Isolation and Structural Characterization of. Echinocystic Acid Triterpenoid Saponins from the. Australian Medicinal and Food Plant Acacia ligulata

Transcription

1 Isolation and Structural Characterization of Echinocystic Acid Triterpenoid Saponins from the Australian Medicinal and Food Plant Acacia ligulata Diana Jæger,, Chi P. Ndi, Christoph Crocoll, Bradley S. Simpson, Bekzod Khakimov, Ruth Marian Guzman-Genuino,,,O John D. Hayball,,,O Xiaohui Xing, Δ,# Vincent Bulone, Δ,# Philip Weinstein, Birger L. Møller, and Susan J. Semple*, Sansom Institute for Health Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, 5000, SA, Australia Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Flinders Centre for Innovation in Cancer, Flinders University, Bedford Park, South Australia, 5042, Australia Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26, DK-1958 Frederiksberg C, Denmark 1

2 Experimental Therapeutics Laboratory, Hanson Institute and Sansom Institute, Adelaide, 5000, SA, Australia O Robinson Research Institute and Adelaide Medical School, University of Adelaide, Adelaide, SA, 5005, Australia Δ ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, Urrbrae, 5064, Australia # Division of Glycoscience, Royal Institute of Technology (KTH), School of Biotechnology, AlbaNova University Centre, Stockholm, SE-10691, Sweden Department of Ecology and Environmental Sciences, School of Biological Sciences, The University of Adelaide, Adelaide 5005, SA, Australia 2

3 Supporting information List of Contents A. Experimental Sample preparation and experimental conditions used for GC-MS Acid Hydrolysis of Saccharides Alkaline Hydrolysis of Saccharides B. Spectra and GC-MS traces Figure S1. 1 H NMR spectrum of ligulataside A (1). Figure S2. 1 H NMR spectrum of ligulataside B (2). Figure S3. 13 C NMR spectrum of ligulataside A (1). Figure S4. 13 C NMR spectrum of ligulataside B (2). Figure S5. APT spectrum of ligulataside A (1). Figure S6. APT spectrum of ligulataside B (2). Figure S7. HSQC spectrum of ligulataside A (1). Figure S8. HSQC spectrum of ligulataside B (2). Figure S9. COSY spectrum of ligulataside A (1). Figure S10. COSY spectrum of ligulataside B (2). Figure S11. HMBC spectrum of ligulataside A (1). Figure S12. HMBC spectrum of ligulataside B (2). Figure S13. TOCSY spectrum of ligulataside A (1). Figure S14. TOCSY spectrum of ligulataside B (2). Figure S15. ROESY spectrum of ligulataside A (1). Figure S16. ROESY spectrum of ligulataside B (2). Figure S17. Mass Spectrometry of ligulataside A (1). Figure S18. Fragmentation of ligulataside A (1). Figure S19. Mass Spectrometry of ligulataside B (2). 3

4 Figure S20. Fragmentation of ligulataside B (2). Figure S21. Aglycone identification by GC-MS after acid hydrolysis (Part 1). Figure S22. Aglycone identification by GC-MS after acid hydrolysis (Part 2). Figure S23. GC-MS traces of saccharide fractions from acid hydrolysis. Figure S24. GC-MS traces of saccharide fractions from acid hydrolysis. Figure S25. MS and MS/MS fragmentation the major peak in LC-MS traces after alkaline hydrolysis. Figure S26. MS fragmentation pattern of PMAAs from the two ligulataside samples. Figure S27. Total ion current (TIC) chromatogram of PMAAs from the two ligulataside compounds A (1) and B (2). Reference 4

5 Experimental Sample preparation and experimental conditions used for GC-MS Samples were derivatized prior to GC-MS injection by addition of 40 μl trimethylsilyl cyanide (TMSCN). 1 All steps involving sample derivatization and injection were automated using a MultiPurpose Sampler (MPS) (Gerstel, Mülheim an der Ruhr, Germany). After reagent addition, the sample was transferred into the agitator of the MPS and incubated at 40 C for 40 min at 750 rpm. Immediately after derivatization, 1 μl of the derivatized sample was injected into a cooled injection system port (CIS4, Gerstel) in the splitless mode. The septum purge flow and purge flow to split vent at 2.5 min after injection were set to 25 and 15 ml/min, respectively. The initial temperature of the CIS4 port was 120 C, and heated at 5 C/s to reach 320 C (after 30 s of equilibrium time), where it was kept for 10 min. After heating, the CIS4 port was gradually cooled to 250 C at 5 C/s, and this temperature was kept constant during the entire run. The GC-MS consisted of an Agilent 7890A GC and an Agilent 5975C series MSD (Agilent Technologies, Glostrup, Denmark). GC separation was performed on an Agilent HP-5MS column (30 m 250 μm 0.25 μm) using hydrogen as carrier gas at a constant flow rate of 1.2 ml/min. The GC oven temperature program was as follows: initial temperature 40 C, equilibration time 2.0 min, heated up to 270 C at 12 C/min, then heated at 6 C/min until 320 C and held for 10 min. Mass spectra were recorded in the range of m/z amu with a scanning frequency of 3.2 scans/s, and the MS detector was switched off during the 6 min of solvent delay time. The transfer line, ion source and quadrupole temperatures were set to 290 C, 230 C and 150 C, respectively. The mass spectrometer was tuned according to the manufacturer s recommendation by using perfluorotributylamine (PFTBA). The MPS and GC-MS was controlled using vendor software Maestro (Gerstel). Acid Hydrolysis of Saccharides Acid hydrolysis was performed by adding 500 µl of 2 M HCl to 1 mg of each compound, vortexing for 30 sec and then incubating the sample at 99 C and 1400 rpm for 1.5 h in a Thermomixer (Eppendorf, Germany). After the mixture had cooled down, the aglycone was separated from the saccharides by adding 900 µl of dimethyl ether followed by vortexing for 30 sec and centrifugation at x g (Eppendorf 5424 Microcentrifuge, Eppendorf, Hamburg, Germany) for 1 min to separate the phases. After centrifugation, 750 µl of the upper dimethyl ether phase was recovered. 5

6 The procedure was repeated three times by the addition of 750 µl dimethyl ether followed by the recovery of 600 µl of the dimethyl ether phase. The recovered dimethyl ether fractions were pooled in a 15 ml reaction tube and washed with twice the volume of MilliQ grade water ( 5 ml) followed by vortexing for 30 sec and centrifugation at 5000 x g for 3 min (Eppendorf 5424 Microcentrifuge). One ml of the dimethyl ether fractions was transferred to a 1.5 ml reaction tube and dried using a SpeedVac (ScanSpeed 32, Scanvac, Denmark) at 35 C for 1 h at 1000 rpm. After the fraction was completely dry, 200 µl of 100% MeOH were added and the tubes were vigorously vortexed prior to centrifugation at x g for 3 min (Eppendorf 5424 Microcentrifuge, Eppendorf, Hamburg, Germany). The acidic H2O fraction (500 µl), containing the hydrolysed saccharides was recovered from below the residual dimethyl ether fraction. The ph was adjusted to 7 by the addition of 1M NaOH. From both the methanol fraction and acidic H2O fraction, 100 µl of the hydrolyzed saponin solution or 10 µl of saccharide standards (L-arabinose, α-d-(+)-glucose, D-(+)-galactose, D-(+)- xylose, D-(+)-mannose, D-glucuronic acid, N-acetyl-D-glucosamine, 2-deoxy-D-glucose, D- galacturonic acid, L-(+)-rhamnose, and D-(-)-ribose from 2 mm stock solutions, all obtained from Sigma-Aldrich) and echinocystic acid standard (Extrasynthese, Genay Cedex, France) were transferred into a 200 µl glass insert, placed in a 2 ml reaction tube with a small hole in the lid and completely dried under reduced pressure using a SpeedVac (ScanSpeed 32, Scanvac, Lynge Denmark) at 35 C for 1.5 h at 1000 rpm. Completely dried samples in glass inserts were placed in GC-MS vials and sealed with air-tight golden magnetic lids and analyzed. Alkaline Hydrolysis of Saccharides. Similar to the acid hydrolysis procedure described above, HPLC-purified compounds 1 and 2 were used for alkaline hydrolysis to hydrolyze the saccharides attached to C-28 of the aglycone backbone via ester bonds. An aliquot containing 1 mg of each saponin was transferred to 1.7 ml screw cap reaction tube and completely dried using a speed vac for 1 h at 35 C, then 200 µl of 1 N NH4OH were added and vortexed for 30 sec. Alkaline hydrolysis was performed by incubation for 24 h at 60 C and 1400 rpm in a Thermomixer (Eppendorf). The reaction was cooled down to room temperature and neutralized by addition of formic acid. The resulting solution was subsequently purified using a C18 96 well extraction plate (Empore C18-SD (Octadecyl) Standard Density, 3M, 6

7 St. Paul, MN, USA). Elution was performed with 100 µl of 100% MeOH, then 10 µl of the eluate were diluted five-fold with MilliQ-grade H2O and subjected to LC-MS/MS analysis. 7

8 PROTON_UniSA_DAL109_cd3od_ _600_01.esp Methanol Normalized Intensity Chemical Shift (ppm) Figure S1. 1 H NMR spectrum of ligulataside A (1). 8

9 PROTON_UniSA_DJ_F127_135_cd3od_ _600_01b.esp Methanol Methanol Normalized Intensity Chemical Shift (ppm) Figure S2. 1 H NMR spectrum of ligulataside B (2). 9

10 CARBON_UniSA_DAL109_cd3od_ _600_01.esp Normalized Intensity Chemical Shift (ppm) Figure S3. 13 C NMR spectrum of ligulataside A (1). 10

11 CARBON_UniSA_DJ_F127_135_cd3od_ _600_01b.esp Normalized Intensity Chemical Shift (ppm) Figure S4. 13 C NMR spectrum of ligulataside B (2). 11

12 APT_UniSA_DAL109_cd3od_ _600_01.esp Normalized Intensity Chemical Shift (ppm) Figure S5. APT spectrum of ligulataside A (1). 12

13 APT_UniSA_DJ_F127_135_cd3od_ _600_01b.esp Normalized Intensity Chemical Shift (ppm) Figure S6. APT spectrum of ligulataside B (2). 13

14 F1 Chemical Shift (ppm) F2 Chemical Shift (ppm) Figure S7. HSQC spectrum of ligulataside A (1). 14

15 F1 Chemical Shift (ppm) F2 Chemical Shift (ppm) Figure S8. HSQC spectrum of ligulataside B (2). 15

16 F1 Chemical Shift (ppm) F2 Chemical Shift (ppm) Figure S9. COSY spectrum of ligulataside A (1). 16

17 F1 Chemical Shift (ppm) F2 Chemical Shift (ppm) Figure S10. COSY spectrum of ligulataside B (2). 17

18 F1 Chemical Shift (ppm) F2 Chemical Shift (ppm) Figure S11. HMBC spectrum of ligulataside A (1). 18

19 F1 Chemical Shift (ppm) F2 Chemical Shift (ppm) Figure S12. HMBC spectrum of ligulataside B (2). 19

20 F1 Chemical Shift (ppm) F2 Chemical Shift (ppm) Figure S13. TOCSY spectrum of ligulataside A (1). 20

21 F1 Chemical Shift (ppm) F2 Chemical Shift (ppm) Figure S14. TOCSY spectrum of ligulataside B (2). 21

22 F1 Chemical Shift (ppm) F2 Chemical Shift (ppm) Figure S15. ROESY spectrum of ligulataside A (1). 22

23 F1 Chemical Shift (ppm) F2 Chemical Shift (ppm) Figure S16. ROESY spectrum of ligulataside B (2). 23

24 Figure S17. Mass Spectrometry of ligulataside A (1). HRESIMS of ligulataside A (1) indicated a mass of m/z [M + H] +. Figure S18. Fragmentation of ligulataside A (1). A + B: MS/MS fragmentation of ligulataside A (1) indicates the loss of saccharide units and an acetyl group of m/z 42. The presence of m/z 366 and m/z 204 suggested the presence of an N-acetylglucosamine linked to a hexose unit (B). Neutral losses of saccharide units are indicated for pentoses [-132], methyl-pentose [-146], hexose [-162] and the acetyl group [-42] with the respective mass error. 24

25 Figure S19. Mass Spectrometry of ligulataside B (2). HRESIMS of ligulataside B (2) indicated a mass of m/z [M + H] +. Figure S20. Fragmentation of ligulataside B (2). A + B: MS/MS fragmentation of ligulataside B (2) indicates the loss of saccharide units. The presence of m/z 366 and m/z 204 suggested the presence of an N-acetylglucosamine linked to a hexose unit (B). Neutral losses of saccharide units are indicated for pentoses [- 132], methyl-pentose [-146], and hexose [-162] with the respective mass error. 25

26 Figure S21. Aglycone identification by GC-MS after acid hydrolysis (Part 1). A: Saponin fraction from acid hydrolysis of ligulataside A (1). B: Saponin fraction from acid hydrolysis of ligulataside B (2). C: Echinocystic acid standard. 26

27 Figure S22. Aglycone identification by GC-MS after acid hydrolysis (Part 2). A: MS spectra after derivatisation of the saponin fractions from the saponin fraction of the acid hydrolysis of ligulataside A (1). B: MS spectra after derivatisation of the saponin fractions from the saponin fraction of the acid hydrolysis of ligulataside B (2). C: Echinocystic acid standard. Identification is based on comparison of fragmentation pattern and retention time to those values from the standard of echinocystic acid. 27

28 Figure S23. GC-MS traces of saccharide fractions from acid hydrolysis. A: ligulataside A (1). B: ligulataside B (2). 28

29 Figure S24. GC-MS traces of saccharide fractions from acid hydrolysis. A: ligulataside A (1). B: ligulataside B (2). Inverted are shown the GC-MS traces of the identified saccharides: D-(+)-xylose (retention time (RT): min), L-arabinose (RT: min), L-(+)-rhamnose (RT: min), D-(+)-glucose (RT: min). N-acetyl- D-glucosamine (RT: min) was indicated to be present by results from fragmentation by LC-MS but could not be detected by GC-MS probably due to degradation. 29

30 Figure S25. MS and MS/MS fragmentation the major peak in LC-MS traces after alkaline hydrolysis. A: MS and MS/MS of m/z [M+H]+ (C44H72NO14) in ligulataside A (1). B: MS and MS/MS of m/z [M+H]+ (C44H72NO14) in ligulataside B (2). Glc = neutral loss corresponding to glucose. 30

31 Figure S26. MS fragmentation pattern of PMAAs from t-xylp, t-araf, 2-Arap, 3,4- Rhmp, t-glcp, and 4-GlcNAcp 31

32 Figure S27. Total ion current (TIC) chromatogram of PMAAs from the two ligulataside compounds A (1) and B (2). PMAAs from neutral saccharide and amino saccharide were tested using the SP2380 column (A and B) and the CP-Sil 5 CB column (C and D), respectively. 32

33 Reference (1) Khakimov, B.; Motawla, M. S.; Bak, S.; Engelsen, S. B. Anal. Bioanal. Chem. 2013, 405,