toad meat, three-phase solvent system, liquid−liquid extraction, high speed counter-current chromatography, epimers of minor bufadienolide, response surface methodology

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INTRODUCTION

Toad meat contains huge amounts of protein and fat, and this made it rather hard to separate minor bufadienolide surrounded by the complex matrix directly. So optimized extraction conditions by response surface methodology (RSM) and a proper pretreatment technique were needed to enrich these minor bufadienolides in toad meat before further separation. Liquid−liquid extraction (LLE) is one of the oldest and most widely used techniques for pretreatment of crude sample due to its low cost, high efficiency, and simple procedure.5−7 However, the repeated extraction with organic solvents of various polarities would lead to serious loss of these minor targets.8,9 A three-phase solvent system could form three mutually immiscible phases composed of hydrophobic upper phase (UP), moderately polar middle phase (MP), and hydrophilic lower phase (LP).10 It showed great advantages to pretreat the biosamples. A highly polar matrix, such as protein and saccharide, could be well distributed in the hydrophilic lower phase. Meanwhile, a nonpolar matrix, such as

Toad is a widely distributed amphibian species throughout the agricultural landscapes all around the world.1 Despite the great medicinal value of toad venom and toad skin, the meat of toad is also a kind of famous and delicious food in some parts of the world, such as China, Australia, Peru, Cambodia, and Thailand. A scientist from Australia suggested that toads were sources of healthy food, rich in protein and essential omega 3.2 Dishes named Smoked toad, Toad leg, Clay pot, and LaSi, where the main ingredient is toad meat, are rather popular in China and Australia. However, due to the toxic bufadienolide in their back and skin, there were some cases in which people who ate toad meat and related dishes had some adverse reactions, such as vomiting, diarrhea, convulsion, and even death.3 The toxicity of bufadienolide has been attributed mainly to the inhibitory effect on the Na+,K+- ATPase (NKA) and the alteration of intracellular calcium stores.4 Our preliminary analysis revealed that minor bufadienolide also exists in carefully handled toad meat. So it is rather important and essential to characterize the corresponding minor bufadienolide of toad meat with an efficient enrichment and separation method for further quantitative analysis and safety evaluation. © 2018 American Chemical Society

Received: Revised: Accepted: Published: 1008

November 15, 2017 December 30, 2017 January 4, 2018 January 4, 2018 DOI: 10.1021/acs.jafc.7b05310 J. Agric. Food Chem. 2018, 66, 1008−1014

Article

Journal of Agricultural and Food Chemistry

the coefficient of determination R2, and F-test at a probability (p) of 0.05 was employed to evaluate its statistical significance.38,39 Surface plots derived from the optimized model could reveal the interactive effects of each factors clearly. Finally, the optimization of reflux extraction process was conducted. The optimum goal was to obtain a maximum response value under the same operating conditions, and the desirability values (range from 0 to 1) were employed to evaluate the credibility of the optimum conditions.40,41 Reflux Extraction. The toad meat (5 kg) were obtained by removing head, skin, viscera, and other tissues from Bufo gargarizans. It was treated by mince machine and underwent three reflux extractions using 83% ethanol at 79 °C. The extraction time was 2.1 h, and liquid/ solid ratio was 20:1. All filtrates were combined and concentrated at 45 °C under reduced pressure. The residue was stored in a refrigerator for use in the subsequent liquid−liquid extraction. High Performance Liquid Chromatography (HPLC) Analysis. The apparatus used was an Agilent 1200 system (Agilent Technologies Co. Ltd., USA). The Agilent 1200 system consists of a G1312A solvent delivery unit, a G1315D DAD unit, a G1316A column thermostat, a G1329A autosampler, and an Agilent ChemStation. The column was a Phenomenex Luna-C18 analytical column (250 mm × 4.6 mm, 5 μm). The mobile phase was composed of 0.1% formic acid water (A) and acetonitrile (B). The gradient elution program was 10−70% B in 0−40 min. The flow rate was 1.0 mL/min, and the detection wavelength was set at 296 nm. Optimization of Suitable Three-Phase Solvent System for Enriching the Target Compounds. Toad meat crude extract was screened with a series of three-phase solvent systems composed of nhexane/methyl acetate/acetonitrile/water to obtain a maximum partition coefficient KM/U (KM/U = AMP/AUP, A means the peak area of the HPLC chromatogram) and KM/L (KM/L = AMP/ALP) of the minor bufadienolide.42 The KM/U and KM/L values were determined using HPLC as the following partition coefficient K of HSCCC. Selection of Two-Phase Solvent System. The two-phase solvent system was screened on the basis of the partition coefficient (K) determined by HPLC. In brief, 5 mg of enriched sample was added in the equilibrated two-phase solvent system and shaken violently to achieve equilibration of target compounds between lower and upper phases, and kept still for 10 min. 2 mL of each phase was evaporated and analyzed by HPLC. The K value was determined as the peak area of each target compound in stationary phase divided by that in mobile phase. HSCCC Separation Procedure. The HSCCC experiment was conducted on a TBE-300B high-speed counter-current chromatography system (Shanghai Tauto Biotech, Shanghai, China) with a total coil volume of 280 mL used (tube diameter = 1.6 mm). The system was equipped with a TBP-5002 constant-flow pump, a UV500 detector module, a N2000 workstation, and a DC-0506 constant temperature circulating implement. The column was washed with ethanol to remove any impurity before the separation procedure. The column was first filled with the stationary phase. After the apparatus was rotated at 900 rpm, the mobile phase was pumped into the column at a flow rate of 2.0 mL/min. After hydrodynamic equilibration was achieved, enriched sample solution (200 mg of the sample was dissolved in 10 mL of both phases) was injected into the separation column, the chromatogram was recorded at a wavelength of 296 nm, and target fractions were collected manually according to the HSCCC chromatogram. Identification of Target Compounds. Identification of the HSCCC peak fractions was performed by spectroscopic analysis. 1H NMR and 13C NMR spectra were measured in methanol-d4 on a Bruker 300 NMR spectrometer using tetramethylsilane (TMS) as the internal standard. NKA Inhibition Assay. The NKA inhibitory activities of four epimers were determined by colorimetry as previously reported.43,44 In brief, NKA was preincubated in incubation medium (3 mM MgCl2, 3 mM Na-phosphate, 40 mM Tris, pH 7.0) with increasing concentrations of four epimers at 37 °C for 2 h. The NKA activity is subsequently determined by liberated Pi for ATP hydrolysis after

fatty acid, could be well distributed in the hydrophobic upper phase.11,12 This integrative process would lead to remarkable enrichment of moderately polar targets in the middle phase with slight loss. Thus, an optimized three-phase solvent system with proper partition coefficient was a unique way to enrich these minor bufadienolides from toad meat full of complex matrix. Bufadienolide is typically a C-24 steroid with a characteristic α-pyrone ring at the C-17 position and has traditionally been separated and purified from toad venom by column chromatography.13 However, the performance of these conventional methods are severely restricted by labor consumption, solvent consumption, and serious sample loss.14,15 As to efficient separation technique with less sample loss for the minor bufadienolide, high speed counter-current chromatography (HSCCC) would be a good choice.16−18 HSCCC is a kind of solid support free liquid chromatography based on liquid−liquid partition, in which samples have complete recovery for no irreversible adsorption caused by solid matrix occurred in the conventional chromatographic techniques.19−22 High-performance separation, online detection, and automatic control make it rather easy to realize efficient preparation of target compounds. In addition, components with similar skeleton and polarity could be separated easily and efficiently by HSCCC.23 It has been widely applied for separation of bioactive compounds from natural products and biosamples.20,24−27 In the current paper, an efficient strategy based on liquid− liquid extraction with three-phase solvent system and high speed counter-current chromatography was established to rapidly enrich and separate four epimers of minor bufadienolide from toad meat. We also evaluated NKA inhibitory effect of the purified epimers.

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MATERIALS AND METHODS

Materials. All solvents used in extraction and HSCCC were of analytical grade (Guangdong Guanghua Sci-Tech Co., Ltd., China).The acetonitrile used for HPLC analysis was of chromatographic grade and was purchased from Mreda Technology Inc. (USA). All reagents used in the NKA inhibition test were purchased from Sigma-Aldrich. Methanol-d4 was employed for NMR analysis. Toad meat was purchased from GuangZhou (GuangDong, China). Optimization of Reflux Extraction Conditions. RSM was employed to optimize the reflux extraction conditions of the minor bufadienolide on the basis of single-factor experiments.28−31 A Box− Behnken design (Design-Expert v10.0.1) with three independent variables was employed to obtain the best combination of extraction variables for the yield of minor bufadienolide.32 The variables used were as follows: ethanol concentration (X1), extraction time (X2), extraction temperature (X3). The W (W = ∑{Ai(peak area)}, where Ai(peak area) was the proportion of peak area of each target in HPLC analysis) was chosen as the response value. Seventeen experiments composed of 12 factorial experiments and five replicates at the center point (Table S1) were designed in the RSM optimization process.33,34 The relationship between the independent and dependent variables was developed by the least-squares methodology.35−37 The secondorder polynomial equation was employed to fit the experimental data as follows: 3

Y = β0 +

3

2

3

∑ βi Xi + ∑ βiiXi 2 + ∑ ∑ i=1

i=1

βijXij

i=1 j=i+1

where Y represents the predicted response, β0, βi and βii, βij are the regression coefficients of variables for intercept, linear, quadratic, and interaction terms, respectively, and Xi and Xj are independent variables (i ≠ j). The fitness of the polynomial model equation is expressed by 1009

DOI: 10.1021/acs.jafc.7b05310 J. Agric. Food Chem. 2018, 66, 1008−1014

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Journal of Agricultural and Food Chemistry

Figure 1. Response surface showing the interactive effect of the variables on W: (I) extraction time vs ethanol concentration; (II) extraction temperature vs ethanol concentration; (III) extraction temperature vs extraction time.

Table 1. KM/U and KM/L Values Given by the Liquid−Liquid Extraction with Three-Phase Solvent Systems three-phase solvent systems

A

B

C

D

no.

n-hexane

methyl acetate

acetonitrile

water

KM/U

KM/L

KM/U

KM/L

KM/U

KM/L

KM/U

KM/L

1 2 3 4 5 6

5 5 5 4 3 2

5 6 7 6 6 6

5 5 5 5 5 5

5 5 5 5 5 5

7.76 10.20 9.06 12.57 16.91 25.36

19.79 21.36 22.23 18.87 18.03 16.56

6.80 8.61 7.78 10.23 13.09 20.01

20.81 22.54 24.22 20.04 19.09 17.64

5.78 7.04 6.47 8.09 10.63 17.51

23.87 27.05 29.51 23.52 21.64 20.54

5.35 6.41 5.94 7.27 9.78 15.31

27.98 31.21 32.46 25.76 23.87 22.54

incubation for a further 10 min in standard assay medium (130 mM NaCl, 20 mM KCl, 4 mM MgCl2, and 3 mM ATP).

of relative yield of four epimers led to the subsequent decrease of W, and these results were consistent with the single-factor experiments. According to the RSM optimization, the maximum W (3.28%) could be predicted under the optimal conditions (ethanol concentration of 83%, extraction time of 2.1 h, and extraction temperature of 79 °C). The reflux extraction of toad meat was conducted under the optimal conditions, and it led to a satisfactory result with W = 3.25%. Optimization of Suitable Three-Phase Solvent System for Enriching the Target Compounds. Theoretically, LLE could be used to enrich the target compounds by a solvent system with proper partition coefficient, and good results could be obtained in LLE when the target compounds distributed in only one phase.9,47,48 As for two-phase solvent system, the extract of biosamples needed to be treated with organic solvent of various polarities, and these repeated extractions with different solvents would lead to serious loss of minor target components. Three-phase solvent system could form three mutually immiscible phases composed of hydrophobic UP, moderately polar MP, and hydrophilic LP.49 Hdrophobic UP and hydrophilic LP could dramatically remove the high polar and nonpolar matrix from crude extract. The minor target components could be enriched significantly in moderately polar MP with a proper three-phase solvent system of maximum KM/U and KM/L values. A series of solvent systems composed of n-hexane/methyl acetate/acetonitrile/water (HMAW) were screened, the KM/U and KM/L values of the minor bufadienolde were measured, and the results have been summarized in Table 1. First, HMAW (5:5:5:5, v/v) was used to test the distribution of the target compounds. It was found that the target compounds mainly distributed in MP and UP. Considering the moderate polarity of target compounds, methyl acetate was added to promote the target compounds transferring from UP to MP. However, it is not very significant, the KM/U values began to decrease slowly when the ratio of methyl acetate exceeded 7. So the ratio of n-hexane was decreased to adjust the

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RESULTS AND DISCUSSION Optimization of Reflux Extraction Conditions. According to the designed experiments and data fitness, the full quadratic models (R2 = 98.31) were more efficient for W compared with other model. The model is Y = −44.03 + 0.68X1 + 5.31X 2 + 0.34X3 + (2.05 × 10−2)X1X 2 + (5.01 × 10−4)X1X3 − (2.25 × 10−2)X 2X3 − (4.59 × 10−3)X12 − 1.24X 2 2 − (2.14 × 10−3)X32

The significance of the model was confirmed by analysis of variance (ANOVA). Each coefficient corresponding to the above equation indicated the interaction strength between each independent variable, and the significance was checked by pvalues.36,45 According to the ANOVA analysis shown in Table S2, the linear terms X1, X2, and X3, the interactive term X23, and the quadratic terms X12, X22, and X32 showed a significant effect (P < 0.05) on W. However, the interactive terms X12 and X13 did not show a significant effect (P > 0.05). The relationship between responses and experimental levels of each variable could be revealed visually by 3D plots, which were the graphic representations of the regression models. This approach also provides a way to evaluate the type of interaction between two test variables.41,46 Figure 1I shows the interaction between extraction time and ethanol concentration. Initially, W increased with increasing of extraction time and ethanol concentration, but eventually began to decrease again. Due to complex matrix in toad meat, the content of impurity and target compounds would increase simultaneously with the increasing of extraction time and ethanol concentration. When extraction time exceeded 2.25 h and ethanol concentration exceeded 85%, the relative increase rate of ∑Ai (Ai refers to peak area of the four epimers) would be slower than the relative increase rate of ∑Aj (Aj refers to peak area of the impurity). Thus, the decrease 1010

DOI: 10.1021/acs.jafc.7b05310 J. Agric. Food Chem. 2018, 66, 1008−1014

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Journal of Agricultural and Food Chemistry

Figure 2. HPLC chromatograms of the crude extract (I) and enriched sample by LLE with three-phase solvent system (II). Conditions: column, Phenomenex Luna-C18 analytical column (250 mm × 4.6 mm, 5 μm); mobile phase, 0.1% acid water (A) and acetonitrile (B), gradient elution program 10−70% B in 0−40 min; flow rate, 1.0 mL/min; detection wavelength, 296 nm.

Table 2. K Values of the Target Compounds

distribution of these minor bufadienolides between UP and MP. As can be seen in Table 1, the target compounds could greatly transfer from UP to MP with the ratio of n-hexane decreasing from 5 to 2. However, the settling time would increase significantly when the ratio of n-hexane was less than 2. Thus, HMAW (3:6:5:5, v/v) was used with large enough KM/U and KM/L values to enrich the minor bufadienolide from crude extract. LLE time was also optimized; it revealed that these minor bufadienolides could be well enriched in MP after 3 extraction times. As a result, 2.8 g of enriched sample was obtained. The total content of these minor bufadienolides was increased from 3.25% to 46.23% (Figure 2). Selection of HSCCC Experimental Conditions. The selection of solvent system is the most important step for a successful HSCCC separation. A suitable two-phase solvent system should provide an ideal range of partition coefficients guided by the chemical nature of target compounds.50,51 Many important characteristics, such as the sample polarity (evaluated from partition coefficient values), solubility, ionic form, and the capability to form complexes to consider, should be taken into account when we evaluate a solvent system. A suitable partition coefficient (K) should generally be in the range of 0.2−5,51,52 and the separation factor (α = K2/K1, K2 > K1) should be greater than 1.5 theoretically.41,53 A considerably smaller or larger K value would give lower resolution due to eluting the solute closer to the solvent front, or tends to give broader and more dilute peaks leading to a longer elution time. Furthermore, the solvent system must separate clearly and quickly into two phases as well in acceptable settling time.54,55 We optimized the solvent system for HSCCC separation based on the above stated principles. A series of two-phase solvent systems composed of chloroform/methanol/water were screened, the K values of target compounds were measured, and results have been summarized in Table 2. It was found that the ratio change of methanol would greatly affect the

K values

solvent system no.

chloroform

methanol

water

A

B

C

D

1 2 3 4 5 6 7

4 4 4 4 4 4 4

4 3 2 1 2 2 2

4 4 4 4 3 2 1

0.08 0.17 0.47 0.74 0.39 0.33 0.26

1.28 1.45 1.58 1.89 0.84 0.52 0.31

0.47 0.82 1.47 2.57 1.21 1.03 0.89

3.41 3.89 4.21 4.96 2.98 1.77 1.04

distribution of targets A and C between two phases, and the distribution of targets B and D would be affected significantly with the ratio change of water. Eventually, the system of chloroform/methanol/water (4:2:2, v/v/v) was selected for suitable K values and α values. In addition to the solvent system, the flow rate of the mobile phase and the revolution speed of the apparatus were also studied.56 Based on peak resolution, separation time, and retention rate of the stationary phase, a flow rate of 2.0 mL/min and a revolution speed of 900 rpm with 68% of retention rate of stationary phase were selected in the subsequent HSCCC separation procedures. Under the selected conditions, 23 mg of A, 26 mg of B, 7 mg of C, and 6 mg of D were obtained from 200 mg of enriched sample in less than 240 min (Figure 3). HPLC analysis showed that the purity of the whole target compounds was over 91%. Structural Identification. The chemical structures of the four epimers were elucidated by 1H NMR analysis, and results are as follows (Figure 4): 3-epi-Arenobufagin (A): HR-ESI-MS (m/z) 417.2284 [M + H]+; 1H NMR (300 MHz) 7.89 (1H, dd, J = 9.7, 2.6 Hz, H-22), δ 7.50 (1H, d, J = 2.6 Hz, H-21), 6.29 (1H, d, J = 9.7 Hz, H23), 4.35 (1H, d, J = 11.2 Hz, H-11), 4.12(1H, dd, J = 9.5, 7.1 Hz, H-17), 3.56 (1H, m, H-3), 1.14 (3H, s, H-19), 0.89 (3H, s, 1011

DOI: 10.1021/acs.jafc.7b05310 J. Agric. Food Chem. 2018, 66, 1008−1014

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Journal of Agricultural and Food Chemistry

direct consequence of their binding is an inhibition of the enzyme. It is the NKA inhibitory effect that is attributed to toxicity of bufadienolide for its alteration of intracellular calcium stores.4 According to the dose−response curves revealing inhibition of NKA by the four epimers from toad meat (Figure 5), 3β-

Figure 3. HSCCC chromatogram of the enriched sample using chloroform/methanol/water (4:2:2, v/v/v). Conditions: stationary phase, lower phase; flow rate, 2.0 mL/min; revolution speed, 900 rpm; sample amount, 200 mg; separation temperature, 25 °C; detection wavelength, 296 nm; retention of the stationary phase: 68%.

Figure 5. Inhibitory activity of four epimers against NKA at 37 °C.

isomers of bufadienolide showed stronger (>8-fold) inhibitory activity than 3α-isomers (IC50-A = 29.75 μM, IC50-B = 3.39 μM, IC50-C = 23.55 μM, IC50-D = 2.63 μM); this may mean that 3β-isomers were more toxic than 3α-isomers. It was also in accordance with the phenomenon that only the 3βbufadienolide was secreted in toad venom for chemical defense.60 This significant difference of inhibitory effect on NKA laid a foundation for the further safety evaluation of toad meat as a food source. In conclusion, this study presents an efficient strategy based on liquid−liquid extraction with three-phase solvent system and high speed counter-current chromatography for rapid enrichment and separation of epimers of minor bufadienolide from toad meat. LLE with three-phase solvent system could greatly enrich minor constituents from complex matrix, and it can minimize the loss of minor targets induced by repeated extraction with different kinds of organic solvents occurring in the classical liquid two-phase extraction. It also revealed that there existed significant difference of the inhibitory activity against NKA between the purified epimers. The results also demonstrated that HSCCC could be a powerful technique for separation of epimers from biosamples and natural products. The established strategy for enrichment and separation of four epimers would promote the characterization of minor bufadienolide of toad meat for further quantitative analysis and safety evaluation.

Figure 4. Chemical structures of 3-epi-arenobufagin (A), arenobufagin (B), 3-epi-bufalin (C), and bufalin (D).

H-18). These data are in agreement with earlier published data for 3-epi-arenobufagin.57 Arenobufagin (B): HR-ESI-MS (m/z) 417.2278 [M + H]+; 1 H NMR (300 MHz) 7.91 (1H, dd, J = 9.7, 2.6 Hz, H-22), δ 7.52 (1H, d, J = 2.6 Hz, H-21), 6.31 (1H, d, J = 9.7 Hz, H-23), 4.36 (1H, d, J = 11.2 Hz, H-11), 4.14(1H, dd, J = 9.7, 6.8 Hz, H-17), 4.03 (1H, s, H-3), 1.18 (3H, s, H-19), 0.90 (3H, s, H18). These data are in agreement with earlier published data for arenobufagin.4 3-epi-Bufalin (C): HR-ESI-MS (m/z) 387.2530 [M + H]+; 1 H NMR (300 MHz) δ 8.00 (1H, dd, J = 9.7, 2.6 Hz, H-22), 7.43 (1H, d, J = 2.6 Hz, H-21), 6.28 (1H, d, J = 9.7 Hz, H-23), 3.56(1H, m, H-3), 0.93 (3H, s, H-19), 0.70 (3H, s, H-18). These data are similar to earlier published data for 3-epibufalin.4 Bufalin (D): HR-ESI-MS (m/z) 387.2524 [M + H]+; 1H NMR (300 MHz) δ 7.99 (1H, dd, J = 9.7, 2.5 Hz, H-22), 7.43 (1H, d, J = 2.5 Hz, H-21), 6.28 (1H, d, J = 9.7 Hz, H-23), 4.05(1H, s, H-3), 0.96 (3H, s, H-19), 0.71 (3H, s, H-18). These data are similar to earlier published data for Bufalin.58 NKA Inhibition Activity of Four Epimers. NKA is the ion pump responsible for maintenance of the electrochemical gradients of Na+ and K+ across the membrane of animal cells.59 It is widely recognized that bufadienolides target NKA, and a

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b05310. Box−Bohnken design matrix and experimental response, analysis of variance, and 1H and 13C NMR data (PDF) 1012

DOI: 10.1021/acs.jafc.7b05310 J. Agric. Food Chem. 2018, 66, 1008−1014

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Journal of Agricultural and Food Chemistry

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86-20-85221016. *E-mail: [email protected]. ORCID

Renwang Jiang: 0000-0002-2163-1683 Funding

This work was supported by the General Program of Natural Science Foundation of Qinghai Province (2016-ZJ-911) and the Basic Application Research Plan of Qinghai Province (2012-Z-714). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED NKA, Na+,K+-ATPase; LLE, liquid−liquid extraction; RSM, response surface methodology; UP, upper phase; MP, moderately polar middle phase; LP, hydrophilic lower phase; HSCCC, high speed counter-current chromatography; HMAW, n-hexane/methyl acetate/acetonitrile/water

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