1

COUNTERCURRENT CHROMATOGRAPHY SEPARATION OF SAPONINS BY 1

SKELETON TYPE FROM Ampelozizyphus amazonicus FOR OFF-LINE UHPLC-2

HRMS ANALYSIS AND CHARACTERISATION. 3

4

Fabiana de Souza Figueiredoa, Rita Celano

b, Danila de Sousa Silva

c, Fernanda das 5

Neves Costaa, Peter Hewitson

d, Svetlana Ignatova

d, Anna Lisa Piccinelli

b, Luca 6

Rastrellib, Suzana Guimarães Leitão

c, Gilda Guimarães Leitão

a* 7

8 aUniversidade Federal do Rio de Janeiro, Instituto de Pesquisas de Produtos Naturais, 9

CCS, bloco H, Ilha do Fundão, 21941-590, RJ, Brazil 10 bUniversità di Salerno, Dipartimento di Farmacia, Via Giovanni Paolo II 132, 84084 11

Fisciano, Italy 12 cUniversidade Federal do Rio de Janeiro, Departamento de Produtos Naturais e 13

Alimentos, Faculdade de Farmácia, CCS, bloco A2, Ilha do Fundão, 21941-590, RJ, 14

Brazil 15 dAdvanced Bioprocessing Centre, Institute of Environment, Health & Societies, 16

CEDPS, Brunel University London, Middlesex UB8 3PH, UK 17

18

Corresponding author: *ggleitao@nppn.ufrj.br 19

20

Keywords: Ampelozizyphus amazonicus, Rhamnaceae, saponin skeleton type, triterpene 21

saponins, countercurrent chromatography, HPLC-MS 22

23

24

Abstract 25

26

Ampelozizyphus amazonicus Ducke (Rhamnaceae), a medicinal plant used to prevent 27

malaria, is a climbing shrub, native to the Amazonian region, with jujubogenin 28

glycoside saponins as main compounds. The crude extract of this plant is too complex 29

for any kind of structural identification, and HPLC separation was not sufficient to 30

resolve this issue. Therefore, the aim of this work was to obtain saponin enriched 31

fractions from the bark ethanol extract by countercurrent chromatography (CCC) for 32

further isolation and identification/characterisation of the major saponins by HPLC and 33

MS. The butanol extract was fractionated by CCC with hexane - ethyl acetate - butanol - 34

ethanol - water (1:6:1:1:6; v/v) solvent system yielding 4 group fractions. The collected 35

fractions were analyzed by HPLC-HRMS and MSn. Group 1 presented mainly oleane 36

type saponins, and group 3 showed mainly jujubogenin glycosides, keto-dammarane 37

type triterpene saponins and saponins with C31 skeleton. Thus, CCC separated saponins 38

from the butanol-rich extract by skeleton type. A further purification of group 3 by CCC 39

(ethyl acetate - ethanol - water (1:0.2:1; v/v)) and HPLC-IR was performed in order to 40

obtain these unusual aglycones in pure form. 41

42

2

1. Introduction 43

44

Ampelozizyphus amazonicus Ducke (Rhamnaceae) is a climbing shrub native to 45

the Amazonian region, where its barks and roots are used in the folk medicine to 46

prepare a beverage to cure and prevent malaria, as well as a tonic and fortifier [1, 2]. 47

The literature cites triterpenes (ursolic acid, betulinic acid, lupenone, betulin, lupeol, 48

melaleucic acid, 3β-hydroxylup-20(29)-ene-27,28-dioic acid, 2α,3β-dihydroxylup-49

20(29)-ene-27,28-dioic acid and 3β,27α-dihydroxylup-20(29)-en-28β-oic acid), 50

jujubogenin glycoside saponins (3-O-β-D-glucopyranosyl-20-O-α-L-51

rhamnopyranosyljujubogenin and 3-O-[β-D-glucopyranosyl(l2)α-L-52

arabinopyranosyl]-20-O-α-L-rhamnopyranosyljujubogenin) as well as, C30 and C31 53

dammarane-type triterpene saponins (ampelozigenin-l5α-O-acetyl-3-O-α-L-54

rhamnopyranopyranosyl-(12)-D-glucopyranoside) as main compounds in these 55

preparations [3-6]. Saponins are usually produced by plants as a complex mixture with 56

very similar structures and polarities. Each saponin is biosynthesized at low 57

concentration, which makes difficult their direct identification and isolation [7]. 58

Therefore, traditionally, multidimensional chromatography has been used, for example, 59

with column chromatography as the first dimension and countercurrent chromatography 60

(CCC) as the second dimension [8] or CCC as the first dimension and liquid 61

chromatography (LC) as the second one [9]. CCC is a type of liquid-liquid partition 62

chromatography technique with no solid support [10]. The use of liquid stationary phase 63

is advantageous for preparative separations because there is no irreversible adsorption, 64

and it allows a high sample loading, and good reproducibility with the scale-up [11, 12]. 65

For the structural characterisation of saponins mass spectrometry is the most often used 66

technique as it provides important information about skeleton type and number of 67

sugars but not sugar identity or linkage [7]. 68

Our previous studies revealed the presence of dammarane saponins with 69

jujubogenin and keto-dammarane skeletons in Ampelozizyphus amazonicus bark extract 70

[1, 2, 6]. A high complexity of the crude extract, due to the variability and similar 71

structures of saponins, however, did not allow a complete chromatographic separation 72

and identification of individual saponins by UHPLC-HRMS and MSn

[6]. Therefore, a 73

series of pre-purification steps were undertaken starting from consequent liquid-liquid 74

extraction (LLE) of the crude ethanol extract with hexane, ethyl acetate and butanol. 75

Analyses by TLC and HPLC-HRMS revealed the presence of saponins in the butanol 76

3

extract (Figure 1A). The next step was CCC separation to produce less complex 77

samples for HPLC-HRMS characterisation and further isolation of saponins from 78

Ampelozizyphus amazonicus bark by CCC and semi-preparative HPLC. 79

80

Insert Figure 1 here 81

82

2. Materials and methods 83

84

2.1. Chemical reagents and solvents 85

86

Organic solvents used to prepare extracts were analytical grade from Tedia 87

(Tedia Brazil, Rio de Janeiro, Brazil). Organic solvents used for TLC analyses and CCC 88

separations were analytical grade from Fisher Chemicals (Loughborough, UK). MS-89

grade acetonitrile and water were supplied by Romil (Cambridge, UK). Organic 90

solvents used in HPLC-IR separations were HPLC grade from Sigma Aldrich (Milan, 91

Italy) and ultrapure water (18 MΩ) was prepared by a Milli-Q purification system 92

(Millipore, Bedford, USA). 93

94

2.2. Plant material and preparation of the extracts 95

96

The stem barks of A. amazonicus were collected in the Brazilian Amazon, at 97

“quilombola” communities of Oriximiná (State of Pará) [1, 2]. Plants were identified by 98

Mr. José Ramos (parataxonomist) and a voucher specimen, INPA 224161, was 99

deposited at the herbarium of Instituto Nacional de Pesquisas da Amazônia (INPA) 100

(Manaus, AM, Brazil) [1,2]. We received authorization for this study from the Brazilian 101

Directing Council for Genetic Heritage (Conselho de Gestão do Patrimonio Genético) 102

through Resolution n.213 (6.12.2007) published in the Federal Official Gazette of 103

|Brazil on December 27, 2007. 104

The stem barks were dried in a ventilated oven (Marconi, model MA037) and 105

ground in a hammer mill (Marconi, model MA340, serial 9304176). The powder 106

material of bark was extracted by percolation with ethanol. The extract was filtrated and 107

the ethanol was removed by rotary evaporation at 40 ºC under reduced pressure. Then 108

the bark ethanol extract (346.5 g) was sequentially partitioned by hexane/ water, ethyl 109

acetate/ water and butanol/ water in a separatory funnel. The solvents were removed by 110

4

rotary evaporation. The liquid-liquid extraction afforded 0.2 g of hexane, 44.7 g of ethyl 111

acetate and 72.5 g of butanol partitions. 112

113

2.3. Countercurrent chromatography apparatus 114

115

Two high performance countercurrent chromatography (HPCCC) centrifuges 116

were used for CCC separations, a Spectrum (semi-preparative) and a MIDI 117

(preparative), both from Dynamic Extractions Ltd. (DE, Tredegar, UK). The Spectrum 118

was equipped with a polytetrafluorethylene (PTFE) column of 143.5 ml and 1.6 mm 119

tubing I.D. The MIDI had a PTFE column of 912.5 ml and 4.0 mm tubing I.D. All 120

separations were performed at the maximum rotation speed of both instruments, 1600 121

rpm (Revolution radius (R) = 85 mm) and 1400 rpm (R = 110 mm) respectively. The 122

semi-preparative set up had a HPLC pump Agilent HP1200 (Santa Clara, California, 123

USA) and a fraction collector Agilent HP1200 (Santa Clara, California, USA). The 124

preparative chromatographic system had a HPLC pump Knauer K-1800 (Berlin, 125

Germany) and a fraction collector Gilson FC202 (Villiers-le-Bel, France). 126

127

2.4. Thin layer chromatography 128

129

Analyses of A. amazonicus bark extracts, solvent systems and CCC fractions 130

were done by thin layer chromatography (TLC) with silica gel TLC Plates 60F254 131

(Merck Art. 05554, Darmstadt, Germany). The mobile phase used for TLC analyses 132

was butanol – acetic acid – water (8:0.5:1.5; v/v). To visualize the compounds spots, the 133

universal spray-reagent, H2SO4 in methanol (5%, v/v) with vanillin in methanol (1%, 134

v/v), and Komarovisky specific spray-reagent for saponins [3,4] with subsequent 135

heating at 105 ºC on a hot plate were used. 136

137

2.5. Solvent system tests 138

139

The solvent systems tests were performed as follows: small amounts of a sample 140

extract were dissolved in a test tube containing a two-phase solvent system. After 141

shaking and allowing compounds to partition between the two phases, equal aliquots of 142

each phase were spotted beside each other separately on silica gel TLC plates. 143

Distribution coefficients (KD) were determined visually. 144

5

145

2.6. CCC separations 146

147

Solvent systems used in all separations by CCC were prepared in a separatory 148

funnel at room temperature. After the equilibrating, the two phases were separated and 149

degassed by sonication for 5 min. In each separation run, a CCC column was first filled 150

with the stationary phase, after set the rotation, the mobile phase was pumped in. 151

Samples were dissolved in equal volumes of each phase and were injected after the 152

hydrodynamic equilibrium inside the column was reached. The column temperature was 153

maintained at 30° C. 154

155

2.6.1. CCC fractionation of the butanol extract of A. amazonicus 156

157

The solvent system chosen for fractionation of the butanol extract of A. 158

amazonicus was hexane – ethyl acetate – butanol – ethanol – water (1:6:1:1:6; v/v). 159

160

Semi-preparative fractionation: 161

The fractionation was performed on the Spectrum machine with the organic 162

upper phase as stationary phase and the aqueous lower phase as mobile phase (reversed 163

phase mode). Fractions of 4 ml were collected during elution (72 fractions, 2 ml/min, 2 164

Vc) and extrusion (36 fractions, 20 ml/min, 1 Vc). The sample was injected using an 165

Upchurch low pressure injection port (Model V-450, with 1/16 in. fittings) and a loop of 166

7.2 ml. The sample concentration was 100 mg/ml. The stationary phase retention (Sf) 167

before sample injection was 62%. Fractions were analysed by TLC and HPLC-HRMS 168

and MSn analyses (Figure 2). 169

170

Preparative fractionation: 171

The preparative fractionation of the butanol extract of A. amazonicus was 172

performed on the MIDI machine. Fractions of 24 ml were collected during elution (76 173

fractions, 12 ml/min, 2 column volume (Vc)) and extrusion (38 fractions, 24 ml/min, 1 174

Vc). The sample was injected using an Upchurch low pressure injection port (Model V-175

450, with 1/16 in. fittings) and loops of 45 ml and 90 ml. The sample concentration was 176

100 mg/ml. The stationary phase retention (Sf) before injection was 67%. After TLC and 177

HPLC-HRMS and MSn analyses, fractions were combined in groups (Figure 2). 178

6

179

2.6.2. CCC fractionation of group 3 from the butanol extract of A. amazonicus 180

181

Purification of group 3 (Frs. 81 – 101 from the first CCC butanol extract 182

fractionation) was done with ethyl acetate – ethanol – water (1:0.2:1; v/v). The aqueous 183

lower phase was used as stationary phase and the organic upper phase as the mobile 184

phase (normal phase mode). The semi-preparative purification of group 3 was first 185

performed on the Spectrum. Fractions of 4 ml were collected during elution (36 186

fractions, 1 ml/min, 1 Vc) and extrusion (36 fractions, 2 ml/min, 1 Vc). The sample was 187

injected using a loop of 3.66 ml. The sample concentration was 27.5 mg/ml. The 188

stationary phase retention (Sf) before injection was 77%. The preparative purification of 189

group 3 was performed on the MIDI machine. Fractions of 24 ml were collected during 190

elution (38 fractions, 12 ml/min, 1 Vc) and extrusion (38 fractions, 24 ml/min, 1 Vc). 191

The sample was injected using a loop of 45 ml. The sample concentration was 27.5 192

mg/ml. The stationary phase retention (Sf) before injection was 90%. After TLC and 193

HPLC-HRMS and MSn analyses, fractions were combined in groups (Figure 2). 194

195

2.7. HPLC separations 196

197

Fractions from Group 3 CCC separation were combined in different groups 198

(Figure 2). Groups C and D2 were separated further by semi-preparative HPLC-IR. The 199

column used was a HyPurity Aquastar, 150 x 10 mm; particle size 5µ (Thermo Electron 200

Corporation). The semi-preparative HPLC system was composed of a pump Knauer 201

Smartline 1000 (Labservice Analytica, Bologna, Italy) and a refraction index (RI) 202

detector Knauer Smartline 2300 (Labservice Analytica). The mobile phase used was 203

aqueous methanol, 5.9:4.1; v/v, in isocratic mode. For group C, the flow rate was 3.5 204

ml/min, the sample was dissolved in methanol (0.1 mg/µl) and the sample solution 205

injected in each run was 35 µl. For group D2, the flow rate was 2.5 ml/min, the sample 206

was dissolved in methanol (0.1 mg/µl) and the sample solution injected in each run was 207

50 µl. All fractions were analysed by HPLC-HRMS and MSn. 208

209

Insert Figure 2 here 210

211

2.8. UHPLC-HRMS analyses 212

7

213

The butanol extract, fractions from CCC and HPLC-IR separations were 214

analysed on an LTQ OrbiTrap XL mass spectrometer (LTQ OrbiTrap XL, 215

ThermoFisher Scientific) connected to a Platin Blue UHPLC system (KNAUER GmbH, 216

Berlin, Germany). This UHPLC system was composed by two ultra-high-pressure 217

pumps, an auto sampler, a diode array detector and a column temperature manager. The 218

LC parameters used were: a Kinetex C18 column (2.1 x 50 mm, 1.7 µm; Phenomenex, 219

Bologna, Italy), flow rate of 0.5 mL min–1

, column temperature of 30 °C and, water (A) 220

and ACN (B), both containing 0.1% formic acid, as mobile phase. The gradient elution 221

program used was: 10-20% B in 3 min, 20–25% B in 4 min, 25–30% B in 13 min and 222

30–50% B in 5 min. After each injection, the column was washed with 98% B for 4 min 223

and re-equilibrated for 4 min. The mass spectrometer, with ESI source, was operated in 224

negative mode. High purity nitrogen (N2) was used as sheath gas (40 arbitrary units) and 225

auxiliary gas (arbitrary units). High purity helium (He) was used as collision gas. Mass 226

spectrometer parameters used were: 3.5 KV of source voltage, -37 V of capillary 227

voltage, –225 V of tube lens voltage and 280 ºC of capillary temperature. Full scan data 228

acquisition (mass range: m/z 350 – 2000) and data dependant MS scan were performed. 229

The resolution was 60000 and 7500 for the full mass and the data dependant MS scan, 230

respectively. The normalised collision energy of the collision-induced dissociation 231

(CID) cell was set at 35 eV and the isolation width of precursor ions was set at 2.0. 232

Saponins were characterized according to the corresponding spectral characteristics: 233

mass spectra, accurate mass, characteristic fragmentation, and retention time. Xcalibur 234

software (version 2.2) was used for instrument control, data acquisition and data 235

analysis. 236

237

3. Results and discussion 238

239

3.1. Butanol extract separation by CCC 240

241

Previous studies on separation of dammarane saponins by CCC used ethyl 242

acetate – butanol – water (1:1:2; v/v) and hexane – ethyl acetate – ethanol – water 243

(1:1:1:1; v/v) solvent systems [13-14]. Therefore, they were selected for preliminary 244

tests. In search for the best solvent system showing a good distribution of compounds 245

between the two phases (K visually close to 1), different solvent proportions were tested 246

8

in order to change system’s polarity and polarity difference between phases. Some 247

solvents were added or replaced, in order to change the selectivity of systems [15]. 248

Table 1 lists all solvent systems, i-iv, tested for the butanol extract purification. The 249

distribution of compounds between the two phases in each solvent system was analysed 250

by TLC [16]. 251

In the first solvent system, (i) ethyl acetate – butanol – water (1:1:2; v/v), 252

compounds were practically all concentrated in upper phase and in the second system, 253

(ii) hexane – ethyl acetate – ethanol – water (1:1:1:1; v/v), compounds were 254

concentrated mainly in the lower phase. To increase polarity of the second system, ii, 255

the proportions of hexane and ethanol were changed to (iii) hexane – ethyl acetate – 256

ethanol – water (5:6:5:6; v/v), causing a drop in the sample solubility. To resolve this 257

issue and to increase polarity, other solvents such as acetone were added to a solvent 258

system, (iv) hexane - ethyl acetate - acetone - ethanol - water (1:1:0.5:1:1; v/v), and also 259

systems i and ii were combined , (v) hexane - ethyl acetate - butanol - ethanol - water 260

(6:6:1:6:6; v/v). In (iv), the sample has limited solubility and in (v) it was soluble and 261

compounds were more concentrated in lower phase like system (ii). After testing 262

various solvent ratios aiming to achieve a K visually close to 1, the best solvent system 263

appeared to be (vi) hexane - ethyl acetate - butanol - ethanol - water (1:6:1:1:6; v/v). 264

Every other CCC fractions were analysed by TLC and HPLC-MS before being 265

combined in groups (Figure 2) according to TLC profile and mass distribution. 266

267

Insert Table 1 here 268

269

UHPLC-HRMS analyses of CCC groups 1-4 and their fractions revealed the 270

presence of saponins mainly in groups 1 and 3. Although UHPLC-HRMS analyses 271

helped to characterize the groups from CCC separation of butanol extract, the groups 1 272

and 3 still showed a high complexity (Figure 1B and 1C). Thus, CCC fractionation of 273

the butanol extract was scaled-up in order to obtain a higher amount of each group 274

fraction for subsequent purification steps. 275

The scale-up factor (6.36) was calculated as the ratio between the column 276

volumes of Spectrum (143.5 ml) and MIDI (912.5 ml), according to CCC volumetric 277

scale-up [17,18]. This scale-up factor was used to adjust the flow rate and the sample 278

volume. Stationary phase retention (Sf) before injection were 62% and 67% 279

respectively. Based on Sutherland and co-workers (2005) theory, which stated that 280

9

larger tubing bore provides a better stationary phase retention and therefore, larger 281

scale-up factors can be reached, the sample loading was increased by doubling the 282

sample volume. The reproducibility of runs was analysed by TLC. 283

284

3.2. UHPLC-HRMS analyses of butanol extract of CCC groups 285

286

The combination of high resolution mass spectrometry and MSn experiments 287

was employed to identify the main constituents of groups 1 and 3. 288

Group 1 showed a complex profile with two main metabolite classes (Figure 289

1B). The first consisted of polar phenolic compounds (0-5 min), while triterpene 290

glycosides, possibly with the oleane skeleton as aglycone, were inferred as the second 291

metabolite class (7-16 min) (data not shown). A complete elucidation of the group 1 292

saponin structures is currently in progress. 293

Dammarane saponins are the major constituents of the group 3 (Figure 1C). 294

Table 2 report the HRMS data of the main saponins of this CCC group and their 295

proposed molecular formulas. HRMS and MSn data (Table 2) allowed to identify 296

saponins with C30 and C31 keto-dammarane and jujubogenin skeletons (Figure 3), 297

according to our previous studies [2, 6], and dammarane-types saponins. Three saponins 298

(2−4) were tentatively identified as 16-keto-tetrahydroxydammar-23-ene triglycosides 299

(C30H50O5, Figure 3A), based on the presence in MS/MS spectra of the diagnostic 300

product ion [M−C8H14O2]− due to the loss of the side chains by a McLafferty 301

rearrangement [6,20]. Comparison with literature data suggested for the compounds 3 302

and 4 structures superimposable to those of hoduloside VIII and VII, respectively, 303

isolated from Rhamnaceae [21]. Also 5, 9-10, 12−13 and 16 produced a similar 304

fragmentation pathway of 2−4 yielded by McLafferty rearrangement. The dominant 305

product ions [M−C9H16O2]− correspond to the loss of an alkyl side chain with an 306

additional methylene group than to 16-keto-tetrahydroxydammar-23-ene glycosides. 307

Based on this fragmentation pathway and the occurrence of a C31 dammarane-type 308

saponin in A. amazonicus [4], the 16-keto-tetrahydroxydammar-24-methylene structure 309

(C31H52O5, Figure 3B) was proposed as aglycon of saponins 5, 9-10, 12−13 and 16. 310

This aglycone is not reported in the literature and further studies are needed to confirm 311

unambiguously the proposed structure. 312

Compounds 1, 7, 11, 14−15 and 17 were tentatively characterized as glycosides 313

of jujubogenin (C30H48O4, Figure 3C), primarily due to the presence of the product ion 314

10

at m/z 471.3469 in MSn spectra, corresponding to the deprotonated jujubogenin 315

(C30H47O4). Compounds corresponding to the structure proposed for 1 and 17 were 316

previously reported in A. amazonicus [3], whereas the isomers 7 and 11 corresponded 317

presumably to hoduloside IV [22] and bacoside A3 [23], respectively, and the isomers 318

14 and 15 to bacopasaponin C [24]. In addition, the structure of hydroxymethylglutaryl 319

(HMG) jujubogenin glycosides [25] was established for 23 and 24 by the diagnostic 320

neutral loss of −144 Da and molecular formula of product ions [M−HMG]−. Finally, one 321

dammarane-types saponin, 6, and four acetylated derivatives, 20, 22, 25 and 28, were 322

detected in group 3. Their MS/MS spectra (Table 2) suggested the structure of acylated 323

tetrahydroxydammar-24-ene triglycosides, structurally related to ginseng saponins with 324

protopanaxatriol (C30H52O4) as aglycone [26, 27]. 325

The sugar residues of all identified saponins were established by characteristic 326

neutral losses (hexose −162 Da, deoxyhexose −146 Da, pentose −132 Da) and accurate 327

mass of corresponding product ions. Particularly, in the case of C30 (2−4) and C31 keto-328

dammarane saponins (5, 9-10, 12−13 and 16) the product ions at 479.3003 or 509.3109 329

in MSn spectra allowed to establish the nature of the sugar residue (pentose or hexose, 330

respectively) directly attached to the aglycone skeleton. 331

Other minor compounds (7, 8, 18, 19, 21, 26 and 27) detected in group 3 were 332

tentatively identified as saponins, but further studies are required to their detailed 333

characterization and identification of aglycones. 334

335

Insert Figure 3 here 336

Insert Table 2 here 337

338

3.3. Separation of Group 3 by CCC and HPLC-IR 339

340

UHPLC-HRMS analysis of butanol extract CCC groups showed the presence of 341

dammarane saponins only in the group 3. This saponin class is characteristic of A. 342

amazonicus [2-4] and it includes unusual aglycones as C30 and C31 keto-dammarane [4, 343

6, 20]. Thus, a further purification of group 3 by CCC and HPLC-IR was performed in 344

order to obtain as pure as possible these unusual compounds. 345

The same approach, as used for butanol extract, was applied to choose a suitable 346

solvent system for group 3 (Table 1; 1-9). Based on a previous work, where 347

dammarane saponins were isolated from Panax ginseng, the solvent system (1) 348

11

dichloromethane - isopropanol – methanol – 5mM aqueous ammonium acetate (6:3:2:4; 349

v/v) was tested as a start [28]. Compounds were more concentrated in lower phase. 350

Further tests showed that in systems (2) ethyl acetate – butanol – methanol – water 351

(1:0.5:0.2:1; v/v) and (3) ethyl acetate – butanol – ethanol – water (1:0.5:0.2:1; v/v) 352

practically all compounds were in upper phase and any difference between system 353

selectivity was observed [29]. In (4) ethyl acetate – methanol – water (1:0.2:1; v/v) [30] 354

and (5) ethyl acetate – ethanol – water (1:0.2:1; v/v), was achieved a good distribution 355

of compounds between the two phases and a slight difference between compounds 356

selectivity. The system 5 showed visually slightly better selectivity than the system 4. 357

The replacement of ethanol with methanol in (6) ethyl acetate – isopropanol – methanol 358

– water (1:0.5:0.2:1; v/v) and (8) ethyl acetate – propanol – methanol – water 359

(1:0.5:0.2:1; v/v), provided a better distribution between two phases as in (7) ethyl 360

acetate – isopropanol – ethanol – water (1:0.5:0.2:1; v/v) and (9) ethyl acetate – 361

propanol – ethanol – water (1:0.5:0.2:1; v/v), because in ethanol containing systems 362

compounds were slightly more concentrated in upper phase due to ethanol polarity in 363

comparison with methanol. For the same reason, addition of propanol or isopropanol to 364

a solvent system reduced the its selectivity (Ks visually similar). Thus, the solvent 365

system selected for the purification of this group was (5) ethyl acetate – ethanol – water 366

(1:0.2:1; v/v). 367

Every two CCC fractions were analysed by TLC and HPLC-MS before being 368

combined in groups (Figure 2) according to TLC profile and mass distribution. 369

Betulinic acid was identified as main compounds of group A, while dammarane 370

saponins were detected in the other group 3 of CCC fractions. As shown in the 371

chromatograms reported in Figure 4, C, D1 and D2 groups were the most saponin-372

enriched groups. The main components of group C were the C31 dammarane-type 373

saponins 10, 13 and 16, whereas D groups were rich in C30 dammarane saponins 3-4 374

and 10, jujubogenin glycosides 11, 14, 15 and 17 and compound 9. 375

The fractionation of group 3 was also scaled-up to MIDI to obtain larger 376

amounts of enriched fractions for the successive purification by semi-preparative 377

HPLC-IR. Testing four different flow rates, 10, 12, 20 and 40 ml/min resulted in 378

stationary phase retention (Sf) of 86%, 90%, 81% and none, respectively. Therefore, 379

flow rate of 12 ml/min was selected for MIDI runs. The scale-up factor (6.36), applied 380

in CCC separation of butanol extract, was used to adjust the sample volume. 381

12

In order to isolate the main dammarane saponins of A. amazonicus bark, 382

particularly saponins with C31 keto-dammarane-type skeleton, groups C and D2 from 383

the CCC purification of group 3 were selected for a subsequent purification by semi-384

preparative HPLC-IR. This isolation procedure allowed to obtain the jujubogenin 385

glycosides 1, 11 and 14−15, C31 dammarane saponins 9-10 and 13 and C30 dammarane 386

saponins 3 and 4, with a suitable purity grade (checked by NMR) for a detailed 387

characterization of their structures. 388

389

Insert Figure 4 here 390

391

4. Conclusions 392

393

The preparative purification procedure, based on CCC and HPLC-IR 394

separations, was successfully developed to isolate the main constituents of A. 395

amazonicus bark. The CCC reduced the complexity of butanol extract allowing a 396

characterization by HPLC-HRMS of saponins and allowed to isolate unusual C31 397

saponins by HPLC. CCC was able to separate saponins by skeleton type, mainly oleane 398

in group 1 and dammarane in group 3. The demonstrated scale-up methodology enables 399

more detailed chemical studies of compounds via future structure elucidation by NMR. 400

401

Acknowledgement 402

403

F.S. Figueiredo is indebted to Coordenação de Aperfeiçoamento de Pessoal de Nível 404

Superior (CAPES, Brazil) for the Ph.D scholarship. 405

F.N. Costa and S. Ignatova would like to thank Newton Advanced Fellowship project 406

funded by the Royal Society of the United Kingdom. 407

S.G. Leitão and G.G. Leitão are indebted to FAPERJ and CNPq for financial support. 408

The authors are deeply indebted to ARQMO (Associação de Comunidades 409

Remanescentes de Quilombolas do Município de Oriximiná), Oriximiná-PA, Brazil, for 410

supervising plant collection and for providing housing during the field trips. 411

412

413

414

13

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530

16

531 Figure 1. UHPLC-HRMS profiles of butanol extract (A) and its HPCCC groups 1 (B) 532

and 3 (C). 533

534

RT: 0.00 - 23.00 SM: 7G

0 5 10 15 20

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

NL: 4.69E6

Base Peak m/z= 350.0000-1500.0000 F: FTMS - p ESI Full ms [350.00-1500.00] MS BUOH-2MGML

RT: 0.00 - 23.00 SM: 7G

0 5 10 15 20

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

NL: 5.51E6

Base Peak m/z= 350.0000-1500.0000 F: FTMS - p ESI Full ms [350.00-1500.00] MS i-2mgml

RT: 0.00 - 23.00 SM: 7G

0 2 4 6 8 10 12 14 16 18 20 22

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

NL: 4.60E6

Base Peak m/z= 350.0000-1500.0000 F: FTMS - p ESI Full ms [350.00-1500.00] MS iva-2mgml

1

2

34 5

9

10

11

13

14,

15

16

18

20

67

12

17

19

21

24

22,

23

25

27

28

A B

C

17

535 Figure 2. Separation by HPCCC of butanol extract and group 3. This scheme was based on information from a MIDI run. In Spectrum runs only 536

total number of fractions change. 537

538

18

HO

C30H48O4

C

O

HOOH

O

OH

OH

C30H50O5

A

HOOH

O

OH

OH

C31H52O5

B

O

OH

539 Figure 3. Proposed aglycone structures of saponins in group 3: (A) 16-keto-540

tetrahydroxydammar-23-ene, (B) 16-keto-tetrahydroxydammar-24-methylene and (C) 541

jujubogenin. 542

543

544

545

546 Figure 4. UHPLC-HRMS profiles of group 3 HPCCC groups (B, C, D1-2). 547

548

2 18 21 2423

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

NL: 6.61E6

Base Peak m/z= 560.0000-1500.0000 F: FTMS - p ESI Full ms [350.00-1500.00] MS ivac-1mgml

Group 3 CCC-C

1 3 4

10

17 28

14

27

16

13

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0

20

40

60

80

100

Rela

tive

Ab

un

da

nce

NL: 5.24E6

Base Peak m/z= 560.0000-1500.0000 F: FTMS - p ESI Full ms [350.00-1500.00] MS ivad1-1mgml

Group 3 CCC-D11 3

49

10

11

13 14, 15

17

2 518 21 25

28

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0

20

40

60

80

100

Rela

tive

Ab

un

da

nce

NL: 6.73E6

Base Peak m/z= 560.0000-1500.0000 F: FTMS - p ESI Full ms [350.00-1500.00] MS ivad2-1mgml

Group 3 CCC-D2

1

3

4

9

11

14, 15

17

18

255

28

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0

20

40

60

80

100

Rela

tive

Ab

un

da

nce

NL: 3.81E6

Base Peak m/z= 560.0000-1500.0000 F: FTMS - p ESI Full ms [350.00-1500.00] MS ivab-1mgml

Group 3 CCC-B13

16

19

Table 1. Solvent systems tested with butanol extract and group 3. 549

Hex DCM EtOAc Acetone BuOH PrOH iPrOH EtOH MeOH H20 CH3COONH4

5 mM

i - - 1 - 1 - - - - 2 -

ii 1 - 1 - - - - 1 - 1 -

iii 5 - 6 - - - - 5 - 6 -

iv 1 - 1 0.5 - - - 1 - 1 -

v 6 - 6 - 1 - - 6 - 6 -

vi 1 - 6 - 1 - - 1 - 6 -

1 - 6 - - - - 3 - 2 - 4

2 - - 1 - 0.5 - - - 0.2 1 -

3 - - 1 - 0.5 - - 0.2 - 1 -

4 - - 1 - - - - - 0.2 1 -

5 - - 1 - - - - 0.2 - 1 -

6 - - 1 - - - 0.5 - 0.2 1 -

7 - - 1 - - - 0.5 0.2 - 1 -

8 - - 1 - - 0.5 - - 0.2 1 -

9 - - 1 - - 0.5 - 0.2 - 1 -

Hex: hexane. DCM: dichloromethane. EtOAc: ethyl acetate. BuOH: butanol. PrOH: 550

propanol. iPrOH: isopropanol. EtOH: ethanol. MeOH: methanol. 551

552

20

553

Table 2. UHPLC-HRMS data of saponins detected in butanol extract HPCCC group 3. 554

Peak tR

(min)

[M-H]-

(m/z)

Molecular

Formula

ppm Diagnostic product ion a

(m/z)

Aglycone b

Sugar residue c

1 8.2 779.4577 C42H68O13 0.2 633 (-dHex), 617 (-Hex), 471 (C30H48O4) C30H48O4 1 Hex, 1 dHex

2 11.4 959.5211 C48H80O19 0.1 817 (-C8H14O2), 655 (-C8H14O2-Hex), 509d (-C8H14O2-Hex-dHex) C30H50O5 2 Hex, 1 dHex

3 12.2 915.4956 C46H76O18 0.9 773 (-C8H14O2), 611 (-C8H14O2-Hex), 641 (-C8H14O2-Pen), 479d (-

C8H14O2-Hex-Pen)

C30H50O5 2 Pen, 1 Hex

4 12.7 929.5113 C47H78O18 0.9 787 (-C8H14O2), 625(-C8H14O2-Hex), 479d (-C8H14O2-Hex-dHex) C30H50O5 1 Hex, 1 dHex,

1 Pen

5 13.0 959.5217 C48H80O19 0.8 803 (-C9H16O2) 641 (-C9H16O2-Hex), 479d (-C9H16O2-2Hex) C31H52O5 2 Hex, 1 Pen

6 13.6 931.5266 C47H80O18 0.6 799 (-Pen), 769 (-Hex), 637(-Hex-Pen) C30H52O4 2 Hex, 1 Pen

7 13.7 927.4951 C47H76O18 0.3 765 (-Hex-), 603(-2Hex) C30H48O4 2 Hex, 1 Pen

8 13.8 955.4901 C48H76O19 0.5 823 (-Pen), 793(-Hex), 661 (-Pen-Hex) 1 Pen, 1 Hex

9 13.9 959.5217 C48H80O19 0.7 803 (-C9H16O2), 641 (-C9H16O2-Hex), 479d (-C9H16O2-2Hex) C31H52O5 2 Hex, 1 Pen

10 14.4 929.5108 C47H78O18 0.4 773 (-C9H16O2), 611 (- C9H16O2-Hex), 479d (-C9H16O2-Hex-Pen) C31H52O5 1 Hex, 2 Pen

11 14.9 927.4957 C47H76O18 1 795 (-Pen), 765 (-Hex), 633(-Hex-Pen) C30H48O4 2 Hex, 1 Pen

12 15.3 929.5106 C47H78O18 0.2 773 (-C9H16O2), 611 (- C9H16O2-Hex), 479d (-C9H16O2-Hex-Pen) C31H52O5 1 Hex, 2 Pen

13 15.6 929.511 C47H78O18 0.6 773 (-C9H16O2), 611 (- C9H16O2-Hex), 479d (-C9H16O2-Hex-Pen) C31H52O5 1 Hex, 2 Pen

14 15.7 897.4835 C46H74O17 -0.9 765 (-Pen), 735 (Hex), 603(-Pen-Hex), 471d (C30H48O4) C30H48O4 1 Hex, 2 Pen

15 16.0 897.4854 C46H74O17 1.3 765 (-Pen), 735 (Hex), 603(-Pen-Hex), 471d (C30H48O4) C30H48O4 2 Hex, 2 Pen

16 16.2 943.5258 C48H80O18 -0.3 787 (-C9H16O2), 625 (-C9H16O2-Hex), 479d (-C9H16O2-Hex-dHex) C31H52O5 1 Hex, 1 dHex,

1 Pen

17 16.5 911.5001 C47H76O17 0.2 749 (-Hex), 603 (-Hex-dHex) C30H48O4 1 Hex, 1 dHex,

1 Pen

18 16.7 955.5257 C49H80O18 -0.3 793 (-Hex), 647 (-Hex-dHex) 1 Hex, 1 dHex

19 17.4 1013.532 C51H82O20 0.1 851 (-Hex), 705 (-Hex-dHex) 1 Hex, 1 dHex

20 18.0 1003.547 C50H84O20 -0.1 943(-C2H4O2), 841 (-Hex), 781 (-C2H4O2-Hex) C30H52O4 3 Hex

21

21 18.2 1013.532 C51H82O20 0.3 851 (-Hex), 705 (-Hex-dHex) 1 Hex, 1 dHex

22 19.0 1003.548 C50H84O20 0.7 943(-C2H4O2), 841 (-Hex), 781 (-C2H4O2-Hex) C30H52O4 3 Hex

23 19.0 1041.527 C52H82O21 0.1 897 (-HMG), 879 (-Hex), 765 (-HMG-Pen), 735 (-HMG-Hex) C30H48O4 1 Hex, 2 Pen

24 19.3 1041.527 C52H82O21 0 897 (-HMG), 879 (-Hex), 765 (-HMG-Pen), 735 (-HMG-Hex) C30H48O4 2 Hex, 2 Pen

25 20.1 973.5371 C49H82O19 0.5 913 (-C2H4O2), 811 (-Hex), 781 (-C2H4O2-Pen), 751 (-C2H4O2-Hex),

619 (-C2H4O2-Pen-Hex)

C30H52O4 2 Hex 1 Pen

26 20.1 969.5062 C49H78O19 1.0 837 (-Pen), 807 (-Hex), 675 (-Pen-Hex) 1 Hex, 1 Pen

27 20.9 969.5058 C49H78O19 0.4 837 (-Pen), 807 (-Hex), 675 (-Pen-Hex) 1 Hex, 1 Pen

28 21.2 973.5372 C49H82O19 0.6 913 (-C2H4O2), 811 (-Hex), 781 (-C2H4O2-Pen), 751 (-C2H4O2-Hex),

619 (-C2H4O2-Pen-Hex)

C30H52O4 2 Hex 1 Pen

a Hex: hexose; dHex: deoxyhexose; Pen: pentose; HMG: hydroxymthylglutaryl;

b strucutures reported in Figure 3;

c in bold the sugar residue 555

attached to aglycone skeleton; d product ions detected in MS

3 spectra. 556

557

22

558