high resolution accurate mass spectrometry analysis and characterisation

Journal of Chromatography A, 1481 (2017) 92–100

Contents lists available at ScienceDirect

Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Countercurrent chromatography separation of saponins by skeleton type from Ampelozizyphus amazonicus for off-line ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry analysis and characterisation夽 Fabiana de Souza Figueiredo a , Rita Celano b , Danila de Sousa Silva c , Fernanda das Neves Costa a , Peter Hewitson d , Svetlana Ignatova d , Anna Lisa Piccinelli b , Luca Rastrelli b , Suzana Guimarães Leitão c , Gilda Guimarães Leitão a,∗ a

Universidade Federal do Rio de Janeiro, Instituto de Pesquisas de Produtos Naturais, CCS, bloco H, Ilha do Fundão, 21941-590, RJ, Brazil Università di Salerno, Dipartimento di Farmacia, Via Giovanni Paolo II 132, 84084, Fisciano, Italy Universidade Federal do Rio de Janeiro, Departamento de Produtos Naturais e Alimentos, Faculdade de Farmácia, CCS, bloco A2, Ilha do Fundão, 21941-590, RJ, Brazil d Advanced Bioprocessing Centre, Institute of Environment, Health & Societies, CEDPS, Brunel University London, Middlesex, UB8 3PH, UK b c

a r t i c l e

i n f o

Article history: Received 30 September 2016 Received in revised form 13 December 2016 Accepted 17 December 2016 Available online 19 December 2016 Keywords: Ampelozizyphus amazonicus Rhamnaceae Saponin skeleton type Triterpene saponins Countercurrent chromatography HPLC–MS

a b s t r a c t Ampelozizyphus amazonicus Ducke (Rhamnaceae), a medicinal plant used to prevent malaria, is a climbing shrub, native to the Amazonian region, with jujubogenin glycoside saponins as main compounds. The crude extract of this plant is too complex for any kind of structural identification, and HPLC separation was not sufficient to resolve this issue. Therefore, the aim of this work was to obtain saponin enriched fractions from the bark ethanol extract by countercurrent chromatography (CCC) for further isolation and identification/characterisation of the major saponins by HPLC and MS. The butanol extract was fractionated by CCC with hexane - ethyl acetate - butanol - ethanol - water (1:6:1:1:6; v/v) solvent system yielding 4 group fractions. The collected fractions were analysed by UHPLC-HRMS (ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry) and MSn . Group 1 presented mainly oleane type saponins, and group 3 showed mainly jujubogenin glycosides, keto-dammarane type triterpene saponins and saponins with C31 skeleton. Thus, CCC separated saponins from the butanol-rich extract by skeleton type. A further purification of group 3 by CCC (ethyl acetate - ethanol - water (1:0.2:1; v/v)) and HPLC-RI was performed in order to obtain these unusual aglycones in pure form. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Ampelozizyphus amazonicus Ducke (Rhamnaceae) is a climbing shrub native to the Amazonian region, where its barks and roots are used in the folk medicine to prepare a beverage to cure and prevent malaria, as well as a tonic and fortifier [1,2]. The literature cites triterpenes (ursolic acid, betulinic acid, lupenone, betulin, lupeol, melaleucic acid, 3ˇ-hydroxylup-20(29)-ene27,28-dioic acid, 2␣,3ˇ-dihydroxylup-20(29)-ene-27,28-dioic

夽 Selected paper from the 9th International Counter-current Chromatography Conference (CCC 2016), 1–3 August 2016, Chicago, IL, USA. ∗ Corresponding author. E-mail addresses: [email protected], [email protected] (G. Guimarães Leitão). http://dx.doi.org/10.1016/j.chroma.2016.12.053 0021-9673/© 2016 Elsevier B.V. All rights reserved.

acid and 3ˇ,27␣-dihydroxylup-20(29)-en-28ˇ-oic acid), jujubogenin glycoside saponins (3-O-ˇ-dglucopyranosyl-20-O-␣-L-rhamnopyranosyljujubogenin 3-O-[ˇ-d-glucopyranosyl(l → 2)␣-l-arabinopyranosyl]and 20-O-˛-L-rhamnopyranosyljujubogenin) as well as, C30 and C31 dammarane-type triterpene saponins (ampelozigenin-l5␣-O-acetyl-3-O-␣-l-rhamnopyranopyranosyl(1 → 2)-d-glucopyranoside) as main compounds in these preparations [3–6]. Saponins are usually produced by plants as a complex mixture with very similar structures and polarities. Each saponin is biosynthesized at low concentration, which makes difficult their direct identification and isolation [7]. Therefore, traditionally, multidimensional chromatography has been used, for example, with column chromatography as the first dimension and countercurrent chromatography (CCC) as the second dimen-

F. de Souza Figueiredo et al. / J. Chromatogr. A 1481 (2017) 92–100

sion [8] or CCC as the first dimension and liquid chromatography (LC) as the second one [9]. CCC is a type of liquid-liquid partition chromatography technique with no solid support [10]. The use of liquid stationary phase is advantageous for preparative separations because there is no irreversible adsorption, and it allows a high sample loading, and good reproducibility with the scale-up [11,12]. For the structural characterisation of saponins mass spectrometry is the most often used technique as it provides important information about skeleton type and number of sugars but not sugar identity or linkage [7]. Our previous studies revealed the presence of dammarane saponins with jujubogenin and keto-dammarane skeletons in Ampelozizyphus amazonicus bark extract [1,2,6]. A high complexity of the crude extract, due to the variability and similar structures of saponins, however, did not allow a complete chromatographic separation and identification of individual saponins by UHPLCHRMS and MSn [6]. Therefore, a series of pre-purification steps were undertaken starting from consequent liquid-liquid extraction (LLE) of the crude ethanol extract with hexane, ethyl acetate and butanol. Analyses by TLC and HPLC-HRMS revealed the presence of saponins in the butanol extract (Fig. 1A). The next step was CCC separation to produce less complex samples for HPLC-HRMS characterisation and further isolation of saponins from Ampelozizyphus amazonicus bark by CCC and semi-preparative HPLC. 2. Materials and methods 2.1. Chemical reagents and solvents Organic solvents used to prepare extracts were analytical grade from Tedia (Tedia Brazil, Rio de Janeiro, Brazil). Organic solvents used for TLC analyses and CCC separations were analytical grade from Fisher Chemicals (Loughborough, UK). MS-grade acetonitrile and water were supplied by Romil (Cambridge, UK). Organic solvents used in HPLC-RI separations were HPLC grade from Sigma Aldrich (Milan, Italy) and ultrapure water (18 M) was prepared by a Milli-Q purification system (Millipore, Bedford, USA). 2.2. Plant material and preparation of the extracts The stem barks of A. amazonicus were collected in the Brazilian Amazon, at “quilombola” communities of Oriximiná (State of Pará) [1,2]. Plants were identified by Mr. José Ramos (parataxonomist) and a voucher specimen, INPA 224161, was deposited at the herbarium of Instituto Nacional de Pesquisas da Amazônia (INPA) (Manaus, AM, Brazil) [1,2]. We received authorization for this study from the Brazilian Directing Council for Genetic Heritage (Conselho de Gestão do Patrimonio Genético) through Resolution n.213 (6.12.2007) published in the Federal Official Gazette of Brazil on December 27, 2007. The stem barks were dried in a ventilated oven (Marconi, model MA037) and ground in a hammer mill (Marconi, model MA340, serial 9304176). The powder material of bark was extracted by percolation with ethanol. The extract was filtrated and the ethanol was removed by rotary evaporation at 40 ◦ C under reduced pressure. Then the bark ethanol extract (346.5 g) was sequentially partitioned by hexane/water, ethyl acetate/water and butanol/water in a separatory funnel. The solvents were removed by rotary evaporation. The liquid-liquid extraction afforded 0.2 g of hexane, 44.7 g of ethyl acetate and 72.5 g of butanol partitions.

93

Extractions Ltd. (DE, Tredegar, UK). The Spectrum was equipped with a polytetrafluorethylene (PTFE) column of 143.5 ml and 1.6 mm tubing I.D. The MIDI had a PTFE column of 912.5 ml and 4.0 mm tubing I.D. All separations were performed at the maximum rotation speed of both instruments, 1600 rpm (Revolution radius (R) = 85 mm) and 1400 rpm (R = 110 mm) respectively. The semi-preparative set up had a HPLC pump Agilent HP1200 (Santa Clara, California, USA) and a fraction collector Agilent HP1200 (Santa Clara, California, USA). The preparative chromatographic system had a HPLC pump Knauer K-1800 (Berlin, Germany) and a fraction collector Gilson FC202 (Villiers-le-Bel, France). 2.4. Thin layer chromatography Analyses of A. amazonicus bark extracts, solvent systems and CCC fractions were done by thin layer chromatography (TLC) with silica gel TLC Plates 60F254 (Merck Art. 05554, Darmstadt, Germany). The mobile phase used for TLC analyses was butanol − acetic acid − water (8:0.5:1.5; v/v). To visualize the compounds spots, the universal spray-reagent, H2 SO4 in methanol (5%, v/v) with vanillin in methanol (1%, v/v), and Komarovisky specific spray-reagent for saponins [3,4] with subsequent heating at 105 ◦ C on a hot plate were used. 2.5. Solvent system tests The solvent systems tests were performed as follows: small amounts of a sample extract were dissolved in a test tube containing a two-phase solvent system. After shaking and allowing compounds to partition between the two phases, equal aliquots of each phase were spotted beside each other separately on silica gel TLC plates. Distribution coefficients (KD ) were determined visually. 2.6. CCC separations Solvent systems used in all separations by CCC were prepared in a separatory funnel at room temperature. After the equilibrating, the two phases were separated and degassed by sonication for 5 min. In each separation run, a CCC column was first filled with the stationary phase, after set the rotation, the mobile phase was pumped in. Samples were dissolved in equal volumes of each phase and were injected after the hydrodynamic equilibrium inside the column was reached. The column temperature was maintained at 30 ◦ C. 2.6.1. CCC fractionation of the butanol extract of A. amazonicus The solvent system chosen for fractionation of the butanol extract of A. amazonicus was hexane – ethyl acetate – butanol – ethanol – water (1:6:1:1:6; v/v).

2.3. Countercurrent chromatography apparatus

2.6.1.1. Semi-preparative fractionation. The fractionation was performed on the Spectrum machine with the organic upper phase as stationary phase and the aqueous lower phase as mobile phase (reversed phase mode). Fractions of 4 ml were collected during elution (72 fractions, 2 ml/min, 2 Vc ) and extrusion (36 fractions, 20 ml/min, 1 Vc ). The sample was injected using an Upchurch low pressure injection port (Model V-450, with 1/16 in. fittings) and a loop of 7.2 ml. The sample concentration was 100 mg/ml. The stationary phase retention (Sf ) before sample injection was 62%. Fractions were analysed by TLC and UHPLC-HRMS and MSn analyses (Fig. 2).

Two high performance countercurrent chromatography (HPCCC) centrifuges were used for CCC separations, a Spectrum (semi-preparative) and a MIDI (preparative), both from Dynamic

2.6.1.2. Preparative fractionation. The preparative fractionation of the butanol extract of A. amazonicus was performed on the MIDI machine. Fractions of 24 ml were collected during elution (76

94

F. de Souza Figueiredo et al. / J. Chromatogr. A 1481 (2017) 92–100 100

A

90

100

80 Relative Abundance

70 60 50 40 30

70 60 50 40 30

20

20

10

10

0

0

15

20

0

5

100

80

20

C

30

27

25

22, 23

19 20

2

20

24

18

6

40

21

7

50

10

17

60

28

16

70

4 5

Relative Abundance

15

1

3

90

10 Time (min)

15

10 Time (min)

11 13 14,

5

9

0

12

Relative Abundance

B

90

80

10 0 0

2

4

6

8

10

12 Time (min)

14

16

18

Fig. 1. UHPLC-HRMS profiles of butanol extract (A) and its HPCCC groups 1 (B) and 3 (C).

Fig. 2. Separation by HPCCC of butanol extract and group 3. This scheme was based on information from a MIDI run. In Spectrum runs only total number of fractions change.

20

22

F. de Souza Figueiredo et al. / J. Chromatogr. A 1481 (2017) 92–100

95

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

i ii iii iv v vi 1 2 3 4 5 6 7 8 9

Hex

DCM

EtOAc

Acetone

BuOH

PrOH

iPrOH

EtOH

MeOH

H2 OH2 O

CH3 COONH4 5 mM

– 1 5 1 6 1 – – – – – – – – –

– – – – – – 6 – – – – – – – –

1 1 6 1 6 6 – 1 1 1 1 1 1 1 1

– – – 0.5 – – – – – – – – – – –

1 – – – 1 1 – 0.5 0.5 – – – – – –

– – – – – – – – – – – – – 0.5 0.5

– – – – – – 3 – – – – 0.5 0.5 – –

– 1 5 1 6 1 – – 0.2 – 0.2 – 0.2 – 0.2

– – – – – – 2 0.2 – 0.2 – 0.2 – 0.2 –

2 1 6 1 6 6 – 1 1 1 1 1 1 1 1

– – – – – – 4 – – – – – – – –

Hex: hexane. DCM: dichloromethane. EtOAc: ethyl acetate. BuOH: butanol. PrOH: propanol. iPrOH: isopropanol. EtOH: ethanol. MeOH: methanol.

fractions, 12 ml/min, 2 column volume (Vc )) and extrusion (38 fractions, 24 ml/min, 1 Vc ). The sample was injected using an Upchurch low pressure injection port (Model V-450, with 1/16 in. fittings) and loops of 45 ml and 90 ml. The sample concentration was 100 mg/ml. The stationary phase retention (Sf ) before injection was 67%. After TLC and UHPLC-HRMS and MSn analyses, fractions were combined in groups (Fig. 2).

2.6.2. CCC fractionation of group 3 from the butanol extract of A. amazonicus Purification of group 3 (Frs. 81–101 from the first CCC butanol extract fractionation) was done with ethyl acetate – ethanol – water (1:0.2:1; v/v). The aqueous lower phase was used as stationary phase and the organic upper phase as the mobile phase (normal phase mode). The semi-preparative purification of group 3 was first performed on the Spectrum. Fractions of 4 ml were collected during elution (36 fractions, 1 ml/min, 1 Vc ) and extrusion (36 fractions, 2 ml/min, 1 Vc ). The sample was injected using a loop of 3.66 ml. The sample concentration was 27.5 mg/ml. The stationary phase retention (Sf ) before injection was 77%. The preparative purification of group 3 was performed on the MIDI machine. Fractions of 24 ml were collected during elution (38 fractions, 12 ml/min, 1 Vc ) and extrusion (38 fractions, 24 ml/min, 1 Vc ). The sample was injected using a loop of 45 ml. The sample concentration was 27.5 mg/ml. The stationary phase retention (Sf ) before injection was 90%. After TLC and UHPLC-HRMS and MSn analyses, fractions were combined in groups (Fig. 2).

2.7. HPLC separations Fractions from Group 3 CCC separation were combined in different groups (Fig. 2). Groups C and D2 were separated further by semi-preparative HPLC-RI. The column used was a HyPurity Aquastar, 150 × 10 mm; particle size 5 ␮ (Thermo Electron Corporation). The semi-preparative HPLC system was composed of a pump Knauer Smartline 1000 (Labservice Analytica, Bologna, Italy) and a refractive index (RI) detector Knauer Smartline 2300 (Labservice Analytica). The mobile phase used was aqueous methanol, 5.9:4.1; v/v, in isocratic mode. For group C, the flow rate was 3.5 ml/min, the sample was dissolved in methanol (0.1 mg/␮l) and the sample solution injected in each run was 35 ␮l. For group D2, the flow rate was 2.5 ml/min, the sample was dissolved in methanol (0.1 mg/␮l) and the sample solution injected in each run was 50 ␮l. All fractions were analysed by UHPLC-HRMS and MSn . 2.8. UHPLC-HRMS analyses The butanol extract, fractions from CCC and HPLC-RI separations were analysed on an LTQ OrbiTrap XL mass spectrometer (LTQ OrbiTrap XL, ThermoFisher Scientific) connected to a Platin Blue UHPLC system (KNAUER GmbH, Berlin, Germany). This UHPLC system was composed by two ultra-high-pressure pumps, an auto sampler, a diode array detector and a column temperature manager. The LC parameters used were: a Kinetex C18 column (2.1 × 50 mm, 1.7 ␮m; Phenomenex, Bologna, Italy), flow rate of 0.5 ml min−1 , column temperature of 30 ◦ C and, water (A) and ACN (B), both containing 0.1% formic acid, as mobile phase. The gradient elution program used was: 10–20% B in 3 min, 20–25% B in 4 min, 25–30% B in 13 min and 30–50% B in 5 min. After each injection, the column OH

OH OH

OH

OH

O

O

O

HO

OH

OH

HO

O HO

C30H50O5

C31H52O5

C30H48O4

A

B

C

Fig. 3. Proposed aglycone structures of saponins in group 3: (A) 16-keto-tetrahydroxydammar-23-ene, (B) 16-keto-tetrahydroxydammar-24-methylene and (C) jujubogenin.

96

F. de Souza Figueiredo et al. / J. Chromatogr. A 1481 (2017) 92–100

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

tR (min)

[M-H]-(m/z)

Molecular Formula

ppm

Diagnostic product iona (m/z)

Aglyconeb

Sugar residuec

1

8.2

779.4577

C42 H68 O13

0.2

C30 H48 O4

1 Hex, 1 dHex

2

11.4

959.5211

C48 H80 O19

0.1

C30 H50 O5

2 Hex, 1 dHex

3

12.2

915.4956

C46 H76 O18

0.9

C30 H50 O5

2 Pen, 1 Hex

4

12.7

929.5113

C47 H78 O18

0.9

C30 H50 O5

1 Hex, 1 dHex, 1 Pen

5

13.0

959.5217

C48 H80 O19

0.8

C31 H52 O5

2 Hex, 1 Pen

6

13.6

931.5266

C47 H80 O18

0.6

C30 H52 O4

2 Hex, 1 Pen

7 8

13.7 13.8

927.4951 955.4901

C47 H76 O18 C48 H76 O19

0.3 0.5

C30 H48 O4

2 Hex, 1 Pen 1 Pen, 1 Hex

9

13.9

959.5217

C48 H80 O19

0.7

C31 H52 O5

2 Hex, 1 Pen

10

14.4

929.5108

C47 H78 O18

0.4

C31 H52 O5

1 Hex, 2 Pen

11

14.9

927.4957

C47 H76 O18

1

C30 H48 O4

2 Hex, 1 Pen

12

15.3

929.5106

C47 H78 O18

0.2

C31 H52 O5

1 Hex, 2 Pen

13

15.6

929.511

C47 H78 O18

0.6

C31 H52 O5

1 Hex, 2 Pen

14

15.7

897.4835

C46 H74 O17

−0.9

C30 H48 O4

1 Hex, 2 Pen

15

16.0

897.4854

C46 H74 O17

1.3

C30 H48 O4

2 Hex, 2 Pen

16

16.2

943.5258

C48 H80 O18

−0.3

C31 H52 O5

1 Hex, 1 dHex, 1 Pen

17

16.5

911.5001

C47 H76 O17

0.2

633 (-dHex), 617 (-Hex), 471 (C30 H48 O4 ) 817 (-C8 H14 O2 ), 655 (-C8 H14 O2 -Hex), 509d (-C8 H14 O2 -Hex-dHex) 773 (-C8 H14 O2 ), 611 (-C8 H14 O2 -Hex), 641 (-C8 H14 O2 -Pen), 479d (-C8 H14 O2 -Hex-Pen) 787 (-C8 H14 O2 ), 625(-C8 H14 O2 -Hex), 479d (-C8 H14 O2 -Hex-dHex) 803 (-C9 H16 O2 ) 641 (-C9 H16 O2 -Hex), 479d (-C9 H16 O2 -2Hex) 799 (-Pen), 769 (-Hex), 637(-Hex-Pen) 765 (-Hex-), 603(-2Hex) 823 (-Pen), 793(-Hex), 661 (-Pen-Hex) 803 (-C9 H16 O2 ), 641 (-C9 H16 O2 -Hex), 479d (-C9 H16 O2 -2Hex) 773 (-C9 H16 O2 ), 611 (-C9 H16 O2 -Hex), 479d (-C9 H16 O2 -Hex-Pen) 795 (-Pen), 765 (-Hex), 633(-Hex-Pen) 773 (-C9 H16 O2 ), 611 (-C9 H16 O2 -Hex), 479d (-C9 H16 O2 -Hex-Pen) 773 (-C9 H16 O2 ), 611 (-C9 H16 O2 -Hex), 479d (-C9 H16 O2 -Hex-Pen) 765 (-Pen), 735 (Hex), 603(-Pen-Hex), 471d (C30 H48 O4 ) 765 (-Pen), 735 (Hex), 603(-Pen-Hex), 471d (C30 H48 O4 ) 787 (-C9 H16 O2 ), 625 (-C9 H16 O2 -Hex), 479d (-C9 H16 O2 -Hex-dHex) 749 (-Hex), 603 (-Hex-dHex)

C30 H48 O4

18 19 20

16.7 17.4 18.0

955.5257 1013.532 1003.547

C49 H80 O18 C51 H82 O20 C50 H84 O20

−0.3 0.1 −0.1

C30 H52 O4

1 Hex, 1 dHex, 1 Pen 1 Hex, 1 dHex 1 Hex, 1 dHex 3 Hex

21 22

18.2 19.0

1013.532 1003.548

C51 H82 O20 C50 H84 O20

0.3 0.7

C30 H52 O4

1 Hex, 1 dHex 3 Hex

23

19.0

1041.527

C52 H82 O21

0.1

C30 H48 O4

1 Hex, 2 Pen

24

19.3

1041.527

C52 H82 O21

0

C30 H48 O4

2 Hex, 2 Pen

25

20.1

973.5371

C49 H82 O19

0.5

C30 H52 O4

2 Hex 1 Pen

26

20.1

969.5062

C49 H78 O19

1.0

27

20.9

969.5058

C49 H78 O19

0.4

28

21.2

973.5372

C49 H82 O19

0.6

a b c d

Hex: hexose; dHex: deoxyhexose; Pen: pentose; HMG: hydroxymethylglutaryl. Strucutures reported in Fig. 3. In bold the sugar residue attached to aglycone skeleton. Product ions detected in MS3 spectra.

793 (-Hex), 647 (-Hex-dHex) 851 (-Hex), 705 (-Hex-dHex) 943(-C2 H4 O2 ), 841 (-Hex), 781 (-C2 H4 O2 -Hex) 851 (-Hex), 705 (-Hex-dHex) 943(-C2 H4 O2 ), 841 (-Hex), 781 (-C2 H4 O2 -Hex) 897 (-HMG), 879 (-Hex), 765 (-HMG-Pen), 735 (-HMG-Hex) 897 (-HMG), 879 (-Hex), 765 (-HMG-Pen), 735 (-HMG-Hex) 913 (-C2 H4 O2 ), 811 (-Hex), 781 (-C2 H4 O2 -Pen), 751 (-C2 H4 O2 -Hex), 619 (-C2 H4 O2 -Pen-Hex) 837 (-Pen), 807 (-Hex), 675 (-Pen-Hex) 837 (-Pen), 807 (-Hex), 675 (-Pen-Hex) 913 (-C2 H4 O2 ), 811 (-Hex), 781 (-C2 H4 O2 -Pen), 751 (-C2 H4 O2 -Hex), 619 (-C2 H4 O2 -Pen-Hex)

1 Hex, 1 Pen 1 Hex, 1 Pen C30 H52 O4

2 Hex 1 Pen

F. de Souza Figueiredo et al. / J. Chromatogr. A 1481 (2017) 92–100

16

100 Relative Abundance

97

Group 3 CCC-B

13

80 60 40 20 0 6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

2

13 Relative Abundance

100

Group 3 CCC-C

16

80

14

10

60 40

1

2

20

3

4

17 18

21

27 28

2324

0 6

7

9

10

11

12

3

1

100 Relative Abundance

8

14

15

16

40

2

20

11

5

18

19

20

21

22

23

Group 3 CCC-D1

17

10

60

17

13 14, 15

9

4

80

13

28

18

25

21

0 6

7

8

9

10

12

13

14

15

16

17

18

19

20

9

3

100 Relative Abundance

11

60

22

23

Group 3 CCC-D2

14, 15

80

21

11

1

45

40

25

17 18

20

28

0 6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

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

was washed with 98% B for 4 min and re-equilibrated for 4 min. The mass spectrometer, with ESI source, was operated in negative mode. High purity nitrogen (N2 ) was used as sheath gas (40 arbitrary units) and auxiliary gas (arbitrary units). High purity helium (He) was used as collision gas. Mass spectrometer parameters used were: 3.5 KV of source voltage, −37 V of capillary voltage, −225 V of tube lens voltage and 280 ◦ C of capillary temperature. Full scan data acquisition (mass range: m/z 350–2000) and data dependant MS scan were performed. The resolution was 60,000 and 7500 for the full mass and the data dependant MS scan, respectively. The normalised collision energy of the collision-induced dissociation (CID) cell was set at 35 eV and the isolation width of precursor ions was set at 2.0. Saponins were characterized according to the corresponding spectral characteristics: mass spectra, accurate mass, characteristic fragmentation, and retention time. Xcalibur software (version 2.2) was used for instrument control, data acquisition and data analysis. 3. Results and discussion 3.1. Butanol extract separation by CCC Previous studies on separation of dammarane saponins by CCC used ethyl acetate − butanol − water (1:1:2; v/v) and hexane −

ethyl acetate − ethanol − water (1:1:1:1; v/v) solvent systems [13,14]. Therefore, they were selected for preliminary tests. In search for the best solvent system showing a good distribution of compounds between the two phases (K visually close to 1), different solvent proportions were tested in order to change system’s polarity and polarity difference between phases. Some solvents were added or replaced, in order to change the selectivity of systems [15]. Table 1 lists all solvent systems, i-iv, tested for the butanol extract purification. The distribution of compounds between the two phases in each solvent system was analysed by TLC [16]. In the first solvent system, (i) ethyl acetate − butanol − water (1:1:2; v/v), compounds were practically all concentrated in upper phase and in the second system, (ii) hexane − ethyl acetate − ethanol − water (1:1:1:1; v/v), compounds were concentrated mainly in the lower phase. To increase polarity of the second system, ii, the proportions of hexane and ethanol were changed to (iii) hexane − ethyl acetate − ethanol − water (5:6:5:6; v/v), causing a drop in the sample solubility. To resolve this issue and to increase polarity, other solvents such as acetone were added to a solvent system, (iv) hexane − ethyl acetate − acetone − ethanol − water (1:1:0.5:1:1; v/v), and also systems i and ii were combined, (v) hexane − ethyl acetate − butanol − ethanol − water (6:6:1:6:6; v/v). In (iv), the sample has limited solubility and in (v) it was soluble and compounds were more concentrated in lower phase like system

98

F. de Souza Figueiredo et al. / J. Chromatogr. A 1481 (2017) 92–100

(ii). After testing various solvent ratios aiming to achieve a K visually close to 1, the best solvent system appeared to be (vi) hexane − ethyl acetate − butanol − ethanol − water (1:6:1:1:6; v/v). Every other CCC fractions were analysed by TLC and UHPLC–MS before being combined in groups (Fig. 2) according to TLC profile and mass distribution. UHPLC-HRMS analyses of CCC groups 1–4 and their fractions revealed the presence of saponins mainly in groups 1 and 3. Although UHPLC-HRMS analyses helped to characterize the groups from CCC separation of butanol extract, the groups 1 and 3 still showed a high complexity (Fig. 1B and C). Thus, CCC fractionation of the butanol extract was scaled-up in order to obtain a higher amount of each group fraction for subsequent purification steps. The scale-up factor (6.36) was calculated as the ratio between the column volumes of Spectrum (143.5 ml) and MIDI (912.5 ml), according to CCC volumetric scale-up [17,18]. This scale-up factor was used to adjust the flow rate and the sample volume. Stationary phase retention (Sf ) before injection were 62% and 67% respectively. Based on Sutherland and co-workers (2005) theory, which stated that larger tubing bore provides a better stationary phase retention and therefore, larger scale-up factors can be reached, the sample loading was increased by doubling the sample volume [19]. The reproducibility of runs was analysed by TLC.

A3 [23], respectively, and the isomers 14 and 15 to bacopasaponin C [24]. In addition, the structure of hydroxymethylglutaryl (HMG) jujubogenin glycosides [25] was established for 23 and 24 by the diagnostic neutral loss of −144 Da and molecular formula of product ions [M−HMG]− . Finally, one dammarane-types saponin, 6, and four acetylated derivatives, 20, 22, 25 and 28, were detected in group 3. Their MS/MS spectra (Table 2) suggested the structure of acylated tetrahydroxydammar-24-ene triglycosides, structurally related to ginseng saponins with protopanaxatriol (C30 H52 O4 ) as aglycone [26,27]. The sugar residues of all identified saponins were established by characteristic neutral losses (hexose −162 Da, deoxyhexose −146 Da, pentose −132 Da) and accurate mass of corresponding product ions. Particularly, in the case of C30 (2–4) and C31 ketodammarane saponins (5, 9–10, 12–13 and 16) the product ions at 479.3003 or 509.3109 in MSn spectra allowed to establish the nature of the sugar residue (pentose or hexose, respectively) directly attached to the aglycone skeleton. Other minor compounds (7, 8, 18, 19, 21, 26 and 27) detected in group 3 were tentatively identified as saponins, but further studies are required to their detailed characterization and identification of aglycones. 3.3. Separation of group 3 by CCC and HPLC-RI

3.2. UHPLC-HRMS analyses of butanol extract of CCC groups The combination of high resolution mass spectrometry and MSn experiments was employed to identify the main constituents of groups 1 and 3. Group 1 showed a complex profile with two main metabolite classes (Fig. 1B). The first consisted of polar phenolic compounds (0–5 min), while triterpene glycosides, possibly with the oleane skeleton as aglycone, were inferred as the second metabolite class (7–16 min) (data not shown). A complete elucidation of the group 1 saponin structures is currently in progress. Dammarane saponins are the major constituents of the group 3 (Fig. 1C). Table 2 report the HRMS data of the main saponins of this CCC group and their proposed molecular formulas. HRMS and MSn data (Table 2) allowed to identify saponins with C30 and C31 keto-dammarane and jujubogenin skeletons (Fig. 3), according to our previous studies [2,6], and dammarane-types saponins. Three saponins (2–4) were tentatively identified as 16-ketotetrahydroxydammar-23-ene triglycosides (C30 H50 O5 , Fig. 3A), based on the presence in MS/MS spectra of the diagnostic product ion [M−C8 H14 O2 ]− due to the loss of the side chains by a McLafferty rearrangement [6,20]. Comparison with literature data suggested for the compounds 3 and 4 structures superimposable to those of hoduloside VIII and VII, respectively, isolated from Rhamnaceae [21]. Also 5, 9–10, 12–13 and 16 produced a similar fragmentation pathway of 2–4 yielded by McLafferty rearrangement. The dominant product ions [M−C9 H16 O2 ]− correspond to the loss of an alkyl side chain with an additional methylene group than to 16-keto-tetrahydroxydammar-23-ene glycosides. Based on this fragmentation pathway and the occurrence of a C31 dammarane-type saponin in A. amazonicus [4], the 16-ketotetrahydroxydammar-24-methylene structure (C31 H52 O5 , Fig. 3B) was proposed as aglycon of saponins 5, 9–10, 12–13 and 16. This aglycone is not reported in the literature and further studies are needed to confirm unambiguously the proposed structure. Compounds 1, 7, 11, 14–15 and 17 were tentatively characterized as glycosides of jujubogenin (C30 H48 O4 , Fig. 3C), primarily due to the presence of the product ion at m/z 471.3469 in MSn spectra, corresponding to the deprotonated jujubogenin (C30 H47 O4 ). Compounds corresponding to the structure proposed for 1 and 17 were previously reported in A. amazonicus [3], whereas the isomers 7 and 11 corresponded presumably to hoduloside IV [22] and bacoside

UHPLC-HRMS analysis of butanol extract CCC groups showed the presence of dammarane saponins only in the group 3. This saponin class is characteristic of A. amazonicus [2–4] and it includes unusual aglycones as C30 and C31 keto-dammarane [4,6,20]. Thus, a further purification of group 3 by CCC and HPLC-RI was performed in order to obtain as pure as possible these unusual compounds. The same approach, as used for butanol extract, was applied to choose a suitable solvent system for group 3 (Table 1; 1–9). Based on a previous work, where dammarane saponins were isolated from Panax ginseng, the solvent system (1) dichloromethane − isopropanol − methanol − 5 mM aqueous ammonium acetate (6:3:2:4; v/v) was tested as a start [28]. Compounds were more concentrated in lower phase. Further tests showed that in systems (2) ethyl acetate − butanol − methanol − water (1:0.5:0.2:1; v/v) and (3) ethyl acetate − butanol − ethanol − water (1:0.5:0.2:1; v/v) practically all compounds were in upper phase and any difference between system selectivity was observed [29]. In (4) ethyl acetate − methanol − water (1:0.2:1; v/v) [30] and (5) ethyl acetate − ethanol − water (1:0.2:1; v/v), was achieved a good distribution of compounds between the two phases and a slight difference between compounds selectivity. The system 5 showed visually slightly better selectivity than the system 4. The replacement of ethanol with methanol in (6) ethyl acetate − isopropanol − methanol − water (1:0.5:0.2:1; v/v) and (8) ethyl acetate − propanol − methanol − water (1:0.5:0.2:1; v/v), provided a better distribution between two phases as in (7) ethyl acetate − isopropanol − ethanol − water (1:0.5:0.2:1; v/v) and (9) ethyl acetate − propanol − ethanol − water (1:0.5:0.2:1; v/v), because in ethanol containing systems compounds were slightly more concentrated in upper phase due to ethanol polarity in comparison with methanol. For the same reason, addition of propanol or isopropanol to a solvent system reduced the its selectivity (Ks visually similar). Thus, the solvent system selected for the purification of this group was (5) ethyl acetate − ethanol − water (1:0.2:1; v/v). Every two CCC fractions were analysed by TLC and UHPLC–MS before being combined in groups (Fig. 2) according to TLC profile and mass distribution. Betulinic acid was identified as main compounds of group A, while dammarane saponins were detected in the other group 3 of CCC fractions. As shown in the chromatograms reported in Fig. 4, C, D1 and D2 groups were the most saponin-enriched

F. de Souza Figueiredo et al. / J. Chromatogr. A 1481 (2017) 92–100

groups. The main components of group C were the C31 dammaranetype saponins 10, 13 and 16, whereas D groups were rich in C30 dammarane saponins 3–4 and 10, jujubogenin glycosides 11, 14, 15 and 17 and compound 9. The fractionation of group 3 was also scaled-up to MIDI to obtain larger amounts of enriched fractions for the successive purification by semi-preparative HPLC-RI. Testing four different flow rates, 10, 12, 20 and 40 ml/min resulted in stationary phase retention (Sf ) of 86%, 90%, 81% and none, respectively. Therefore, flow rate of 12 ml/min was selected for MIDI runs. The scale-up factor (6.36), applied in CCC separation of butanol extract, was used to adjust the sample volume. In order to isolate the main dammarane saponins of A. amazonicus bark, particularly saponins with C31 keto-dammarane-type skeleton, groups C and D2 from the CCC purification of group 3 were selected for a subsequent purification by semi-preparative HPLC-RI. This isolation procedure allowed to obtain the jujubogenin glycosides 1, 11 and 14–15, C31 dammarane saponins 9–10 and 13 and C30 dammarane saponins 3 and 4, with a suitable purity grade (checked by NMR) for a detailed characterization of their structures. 4. Conclusions The preparative purification procedure, based on CCC and HPLCRI separations, was successfully developed to isolate the main constituents of A. amazonicus bark. The CCC reduced the complexity of butanol extract allowing a characterisation by UHPLC-HRMS of saponins and allowed to isolate unusual C31 saponins by HPLC. CCC was able to separate saponins by skeleton type, mainly oleane in group 1 and dammarane in group 3. The demonstrated scale-up methodology enables more detailed chemical studies of compounds via future structure elucidation by NMR.

[6]

[7]

[8]

[9]

[10] [11] [12]

[13]

[14]

[15]

[16]

[17]

Acknowledgement F.S. Figueiredo is indebted to Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES, Brazil) for the Ph.D scholarship. F.N. Costa and S. Ignatova would like to thank Newton Advanced Fellowship project funded by the Royal Society of the United Kingdom. S.G. Leitão and G.G. Leitão are indebted to FAPERJ and CNPq for financial support. The authors are deeply indebted to ARQMO (Associac¸ão de Comunidades Remanescentes de Quilombolas do Município de Oriximiná), Oriximiná-PA, Brazil, for supervising plant collection and for providing housing during the field trips.

[18]

[19]

[20]

[21]

[22]

References [1] D.R. de Oliveira, A.L.M.A. Costa, G.G. Leitão, N.G. Castro, J.P. dos Santos, S.G. Leitão, Estudo etnofarmacognóstico da saracuramirá (Ampelozizyphus amazonicus Ducke), uma planta medicinal usada por comunidades quilombolas do Município de Oriximiná-PA, Brasil, Acta Amaz. 41 (2011) 383–392, http://dx.doi.org/10.1590/S0044-59672011000300008. [2] L.M.T. Pec¸anha, P.D. Fernandes, T.J.M. Simen, D.R. De Oliveira, P.V. Finotelli, M.V.A. Pereira, F.F. Barboza, T.D.S. Almeida, S. Carvalhal, A.P.T.R. Pierucci, G.G. Leitão, L. Rastrelli, A.L. Piccinelli, S.G. Leitão, Immunobiologic and antiinflammatory properties of a bark extract from Ampelozizyphus amazonicus Ducke, Biomed Res. Int. 2013 (2013), http://dx.doi.org/10.1155/ 2013/451679. [3] M.G.L. Brandao, M.A. Lacaille-Dubois, M.A. Teixera, H. Wagner, Triterpene saponins from the roots of Ampelozizyphus amazonicus, Phytochemistry 31 (1992) 352–354, http://dx.doi.org/10.1016/0031-9422(91)83076-W. [4] M.G.L. Brandao, M.A. Lacaille-Dubois, M.A. Teixera, H. Wagner, A dammarane-type saponin from the roots of Ampelozizyphus amazonicus, Phytochemistry 34 (1993) 1123–1127. [5] L.V. Rosas, M.S.C. Cordeiro, F.R. Campos, S.K.R. Nascimento, A.H. Januário, S.C. Franc¸a, A. Nomizo, M.P.A. Toldo, S. Albuquerque, P.S. Pereira, In vitro evaluation of the cytotoxic and trypanocidal activities of Ampelozizyphus

[23]

[24]

[25]

[26] [27]

[28]

99

amazonicus (Rhamnaceae), Brazilian J. Med. Biol. Res. 40 (2007) 663–670, http://dx.doi.org/10.1590/S0100-879X2007000500009. T.J.M. Simen, P.V. Finotelli, F.F. Barboza, M.V.A. Pereira, A.P.T.R. Pierucci, M.R.L. Moura, D.R. Oliveira, L.G. Abrac¸ado, R. Celano, F.S. Figueiredo, A.L. Piccinelli, L. Rastrelli, G.G. Leitão, L.M.T. Pec¸anha, S.G. Leitão, Spray-dried extract from the Amazonian adaptogenic plant Ampelozizyphus amazonicus Ducke (Saracura-mirá): chemical composition and immunomodulatory properties, Food Res. Int. 90 (2016) 100–110, http://dx.doi.org/10.1016/j.foodres.2016.10. 040. W. Oleszek, Z. Bialy, Chromatographic determination of plant saponins-an update (2002–2005), J. Chromatogr. A 1112 (2006) 78–91, http://dx.doi.org/ 10.1016/j.chroma.2006.01.037. M. Kang, I.J. Ha, J. Chun, S.S. Kang, Y.S. Kim, Separation of two cytotoxic saponins from the roots of Adenophora triphylla var. japonica by high-speed counter-current chromatography, Phytochem. Anal. 24 (2013) 148–154, http://dx.doi.org/10.1002/pca.2394. D. Zhao, M. Yan, Y. Huang, X. Sun, Efficient protocol for isolation and purification of different soyasaponins from soy hypocotyls, J. Sep. Sci. 35 (2012) 3281–3292, http://dx.doi.org/10.1002/jssc.201200531. W.D. Conway, Counter-Current Chromatography: Apparatus, Theory and Applications, VCH Publishers Inc., NY, 1990. A. Berthod, Countercurrent chromatography: from the milligram to the kilogram, Adv. Chromatogr. 32 (2008) 4–35 (2). A. Berthod, T. Maryutina, B. Spivakov, O. Shpigun, I.A. Sutherland, Countercurrent chromatography in analytical chemistry (IUPAC technical report), Pure Appl. Chem. 81 (2009) 355–387, http://dx.doi.org/10.1351/PACREP-08-06-05. X.L. Cao, Y. Tian, T.Y. Zhang, Q.H. Liu, L.J. Jia, Y. Ito, Separation of dammarane-saponins from notoginseng, root of Panax notoginseng (Burk.) F.H. Chen, by HSCCC coupled with evaporative light scattering detector, J. Liq. Chromatogr. Relat. Technol. 26 (2003) 1579–1591, http://dx.doi.org/10.1081/ JLC-120021268. J. Peng, F. Dong, Y. Qi, X. Han, Y. Xu, L. Xu, Q. Xu, K. Liu, Z. Zhu, Preparative separation of four triterpene saponins from Radix Astragali by high-speed counter-current chromatographycoupled with evaporative light scattering detection, Phytochem. Anal. 19 (2008) 212–217, http://dx.doi.org/10.1002/ pca.1011. Y. Ito, Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography, J. Chromatogr. A. 1065 (2005) 145–168, http://dx.doi.org/10.1016/j.chroma.2004.12.044. A. Marston, K. Hostettmann, Developments in the application of counter-current chromatography to plant analysis, J. Chromatogr. A. 1112 (2006) 181–194, http://dx.doi.org/10.1016/j.chroma.2005.10.018. P. Wood, S. Ignatova, L. Janaway, D. Keay, D. Hawes, I. Garrard, I.A. Sutherland, Counter-current chromatography separation scaled up from an analytical column to a production column, J. Chromatogr. A. 1151 (2007) 25–30, http:// dx.doi.org/10.1016/j.chroma.2007.02.014. F.N. Costa, M.N. Vieira, I. Garrard, P. Hewitson, G. Jerz, G.G. Leitão, S. Ignatova, Schinus terebinthifolius countercurrent chromatography (Part II): Intra-apparatus scale-up and inter-apparatus method transfer, J. Chromatogr. A 1466 (2016) 76–83, http://dx.doi.org/10.1016/j.chroma.2016.08.054. I. Sutherland, D. Hawes, S. Ignatova, L. Janaway, P. Wood, Review of progress toward the industrial scale-up of CCC, J. Liq. Chromatogr. Relat. Technol. 28 (2005) 1877–1891, http://dx.doi.org/10.1081/JLC-200063521. J.J. Ma, L.P. Kang, W.B. Zhou, H.S. Yu, P. Liu, B.P. Ma, Identification and characterization of saponins in extract of Ziziphi spinosae semen (ZSS) by ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MS E), J. Med. Plant Res. 5 (2011) 6152–6159, http://dx.doi. org/10.5897/JMPR11.339. K. Yoshikawa, Y. Nagai, M. Yoshida, S. Arihara, Antisweet natural products. VIII. Structures of hodulosides VI-X from Hovenia dulcis Thunb. var. tomentella Makino, Chem. Pharm. Bull. 41 (1993) 1722–1725. K. Yoshikawa, S. Tumura, K. Yamada, S. Arihara, Antisweet natural products. VII. Hodulosides I, II III, IV, and V from the leaves of Hovenia dulcis Thunb, Chem. Pharm. Bull. 40 (1992) 2287–2291. W. Phrompittayarat, W. Putalun, H. Tanaka, K. Jetiyanon, S. Wlttaya-Areekul, K. Ingkaninan, Determination of pseudojujubogenin glycosides from Brahmi based on immunoassay using a monoclonal antibody against bacopaside I, Phytochem. Anal. 18 (2007) 411–418, http://dx.doi.org/10.1002/pca.996. R. Higuchi, S. Kubota, T. Komori, T. Kawasaki, V.B. Pandey, J.P. Singh, A.H. Shah, Triterpenoid saponins from the bark of Zizyphus joazeir, Phytochemistry 23 (1984) 2597–2600, http://dx.doi.org/10.1016/S0031-9422(00)84106-0. A. Plaza, M. Cinco, A. Tubaro, C. Pizza, S. Piacente, New triterpene glycosides from the stems of Anomospermum grandifolium, J. Nat. Prod. 66 (2003) 1606–1610, http://dx.doi.org/10.1021/np030283j. D.G. Lee, Y. Lee, K. Kim, E.J. Cho, S. Lee, Novel dammarane-type triterpene saponins from Panax ginseng root, Chem. Pharm. Bull. 63 (2015) 927–934. W. Yang, M. Ye, X. Qiao, C. Liu, W. Miao, T. Bo, H. Tao, D. Guo, A strategy for efficient discovery of new natural compounds by integrating orthogonal column chromatography and liquid chromatography/mass spectrometry analysis: its application in Panax ginseng, Panax quinquefolium and Panax notoginseng to characterize 437 potencial new ginsenosides, Anal. Chim. Acta 739 (2012) 56–66, http://dx.doi.org/10.1016/j.aca.2012.06.017. X. Qi, S. Ignatova, G. Luo, Q. Liang, F.W. Jun, Y. Wang, I. Sutherland, Preparative isolation and purification of ginsenosides Rf, Re, Rd and Rb1 from the roots of

100

F. de Souza Figueiredo et al. / J. Chromatogr. A 1481 (2017) 92–100

Panax ginseng with a salt/containing solvent system and flow step-gradient by high performance counter-current chromatography coupled with an evaporative light scattering detector, J. Chromatogr. A. 1217 (2010) 1995–2001, http://dx.doi.org/10.1016/j.chroma.2010.01.057. [29] X. Zhang, Y. Ito, J. Liang, Q. Su, Y. Zhang, J. Liu, W. Sun, Preparative isolation and purification of five steroid saponins from Dioscorea zingiberensis C.H. Wright by counter-current chromatography coupled with evaporative light scattering detector, J. Pharm. Biomed. Anal. 84 (2013) 117–123, http://dx.doi. org/10.1016/j.jpba.2013.02.005.

[30] R. Liu, L. Kong, A. Li, A. Sun, Preparative isolation and purification of saponin and flavone glycoside compounds from Clinopodium chinensis (Benth) O. Kuntze by high-speed countercurrent chromatography, J. Liq. Chromatogr. Relat. Technol. 30 (2007) 521–532, http://dx.doi.org/10.1080/ 10826070601093846.