An Efficient Strategy Based on LiquidâLiquid Extraction with Three

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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 Denglang Zou, Xuelin Zhu, Fan Zhang, Yurong Du, Jianbin Ma, and Renwang Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05310 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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Journal of Agricultural and Food Chemistry

1

An Efficient Strategy Based on Liquid-liquid Extraction with Three-phase Solvent

2

System and High Speed Counter-Current Chromatography for Rapid Enrichment and

3

Separation of Epimers of Minor Bufadienolide from Toad Meat

4 5 6 7 ，‡ ,

Denglang Zou†, Xuelin Zhu†, Fan Zhang†, Yurong Du‡, Jianbin Ma*

8

, Renwang

Jiang*,†

9 10 11 12 13

†

14

Pharmacy, Jinan University, Guangzhou 510632, PR China

15

‡

16

of Life and Geography Science, Qinghai Normal University, Xining 810000, PR

17

China

Institute of Traditional Chinese Medicine and Natural Products, College of

Key Laboratory of Medicinal Plant and Animal Resources of Tibet Plateau, School

18 19 20 21 22 23 24 25 1

ACS Paragon Plus Environment


26

27

ABSTRACT: This study presents an efficient strategy based on liquid-liquid

28

extraction with three-phase solvent system and high speed counter-current

29

chromatography for rapid enrichment and separation of epimers of minor

30

bufadienolide from toad meat. The reflux extraction conditions were optimized by

31

response surface methodology firstly and a novel three-phase solvent system

32

composed of n-hexane/methyl acetate/acetonitrile/water (3:6:5:5, v/v) was developed

33

for liquid-liquid extraction of the crude extract. This integrative extraction process

34

could enrich minor bufadienolide from complex matrix efficiently and minimize the

35

loss of minor targets induced by repeated extraction with different kinds of organic

36

solvent occurred in the classical liquid two-phase extraction. As a result, four epimers

37

of minor bufadienolide were greatly enriched in the middle phase and total content of

38

these epimers of minor bufadienolide was increased from 3.25 % to 46.23 %. Then,

39

the enriched four epimers were separated by HSCCC with a two-phase solvent system

40

composed of cholroform/methanol/water (4:2:2, v/v) successfully. Furthermore, we

41

tested Na+, K+- ATPase inhibitory effect of the four epimers. 3β-isomers of

42

bufadienolide showed stronger (>8-fold) inhibitory activity than 3α-isomers. The

43

characterization of minor bufadienolide in toad meat and their significant difference

44

of inhibitory effect on NKA would promote the further quantitative analysis and

45

safety evaluation of toad meat as a food source.

46

Keywords: Toad meat, Three-phase solvent system, Liquid-liquid extraction, High

47

speed counter-current chromatography, Epimers of minor bufadienolide, Response 2


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surface methodology

49

INTRODUCTION

50

Toad is a wide distributed amphibian species through-out the agricultural landscapes

51

all around the world 1. Despite the great medicinal value of toad venom and toad skin,

52

the meat of toad is also a kind of famous and delicious food in some parts of the world,

53

such as China, Australia, Peru, Cambodia and Thailand. The scientist from Australia

54

suggested toads were sources of healthy food, rich in protein and essential omega 32.

55

Dishes named Smoked toad, Toad leg, Clay pot and LaSi, where the main ingredient

56

is toad meat, are rather popular in China and Australia. However, due to the toxic

57

bufadienolide in their back and skin, there were some cases that people who ate toad

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meat and related dishes had some adverse reactions, such as vomiting, diarrhea,

59

convulsion and even death3. The toxicity of bufadienolide has been attributed mainly

60

to the inhibitory effect on the Na+, K+- ATPase (NKA) and the alteration of

61

intracellular calcium stores4. Our preliminary analysis revealed minor bufadienolide

62

also existed in the carefully handled toad meat. So it is rather important and essential

63

to characterize the corresponding minor bufadienolide of toad meat with an efficient

64

enrichment and separation method for further quantitative analysis and safety

65

evaluation.

66

Toad meat contains huge amount of protein and fat, this made it rather hard to

67

separate minor bufadienolide surrounded by the complex matrix directly. So an

68

optimized extraction conditions by response surface methodology (RSM) and a proper

69

pretreatment technique were needed to enrich these minor bufadienolide in toad meat 3



70

before further separation. Liquid-liquid extraction (LLE) is one of the oldest and most

71

widely used techniques for pretreatment of crude sample due to its low cost, high

72

efficiency and simple procedure

73

solvent of various polarity would lead to serious loss of these minor targets8-9.

74

Three-phase solvent system could formed mutually immiscible three phases

75

composed of hydrophobic upper phase (UP), moderately-polar middle phase (MP)

76

and hydrophilic lower phase (LP)

77

bio-samples. High polar matrix, such as protein and saccharide, could be well

78

distributed in hydrophilic lower phase. Meanwhile, non-polar matrix, such as fatty

79

acid, could be well distributed in hydrophobic upper phase11-12. This integrative

80

process would lead to remarkable enrichment of moderately-polar targets in middle

81

phase with slight loss. Thus an optimized three-phase solvent system with proper

82

partition coefficient was a unique way to enrich these minor bufadienolide from toad

83

meat full of complex matrix.

5-7

. However, the repeated extraction with organic

10

. It showed great advantages to pretreat the

84

Bufadienolide is typically C-24 steroid with a characteristic α-pyrone ring at

85

C-17 position, have traditionally been separated and purified from toad venom by

86

column chromatography13. However, the performance of these conventional methods

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are severely restricted by labor consuming, solvents consuming and serious sample

88

loss

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bufadienolide, high speed counter-current chromatography (HSCCC) would be a good

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choice16-18. HSCCC is a kind of solid support-free liquid chromatography based on

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liquid-liquid partition, in which samples have complete recovery for no irreversible

14-15

. As to efficient separation technique with less sample loss for the minor

4


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adsorption caused by solid matrix occurred in the conventional chromatographic

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techniques19-22. High-performance separation, online detection, and automatic control

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make it rather easy to realize efficient preparation of target compounds. In addition,

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components with similar skeleton and polarity could be separated easily and

96

efficiently by HSCCC

97

compounds from natural products and bio-samples 20, 24-27.

23

. It has been widely applied for separation of bioactive

98

In the current paper, an efficient strategy based on liquid-liquid extraction with

99

three-phase solvent system and high speed counter-current chromatography was

100

established to rapidly enrich and separate four epimers of minor bufadienolide from

101

toad meat. And we also evaluated NKA inhibitory effect of the purified epimers.

102

MATERIALS AND METHODS

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Materials. All solvents used in extraction and HSCCC were of analytical grade

104

(Guangdong Guanghua Sci-Tech Co., Ltd., China).The acetonitrile used for HPLC

105

analysis was of chromatographic grade, and was purchased from Mreda Technology

106

Inc. (USA). All reagents used in NKA inhibition test were purchased from

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Sigma-Aldrich. Methanol-d4 was employed for NMR analysis. Toad meat were

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purchased from GuangZhou (GuangDong, China).

109

Optimization of reflux extraction conditions. RSM was employed to optimize

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the reflux extraction conditions of the minor bufadienolide on the basis of

111

single-factor experiments28-31. A Box-Behnken design (Design-Expert v10.0.1) with

112

three independent variables was employed to obtain the best combination of

113

extraction variables for the yield of minor bufadienolide 5


32

. The variables used were


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114

as follows: ethanol concentration (X1), extraction time (X2), extraction temperature

115

(X3). The W (W=∑{Ai (peak area)}, where Ai (peak area) was the proportion of peak

116

area of each target in HPLC analysis) was chosen as the response value. 17

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experiments composed of 12 factorial experiments and five replicates at the center

118

point (Table S1) were designed in the RSM optimization process

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relationship between the independent and dependent variables was developed by the

120

least square methodology35-37. The second-order polynomial equation was employed

121

to fit the experimental data as follows: 3

Y = β0 +

122

∑βi X i + i =1

3

∑βii X i2 + i =1

2

33-34

. The

3

∑ ∑β

ij

X ij

i =1 j =i +1

123

where Y represents the predicted response, β0, βi and βii, βij are the regression

124

coefficients of variables for intercept, linear, quadratic, and interaction terms,

125

respectively, Xi and Xj are independent variables (i ≠ j). The fitness of the polynomial

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model equation is expressed by the coefficient of determination R2, and F-test at a

127

probability (p) of 0.05 was employed to evaluate its statistical significance

128

Surface plots derived from the optimized model could reveal the interactive effects of

129

each factors clearly.

38-39

.

130

Finally, the optimization of reflux extraction process was conducted. The

131

optimum goal was to obtain a maximum response value under the same operating

132

conditions, and the desirability values (range from 0 to 1) were employed to evaluate

133

the credibility of the optimum conditions 40-41.

134

Reflux Extraction. The toad meat (5 kg) were obtained by removing head, skin,

135

viscera and other tissues from Bufo gargarizans. It was treated by mince machine and 6


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136

underwent three reflux extraction using 83% ethanol at 79 ℃. The extraction time was

137

2.1 h and liquid/solid ratio was 20:1. All filtrates were combined and concentrated at

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45 ℃ under reduced pressure. The residue was stored in a refrigerator for use in the

139

subsequent liquid-liquid extraction.

140

High performance liquid chromatography (HPLC) Analysis. The apparatus

141

used was an Agilent 1200 system (Agilent Technologies Co. Ltd., USA). The Agilent

142

1200 system consists of a G1312A solvent delivery unit, a G1315D DAD unit, a

143

G1316A column thermostat, a G1329A autosampler and an Agilent ChemStation. The

144

column was a Phenomenex Luna-C18 analytical column (250 mm×4.6 mm, 5 µm).

145

Mobile phase was composed of 0.1% formic acid water (A) and acetonitrile (B).

146

Gradient elution program was 10-70% B in 0-40 min. Flow rate was 1.0 mL/min and

147

detection wavelength was set at 296 nm.

148

Optimization of suitable three-phase solvent system for enriching the target

149

compounds. Toad meat crude extract was screen with a series of three-phase solvent

150

systems composed of n-hexane/methyl acetate/acetonitrile/water to obtain a max

151

partition coefficient KM/U (KM/U = AMP/AUP, A means the peak area of HPLC

152

chromatogram) and KM/L (KM/L = AMP/ALP) of the minor bufadienolide

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and KM/L value were determined using HPLC as the following partition coefficient K

154

of HSCCC.

42

. The KM/U

155

Selection of two-phase solvent system. The two-phase solvent system was

156

screen on the basis of the partition coefficient (K) determined by HPLC. In brief, 5

157

mg of enriched sample was added in the equilibrated two-phase solvent system and 7



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158

shaken violently to achieve equilibration of target compounds between lower and

159

upper phases, and kept still for 10 min. 2 mL of each phase was evaporated and

160

analyzed by HPLC. The K value was determined as the peak area of each target

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compound in stationary phase divided by in mobile phase.

162

HSCCC separation procedure. The HSCCC experiment was conducted on a

163

TBE-300B high-speed counter-current chromatography system (Shanghai Tauto

164

Biotech, Shanghai, China) with a total coil volume of 280 mL was used (tube

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diameter = 1.6 mm). The system was equipped with a TBP-5002 constant-flow pump,

166

a UV500 detector module, a N2000 workstation and a DC-0506 constant

167

temperature-circulating implement. The column was washed with ethanol to remove

168

any impurity before the separation procedure. The column was firstly filled with the

169

stationary phase. After the apparatus was rotated at 900 rpm, the mobile phase was

170

pumped into the column at a flow rate of 2.0 ml/min.

171

After hydrodynamic equilibration was achieved, enriched sample solution (200

172

mg of the sample was dissolved in 10 mL of both phases) was injected into the

173

separation column, the chromatogram was recorded at wavelength of 296nm and

174

target fractions were collected manually according to the HSCCC chromatogram.

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Identification of target compounds. Identification of the HSCCC peak

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fractions were performed by spectroscopic analysis. 1H NMR and

177

were measured in Methanol-d4 on a Bruker 300 NMR Spectrometer using

178

tetramethylsilane (TMS) as the internal standard.

179

13

C NMR spectra

NKA inhibition assay. The NKA inhibitory activities of four emipers were 8


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43-44

180

determined by colorimetry as previously reported

. In brief, NKA was

181

preincubated in incubation medium (3mM MgCl2, 3 mM Na-phosphate, 40mM Tris,

182

pH 7.0) with increasing concentrations of four epimers at 37 °C for 2 hours. The NKA

183

activity is subsequently determined by liberated Pi for ATP hydrolysis after incubated

184

for a further 10 min in standard assay medium (130 mM NaCl, 20 mM KCl, 4 mM

185

MgCl2 and 3 mM ATP ).

186

RESULTS AND DISCUSSION

187

Optimization of reflux extraction conditions. According to the designed

188

experiments and data fitness, the full quadratic models (R2 = 98.31) were more

189

efficient for W compared with other model. The model is

190

Y = -44.03+0.68X1+5.31X2+0.34X3+(2.05E-2)X1X2+(5.01E-4)X1X3-(2.25E-2)X2X3

191

-(4.59E-3)X12-1.24X22-(2.14E-3)X3

2

192

The significance of the model was conformed by Analysis of Variance (ANOVA).

193

Each coefficient corresponding to the above equation indicated the interaction

194

strength between each independent variable and their significance were checked by

195

p-values

196

X1, X2 and X3, the interactive terms X23, the quadric terms X12, X22, and X32 showed a

197

significant effect (P0.05).

36, 45

. According to the ANOVA analysis shown in Table S2, the linear term

199

The relationship between responses and experimental levels of each variable

200

could be revealed visually by 3D plots, which were the graphic representations of the

201

regression models. It also provides a way to evaluate the type of interactions between 9



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41, 46

202

two test variables

. Fig. 1℃ shows the interaction between extraction time and

203

ethanol concentration. Initially, W increased with increasing of extraction time and

204

ethanol concentration, but eventually began to decrease again. Due to complex matrix

205

in toad meat, the content of impurity and targets compounds would increase

206

simultaneously with the increasing of

207

When extraction time exceeded 2.25 h and ethanol concentration exceeded 85%, the

208

relative increase rate of ∑Ai (Ai refers to peak area of the four epimers) would be

209

slower than the relative increase rate of ∑Aj (Aj refers to peak area of the impurity).

210

Thus the decrease of relative yield of four epimers lead to the subsequent decrease of

211

W and these results were consistent with the single-factor experiments.

extraction time and ethanol concentration.

212

According to the RSM optimization, the maximum W ( 3.28 % ) could be

213

predicted under the optimal conditions (ethanol concentration of 83%, extraction time

214

of 2.1 h and extraction temperature of 79 ℃ ). The reflux extraction of toad meat was

215

conducted under the optimal conditions and it lead to a satisfied result with the W =

216

3.25 %.

217

Optimization of suitable three-phase solvent system for enriching the target

218

compounds. Theoretically, LLE could be used to enrich the target compounds by a

219

solvent system with proper partition coefficient, good results could be obtained in

220

LLE when the target compounds distributed in only one phase

221

two-phase solvent system, the extract of bio-samples needed to be treated with

222

organic solvent of various polarity, these repeated extraction with different solvent

223

would lead to serious loss of minor target components. Three-phase solvent system 10


9, 47-48

. As for

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224

could formed mutually immiscible three phases composed of hydrophobic UP,

225

moderately-polar MP and hydrophilic LP 49. Hdrophobic UP and hydrophilic LP could

226

dramatically remove the high polar and non-polar matrix from crude extract. The

227

minor target components could be enriched significantly in moderately-polar MP with

228

a proper three-phase solvent system of max KM/U and KM/L values. A series of solvent

229

systems composed of n-hexane/methyl acetate/acetonitrile/water (HMAW) were

230

screened, the KM/U and KM/L values of the minor bufadienolde were measured, and the

231

results have been summarized in Table 1. First, HMAW (5:5:5:5, v/v) was used to test

232

the distribution of the target compounds. It was found that the target compounds

233

mainly distributed in MP and UP. Considering the moderate polarity of target

234

compounds, methyl acetate was added to promote the target compounds transferring

235

from UP to MP. However, it is not very significant, the KM/U values began to decrease

236

slowly when the ratio of methyl acetate exceeded 7. So the ratio of n-hexane was

237

decreased to adjust the distribution of these minor bufadienolide between UP and MP.

238

As can be seen in Table 1, the target compounds could greatly transfer from UP to MP

239

with the ratio of n-hexane decreasing from 5 to 2. However, the settling time would

240

increase significantly when the ratio of n-hexane was less than 2. Thus, HMAW

241

(3:6:5:5, v/v) was used with large enough KM/U and KM/L values to enrich the minor

242

bufadienolide from crude extract.

243

LLE times was also optimized, it revealed these minor bufadienolide could be

244

well enriched in MP after 3 extraction times. As a result, 2.8 g of enriched sample was

245

obtained. The total content of these minor bufadienolde was increased from 3.25 % to 11



246

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46.23% (Fig. 2).

247

Selection of HSCCC experimental conditions. The selection of solvent system

248

is the most important step for a successful HSCCC separation. A suitable two-phase

249

solvent system should provide an ideal range of partition coefficients guided by the

250

chemical nature of target compounds 50-51. Many important characteristics, such as the

251

sample polarity (evaluated from partition coefficient values), solubility, ionic form,

252

and the capability to form complexes to consider, should be taken into account when

253

we evaluate a solvent system. A suitable partition coefficient (K) should generally be

254

in the range of 0.2-5

255

greater than 1.5 theoretically

256

give lower resolution due to eluting the solute closer to the solvent front, or tends to

257

give broader and more dilute peaks leading to a longer elution time. Furthermore, the

258

solvent system must separate clearly and quickly into two phases as well in acceptable

259

settling time 54-55.

260

51-52

, and the separation factor (α= K2/K1, K2> K1) should be 41, 53

. A considerably smaller or larger K value would

We optimized the solvent system for HSCCC separation based on above stated

261

principles.

A

series

of

two-phase

solvent

262

chloroform/methanol/water were screened, the K-values of target compounds were

263

measured, and results have been summarized in Table 2. It was found that the ratio

264

change of methanol would greatly affect the distribution of target A and C between

265

two phases, the distribution of target B and D would be affected significantly with the

266

ratio change of water. Eventually, the system of chloroform/methanol/water (4:2:2,

267

v/v/v) was selected for the suitable K-values and α values. 12


systems

composed

of

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268

In addition to the solvent system, the flow rate of the mobile phase and the 56

269

revolution speed of apparatus were also studied

. Based on peak resolution,

270

separation time and retention rate of stationary phase, a flow rate of 2.0 mL/min and a

271

revolution speed of 900 rpm with 68% of retention rate of stationary phase were

272

selected in the subsequent HSCCC separation procedures.

273

Under the selected conditions, 23 mg of A, 26 mg of B, 7 mg of C and 6 mg of D

274

were obtained from 200 mg of enriched sample in less than 240 min (Fig. 3). HPLC

275

analysis showed the purity of the whole target compounds were over 91%.

276 277

Structural identification. The chemical structures of the four epimers were elucidated by 1H NMR analysis, and results are as follows (Fig. 4):

278

3-epi-arenobufagin (A): HR-ESI-MS (m/z) 417.2284 [M+H]+; 1H NMR (300

279

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,

280

J=9.7 Hz, H-23), 4.35 (1H, d, J=11.2 Hz, H-11), 4.12(1H, dd, J=9.5, 7.1Hz, H-17),

281

3.56 (1H, m, H-3), 1.14 (3H, s, H-19), 0.89 (3H, s, H-18). Those data were in

282

agreement with earlier published data for 3-epi-arenobufagin 57.

283

Arenobufagin (B): HR-ESI-MS (m/z) 417.2278 [M+H]+; 1H NMR (300 MHz):

284

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

285

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

286

(1H, s, H-3), 1.18 (3H, s, H-19), 0.90 (3H, s, H-18). Those data were in agreement

287

with earlier published data for Arenobufagin 4.

288

3-epi-bufalin (C): HR-ESI-MS (m/z) 387.2530 [M+H]+; 1H NMR (300 MHz): δ

289

8.00 (1H, dd, J=9.7, 2.6Hz, H-22), 7.43 (1H, d, J=2.6Hz, H-21), 6.28 (1H, d, J=9.7 Hz, 13



290

H-23), 3.56(1H, m, H-3), 0.93 (3H, s, H-19), 0.70 (3H, s, H-18). Those data were

291

similar to earlier published data for 3-epi-bufalin 4.

292

Bufalin (D): HR-ESI-MS (m/z) 387.2524 [M+H]+; 1H NMR (300 MHz): δ 7.99

293

(1H, dd, J=9.7, 2.5Hz, H-22), 7.43 (1H, d, J=2.5Hz, H-21), 6.28 (1H, d, J=9.7 Hz,

294

H-23), 4.05(1H, s, H-3), 0.96 (3H, s, H-19), 0.71 (3H, s, H-18). Those data were

295

similar to earlier published data for Bufalin 58.

296

NKA inhibition activity of four emipers. NKA is the ion pump responsible for

297

maintenance of the electrochemical gradients of Na+ and K+ across the membrane of

298

animal cells59. It is widely recognized that bufadienolide target NKA, and a direct

299

consequence of their binding is an inhibition of the enzyme. It is the NKA inhibitory

300

effect that attributed to toxicity of bufadienolide for its alteration of intracellular

301

calcium stores4.

302

According to the dose-response curves revealing inhibition of NKA by the four

303

emipers from toad meat (figure 5). 3β-isomers of bufadienolide showed stronger

304

(>8-fold) inhibitory activity than 3α-isomers（IC50-A=29.75µM，IC50-B=3.39µM，

305

IC50-C=23.55µM, IC50-D=2.63µM）, this may means 3β-isomers were more toxic than

306

3α-isomers. It was also accordance with the phenomenon only the 3β-bufadienolide

307

was secreted in toad venom for chemical defense60. This significant difference of

308

inhibitory effect on NKA laid a foundation for the further safety evaluation of toad

309

meat as a food source.

310

In conclusion, this study presents an efficient strategy based on liquid-liquid

311

extraction with three-phase solvent system and high speed counter-current 14


Page 14 of 33

Page 15 of 33


312

chromatography for rapid enrichment and separation of epimers of minor

313

bufadienolide from toad meat. LLE with three-phase solvent system could greatly

314

enrich minor constituents from complex matrix, it can minimize the loss of minor

315

targets induced by repeated extraction with different kinds of organic solvent occurred

316

in the classical liquid two-phase extraction. It also revealed there existed significant

317

difference of the inhibitory activity against NKA between the purified epimers. The

318

results also demonstrated HSCCC could be a powerful technique for separation of

319

epimers from bio-samples and natural products. The established strategy for

320

enrichment and separation of four epimers would promote the characterization of

321

minor bufadienolide of toad meat for further quantitative analysis and safety

322

evaluation.

323

ABBREVIATIONS USED

324

NKA, Na+, K+- ATPase; LLE, Liquid-liquid extraction; RSM, Response surface

325

methodology; UP, Upper phase; MP, Moderately-polar middle phase; LP, hydrophilic

326

lower phase; HSCCC, high speed counter-current chromatography; HMAW,

327

n-hexane/methyl acetate/acetonitrile/water.

328

AUTHOR INFORMATION

329

Corresponding Authors

330

*E-mail addresses: [email protected]. Telphone: +86-20-85221016

331

*E-mail addresses: [email protected].

332

Funding

333

This work was supported by the General Program of Natural Science Foundation of 15



334

Qinghai Province (2016-ZJ-911) and the Basic Application Research Plan of Qinghai

335

Province (2012-Z-714).

336

ASSOCIATED CONTENT

337

Supporting Information

338

Table S1 Box-Bohnken design matrix and experimental response.

339

Table S2 The analysis of variance.

340

Table S3 1H and

341

methanol-d4 (J in Hz, δ in ppm).

342

REFERENCES

343

(1) Frost, D. R.; Grant, T.; Faivovich, J.; Bain, R. H.; Haas, A.; Haddad, C. F. B.; De

344

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(Mangifera

Walasek,

indica

M.;

L.)

Kernels

Grzegorczyk,

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Skalicka-Woźniak,

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Counter-Current

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544 545

Figure captions

546

Figure 1. Response surface showing the interactive effect of the variables on W: (℃)

547

extraction time vs. ethanol concentration; (℃) extraction temperature vs. ethanol

548

concentration; (℃) extraction temperature vs. extraction time.

549

Figure 2. HPLC chromatograms of the crude extract (℃) and enriched sample by LLE

550

with three-phase solvent system (℃). Conditions: column, Phenomenex Luna-C18

551

analytical column (250 mm×4.6 mm, 5 µm); mobile phase, 0.1% acid water (A) and

552

acetonitrile (B), gradient elution program: 10-70% B in 0-40 min; flow rate, 1.0

553

mL/min; detection wavelength, 296 nm. 25



554

Figure 3. HSCCC chromatogram of the enriched sample using chloroform/

555

methanol/water (4:2:2, v/v/v). Conditions: stationary phase, lower phase; flow rate,

556

2.0 mL/min; revolution speed, 900 rpm; sample amount, 200 mg; separation

557

temperature, 25 ºC; detection wavelength, 296 nm; retention of the stationary phase:

558

68%.

559

Figure 4. The chemical structures of 3-epi-arenobufagin (A), Arenobufagin (B) and

560

3-epi-bufalin (C) and Bufalin (D).

561

Figure 5. The inhibitory activity of four emipers against NKA at 37 °C.

26


Page 26 of 33

Page 27 of 33


Table 1 The KM/U and KM/L values given by the liquid–liquid extraction with three-phase solventsystems 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

5

5

5

5

7.76

19.79

6.80

20.81

5.78

23.87

5.35

27.98

2

5

6

5

5

10.20

21.36

8.61

22.54

7.04

27.05

6.41

31.21

3

5

7

5

5

9.06

22.23

7.78

24.22

6.47

29.51

5.94

32.46

4

4

6

5

5

12.57

18.87

10.23

20.04

8.09

23.52

7.27

25.76

5

3

6

5

5

16.91

18.03

13.09

19.09

10.63

21.64

9.78

23.87

6

2

6

5

5

25.36

16.56

20.01

17.64

17.51

20.54

15.31

22.54

Table 2 The K-values of the target compounds Solvent system K-values No. Chloroform Methanol Water A B C 1 4 4 4 0.08 1.28 0.47 2 4 3 4 0.17 1.45 0.82 3 4 2 4 0.47 1.58 1.47 4 4 1 4 0.74 1.89 2.57 5 4 2 3 0.39 0.84 1.21 6 4 2 2 0.33 0.52 1.03 7 4 2 1 0.26 0.31 0.89

27


D 3.41 3.89 4.21 4.96 2.98 1.77 1.04


TOC Graphic

28


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Figure 1 172x44mm (300 x 300 DPI)



Figure 2 145x90mm (300 x 300 DPI)


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Figure 3 88x46mm (300 x 300 DPI)



Figure 4 101x108mm (600 x 600 DPI)


Page 32 of 33

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Figure 5 213x178mm (300 x 300 DPI)


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