COUNTERCURRENT CHROMATOGRAPHY SEPARATION OF SAPONINS BY
1
SKELETON TYPE FROM Ampelozizyphus amazonicus FOR OFF-LINE UHPLC-
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HRMS ANALYSIS AND CHARACTERISATION.
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Fabiana de Souza Figueiredoa, Rita Celanob, Danila de Sousa Silvac, Fernanda das
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Neves Costaa, Peter Hewitsond, Svetlana Ignatovad, Anna Lisa Piccinellib, Luca
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Rastrellib, Suzana Guimarães Leitãoc, Gilda Guimarães Leitãoa*
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aUniversidade Federal do Rio de Janeiro, Instituto de Pesquisas de Produtos Naturais,
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CCS, bloco H, Ilha do Fundão, 21941-590, RJ, Brazil
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bUniversità di Salerno, Dipartimento di Farmacia, Via Giovanni Paolo II 132, 84084
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Fisciano, Italy
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cUniversidade Federal do Rio de Janeiro, Departamento de Produtos Naturais e
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Alimentos, Faculdade de Farmácia, CCS, bloco A2, Ilha do Fundão, 21941-590, RJ,
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Brazil
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dAdvanced Bioprocessing Centre, Institute of Environment, Health & Societies,
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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
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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
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
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
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, H2SO4in 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
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
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
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
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
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
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
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
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
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415
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520
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524
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528
Technol. 30 (2007) 521–532. doi:10.1080/10826070601093846.
529 530
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
Relative Abundance
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
Relative Abundance
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
Relative Abundance
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 3
4 5
9 10 11 1314, 1516 18 20
67 12 17 19 21 2422, 23 25 27 28
A B
C
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
HO
C30H48O4 C
O HO
OH O OH
OH
C30H50O5 A
HO
OH 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 2324
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
Relative Abundance
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
Relative Abundance
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-D1
1 3
4 9
10 11
13 14, 15
17
2 5 18 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
Relative Abundance
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 5 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
Relative Abundance
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-B 13
16
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
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
